WO2019099509A1

WO2019099509A1 - Use of a p2x7 receptor antagonist to treat an inflammatory disease or disorder

Info

Publication number: WO2019099509A1
Application number: PCT/US2018/061037
Authority: WO
Inventors: Matthew Wright MCCLURE; Gary Steven DODSON
Original assignee: Second Genome, Inc.
Priority date: 2017-11-14
Filing date: 2018-11-14
Publication date: 2019-05-23

Abstract

The present disclosure relates to a method for the treatment, amelioration, and/or prevention of P2X7 receptor-mediated diseases or disorders in a subject in need thereof. Such disorders include liver fibrosis, nonalcoholic steatohepatitis, cirrhosis and hepatocellular carcinoma. The method includes the administration of a P2X7 receptor antagonist to the subject. Also described are compositions for treatment as well as dosing regimens.

Description

USE OF A P2X₇ RECEPTOR ANTAGONIST TO TREAT AN INFLAMMATORY

DISEASE OR DISORDER

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present Application claims the benefit of priority to U.S. Provisional Application Nos. 62/585,594, filed on November 14, 2017, and 62/655,660, filed on April 10, 2018, the contents of which are hereby incorporated by reference in their entirety.

FIELD OF THE DISCLOSURE

[0002] The present disclosure relates to methods for the treatment of diseases or disorders that are mediated by or associated with activation of the P2X7 receptor (such as liver fibrosis, non alcoholic steatohepatitis (NASH) or cirrhosis).

BACKGROUND OF THE DISCLOSURE

[0003] The worldwide incidence of nonalcoholic fatty liver disease (NAFLD) is rapidly increasing, paralleling the obesity epidemic (Vizuete et al., 2017, J Clin Translational Hepatol, 5:67-75). Although most patients have simple steatosis, some will develop nonalcoholic steatohepatitis (NASH), which is characterized by hepatocellular damage, inflammation and fibrosis. Persons with NASH are more likely to develop cirrhosis, advanced fibrosis and/or hepatocellular carcinoma.

[0004] Cellular injury plays a central role in triggering inflammation in NAFLD and facilitates its progression to the inflammatory phenotype associated with progression to NASH (Choi and Diehl, 2005, Cur Opin Gastroenterol, 21 :702-707; Diehl et al., 2005 Gut, 54:303-306; Tilg and Diehl, 2000, New Eng J Med, 343: 1467-1467). Chatterjee et al. (2012, Free Radic Biol Med, 52: 1666- 1679) describes studies which suggest that P2X7 receptor-mediated NADPH oxidase activation is a primary event in exacerbation of steatohepatitic lesions in obese mice and that in the obese mice, P2X7 receptor stimulation is indirectly involved in exacerbation of inflammatory responses in the liver. Das et al. (2013, Am J Physiol Gastrointest Liver Physiol, 305:G950-G963), e.g., suggest that the P2X7 receptor is a key regulator of autophagy induced by metabolic oxidative stress in NASH, thereby modulating hepatic inflammation. [0005] Activation of the P2X7 receptor induces cytosolic release of oxidized mitochondrial DNA, which promotes NLRP3 inflammasome assembly. NLRP3 inflammasome activation results in the production of IL-1 β, a pro-inflammatory cytokine. The observations that P2X7 receptors are localized on proinflammatory cells and that activation of P2X7 receptors modulates release of IL- 1 is consistent with a role for the P2X7 receptor in inflammatory diseases

[0006] Despite its impact and severity, there is no FDA-approved treatment for NASH. The present disclosure provides P2X7 receptor antagonists, formulations containing P2X7 receptor antagonist and methods of using P2X7 receptor antagonists for the treatment of liver disorders associated with P2X7 receptor activation, including NAFLD and NASH.

SUMMARY OF THE DISCLOSURE

[0007] The present disclosure provides, among other things, methods of treating diseases or disorders that are mediated by or associated with activation of the P2X7 receptor by administering a therapeutically effective amount of a P2X7 receptor antagonist. In some embodiments, the P2X7 receptor antagonist is a compound of Formula I, Formula II, Formula III, Formula Ilia, Formula IV, or Formula IVa or a pharmaceutically acceptable salt thereof.

[0008] In some embodiments, the present disclosure provides a method of treating liver disease associated with P2X7 receptor activation by administering a therapeutically effective amount of a P2X7 receptor antagonist. In some embodiments, the P2X7 receptor antagonist is a compound of Formula I, Formula II, Formula III, Formula Ilia, Formula IV, or Formula IVa or a

pharmaceutically acceptable salt thereof. In some embodiments, the P2X7 receptor antagonist is Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6 or

Compound 7 or a pharmaceutically acceptable salt thereof. In some embodiments, the P2X7 receptor antagonist is Compound 1 or a pharmaceutically acceptable salt thereof.

[0009] In some embodiments, the present disclosure provides a method of treating nonalcoholic steatohepatitis (NASH). In further embodiments, the NASH is Fibrosis stage 1-3 NASH as determined by two-dimensional magnetic resonance elastography (2D MRE) score. [0010] In some embodiments, the present disclosure provides a method of treating Fatty Liver Disease (FLD). In some embodiments, the present disclosure provides a method of treating Nonalcoholic Fatty Liver Disease (NAFLD).

[0011] In some embodiments, the present disclosure provides a method of treating liver disease associated with P2X7 receptor activation where prior to administering the P2X7 receptor antagonist, the patient has advanced liver fibrosis as determined by 2D MRE score.

[0012] In some embodiments, the present disclosure provides a method of treating liver disease associated with P2X7 receptor activation where prior to administering the P2X7 receptor antagonist, the patient has liver cirrhosis as determined by 2D MRE score.

[0013] In some embodiments, the present disclosure provides a method of treating liver disease associated with P2X7 receptor activation where prior to administering the P2X7 receptor antagonist, the patient’s liver stiffness as determined by MRE is between about 2.5 kPa to about 4.65 kPa.

[0014] In some embodiments, the present disclosure provides a method of treating liver disease associated with P2X7 receptor activation where prior to administering the P2X7 receptor antagonist, the patient’s proton density fat fraction (PDFF) as determined by multiparametric magnetic resonance is≥ 8% steatosis.

[0015] In some embodiments, the present disclosure provides a method of treating liver disease associated with P2X7 receptor activation where prior to administering the P2X7 receptor antagonist, the patient’s cTl score as determined by multiparametric magnetic resonance is≥ 827.5 ms.

[0016] In some embodiments, the present disclosure provides a method of treating liver disease associated with P2X7 receptor activation where prior to administering the P2X7 receptor antagonist, the patient’s liver stiffness as determined by ultrasound-based transient elastography is > 8.

[0017] In some embodiments, the present disclosure provides a method of treating liver disease associated with P2X7 receptor activation where prior to administering the P2X7 receptor antagonist, the patient’s controlled attenuation parameter (CAP) as determined by ultrasound- based transient elastography (Fibroscan) is≥300 dB/m.

[0018] In some embodiments, the present disclosure provides a method of treating liver disease associated with P2X7 receptor activation where prior to administering the P2X7 receptor antagonist, the patient’s alanine aminotransferase (ALT) is between about 5 IU/L and 225 IU/L.

[0019] In some embodiments, the present disclosure provides a method of treating liver disease associated with P2X7 receptor activation where prior to administering the P2X7 receptor antagonist, the patient’s Steatosis, Activity, and Fibrosis (SAF) activity score is≥ 2.

[0020] In some embodiments, the present disclosure provides a method of treating liver disease associated with P2X7 receptor activation where prior to administering the P2X7 receptor antagonist, the patient’s SAF activity score is <2.

[0021] In some embodiments, the present disclosure provides a method of treating liver disease associated with P2X7 receptor activation where prior to administering the P2X7 receptor antagonist, the difference in the patient’s spleen and liver Hounsfield units (HUspieen-HUiiver) as determined by CT scan is greater than 0.

[0022] In some embodiments, the present disclosure provides a method of treating liver disease associated with P2X7 receptor activation in a patient diagnosed with at least 1 disease or disorder selected from the group consisting of hepatocellular carcinoma, obesity, insulin resistance diabetes and Type 2 diabetes.

[0023] In some embodiments, the present disclosure provides a method of treating a liver disease associated with P2X7 receptor activation comprising administering for at least 1 week to a patient in need thereof a therapeutically effective amount of compound of Formula (III):

or a pharmaceutically acceptable salt thereof, wherein

R¹ is selected from C₂-C₄ alkyl optionally substituted with -OH, -NH₂, and -CONH₂;

R² is selected from CH₃ and halogen;

R³ is selected from H and CH₃; and

R⁵ andR⁶ are independently selected from H, halogen and CF₃.

[0024] In some embodiments, the compound of Formula (III) has an IC50 against the P2X7 receptor of about 0.1 nM to about 10 nM as determined by an in vitro assay that measures the concentration of IL-1 β in whole blood samples.

[0025] In some embodiments, the compound of Formula (III) has an IC50 against the P2X7 receptor of about 10 nM to about 100 nM as determined by an in vitro assay that measures the concentration of IL-1 β in whole blood samples.

[0026] In some embodiments, the method of treating a liver disease associated with P2X7 receptor activation comprises administering for at least 1 week to a patient in need thereof a total daily dose of about 50 mg to about 750 mg of the compound of Formula (III) or a

pharmaceutically acceptable salt thereof.

[0027] In some embodiments, the method of treating a liver disease associated with P2X7 receptor activation comprises administering for at least 1 week to a patient in need thereof a compound of Formula (III) to provide a mean blood plasma

of about 10,000 to about 50,000 ng h/mL of the compound of Formula (III).

[0028] In some embodiments, the method of treating a liver disease associated with P2X7 receptor activation comprises administering for at least 1 week to a patient in need thereof a compound of Formula (III) to provide a mean steady state blood plasma concentration of greater than about 125 ng/mL of the compound of Formula (III).

[0029] In some embodiments, the compound of Formula (III) is administered in an immediate release tablet. In some embodiments, the compound of Formula (III) is administered with food.

[0030] In some embodiments, the compound of Formula (III) is administered for at least about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 12 weeks, about 18 weeks, about 24 weeks, or about 50 weeks.

[0031] In some embodiments, the present disclosure provides a method of treating a liver disease associated with P2X7 receptor activation comprising administering for at least 1 week to a patient in need thereof a therapeutically effective amount of a compound selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.

[0032] In some embodiments, the present disclosure provides a method of treating a liver disease associated with P2X7 receptor activation comprising administering for at least 1 week to a patient in need thereof a therapeutically effective amount of Compound 1 :

[0033] or a pharmaceutically acceptable salt thereof. In certain embodiments, a therapeutically effective amount of Compound 1 is administered (i.e., not a pharmaceutically acceptable salt).

[0034] In some embodiments, the present disclosure provides methods of treating nonalcoholic steatohepatitis (NASH) comprising administering for at least 1 week to a patient in need thereof a therapeutically effective amount of Compound 1 or a pharmaceutically acceptable salt thereof. In certain embodiments, the method of treating NASH comprises administering for at least 1 week to a patient in need thereof Compound 1 or a pharmaceutically acceptable salt thereof three times daily. In certain embodiments, the method of treating NASH comprises administering for at least 1 week to a patient in need thereof Compound 1 or a pharmaceutically acceptable salt thereof twice daily. In some embodiments, the method of treating NASH comprises

administering for at least 1 week to a patient in need thereof Compound 1 or a pharmaceutically acceptable salt thereof once daily. In certain embodiments, the method of treating NASH comprises administering for at least 1 week to a patient in need thereof a total daily dose of Compound 1 or a pharmaceutically acceptable salt thereof of about 50 mg to about 500 mg. In certain embodiments, the method of treating NASH comprises administering for at least 1 week to a patient in need thereof a total daily dose of Compound 1 or a pharmaceutically acceptable salt thereof of about 500 mg. In certain embodiments, the method of treating NASH comprises administering for at least 1 week to a patient in need thereof a total daily dose of Compound 1 or a pharmaceutically acceptable salt thereof of about 200 mg. In certain embodiments, the method of treating NASH comprises administering for at least 1 week to a patient in need thereof a total daily dose of Compound 1 or a pharmaceutically acceptable salt thereof of about 100 mg. In certain embodiments, the method of treating NASH comprises administering for at least 1 week to a patient in need thereof about 250 mg of Compound 1 or a pharmaceutically acceptable salt thereof twice a day to provide a substantial reduction in NASH. In certain embodiments, the method of treating NASH comprises administering for at least 1 week to a patient in need thereof about 100 mg of Compound 1 or a pharmaceutically acceptable salt thereof twice a day to provide a substantial reduction in NASH. In certain embodiments, the method of treating NASH comprises administering for at least 1 week to a patient in need thereof about 50 mg of

Compound 1 or a pharmaceutically acceptable salt thereof twice a day to provide a substantial reduction in NASH.

[0035] In some embodiments, the method of treating a liver disease associated with P2X7 receptor activation comprises administering for at least 1 week to a patient in need thereof a total daily dose of about 50 mg to about 750 mg of the compound of Compound 1 or a

pharmaceutically acceptable salt thereof.

[0036] According to some embodiments, the method of treating a liver disease associated with P2X7 receptor activation comprises administering for at least 1 week to a patient in need thereof a total daily dose of about 50 mg to about 500 mg of Compound 1.

[0037] In some embodiments, the method of treating a liver disease associated with P2X7 receptor activation comprises administering for at least 1 week to a patient in need thereof a total daily dose of about 500 mg of Compound 1. In some embodiments, the method of treating a liver disease associated with P2X7 receptor activation comprises administering for at least 1 week to a patient in need thereof about 250 mg of Compound 1 twice daily.

[0038] In some embodiments, the method of treating a liver disease associated with P2X7 receptor activation comprises administering for at least 1 week to a patient in need thereof a total daily dose of about 100 mg of Compound 1. In some embodiments, the method of treating a liver disease associated with P2X7 receptor activation comprises administering for at least 1 week to a patient in need thereof about 50 mg of Compound 1 twice daily.

[0039] In some embodiments, the method of treating a liver disease associated with P2X7 receptor activation comprises administering for at least 1 week to a patient in need thereof a compound of Compound 1 to provide a mean blood plasma

of about 10,000 to about 50,000 ng h/mL of the Compound 1.

[0040] In some embodiments, the method of treating a liver disease associated with P2X7 receptor activation comprises administering for at least 1 week to a patient in need thereof a compound of Compound 1 to provide a mean steady state blood plasma concentration of greater than about 125 ng/mL of the Compound 1.

[0041] In some embodiments, the method of treating a liver disease associated with P2X7 receptor activation comprises administering for at least 1 week to a patient in need thereof Compound 1 to provide a mean steady state blood plasma AUC (0-12) hours from about 1500 ng*h/ml to about 10500 ng*h/ml of Compound 1.

[0042] In some embodiments, the method of treating a liver disease associated with P2X7 receptor activation comprises administering for at least 1 week to a patient in need thereof Compound 1 to provide a mean steady state blood plasma Cmin from about 150 ng/mL to about 550 ng/ml of Compound 1.

[0043] In some embodiments, the method of treating a liver disease associated with P2X7 receptor activation comprises administering for at least 1 week to a patient in need thereof Compound 1 to provide a mean steady state blood plasma from about 150 ng/mL to about 550 ng/ml of Compound 1. [0044] In some embodiments, after treatment for a period of at least 1 week, the patient experiences no worsening in liver disease compared to prior to said administering.

[0045] According to some embodiments, after the treatment the patient experiences a substantial reduction in liver disease compared to prior to said administering. In some embodiments, after the treatment the patient experiences a reduction in liver disease that is characterized by an at least 2% reduction in the patient’s cTl score as determined by multiparametric magnetic resonance. In some embodiments, after the treatment the patient experiences a reduction in liver disease that is characterized by an at least 2 point improvement in Nonalcoholic Fatty Liver Disease Activity Score (NAS) value. In some embodiments, after the treatment the patient experiences a reduction in liver disease that is characterized by an at least 50% reduction in NAS value. In some embodiments, after the treatment the patient experiences a reduction in liver disease that is characterized by an at least 10% reduction in alanine aminotransferase (ALT) compared to prior to administering. In some embodiments, after the treatment the patient experiences a reduction in liver disease that is characterized by an at least 10% reduction in aspartate aminotransferase (AST) compared to prior to administering. In some embodiments, after the treatment the patient experiences a reduction in liver disease that is characterized by an at least 10% reduction in liver stiffness as determined by magnetic resonance elastography (MRE) compared to prior to administering. In some embodiments, after the treatment the patient experiences a reduction in liver disease that is characterized by an at least 1 point reduction in Ultrasonographic Steatosis Score (USS). In some embodiments, after the treatment the patient experiences a reduction of liver disease that is characterized by an increase of the patient’s liver Hounsfield units (HU) to at least about 20 HU as determined by CT scan. In some embodiments, after the treatment the patient experiences a reduction of liver disease that is characterized by a hepatic venous pressure gradient (HVPG) of less than about 12 mm Hg. In some embodiments, after the treatment the patient experiences a reduction of liver disease that is characterized by a decrease in blood IL-1 β concentration of at least about 30% compared to prior to the

administering.

[0046] In some embodiments, the present disclosure provides a method of treating a liver disease associated with P2X7 receptor activation comprising administering a compound of Formula (III) and a drug selected from the group consisting of a 5-aminosalicyate agent, a corticosteroid, and an antibiotic, or a combination thereof.

[0047] In some embodiments, after administering for a period of at least 1 week, 2 weeks, 3 weeks or 4 weeks, the patient experiences a decrease in the blood concentration of IL-1 β compared to prior to the administering.

[0048] In some embodiments, the decrease in the patient’s blood concentration of IL-1 β is at least about 30%, about 40%, about 50%, about 60%, about 70% or about 80% compared to prior to the administering.

[0049] According to some embodiments, the present disclosure provides methods of treating liver disease associated with P2X7 receptor activation comprising administering for at least a week to a patient in need thereof a therapeutically effective amount of compound of Formula:

or a pharmaceutically acceptable salt thereof. In certain further embodiments, the liver disease associated with P2X7 receptor activation is NASH.

[0050] According to some embodiments, the present disclosure provides methods of treating a liver disease associated with P2X7 receptor activation comprising administering for at least 1 week to a patient in need thereof a therapeutically effective amount of compound of Formula:

or a pharmaceutically acceptable salt thereof, wherein the administering provides a mean steady state blood plasma AUC (0-12) hours from about 1500 ng*h/ml to about 10500 ng*h/ml of the compound. In certain further embodiments, the liver disease associated with P2X7 receptor activation is NASH.

[0051] According to some embodiments, the present disclosure provides methods of treating a liver disease associated with P2X7 receptor activation comprising administering for at least a week to a patient in need thereof a therapeutically effective amount of compound of Formula:

or a pharmaceutically acceptable salt thereof, wherein the administering provides a mean steady state blood plasma from about 150 ng/mL to about 550 ng/ml of the compound. In certain further embodiments, the liver disease associated with P2X7 receptor activation is NASH.

[0052] According to some embodiments, the present disclosure provides methods of treating a liver disease associated with P2X7 receptor activation comprising administering for at least a week to a patient in need thereof a therapeutically effective amount of compound of Formula:

or a pharmaceutically acceptable salt thereof, wherein about 50 mg to about 500 mg of the compound is administered daily. In certain further embodiments, the liver disease associated with P2X7 receptor activation is NASH.

[0053] According to some embodiments, the patient treated according to the methods of the present disclosure, e.g., treated with Compound 1, experiences no decrease in the size, number, and/or density of hepatic lipid vacuoles as compared to an untreated patient or a patient treated with a placebo.

[0054] According to some embodiments, the present disclosure provides methods for inhibiting P2X7 receptor activity in the cells of a subject in need thereof comprising administering to the subject a dose of about 100 to about 2000 mg of a P2X7 receptor antagonist, wherein the P2X7 receptor antagonist has an IC₅₀ of about 0.1 nM to about 10 nM as determined by an in vitro assay that measures the concentration of IL-1 β in whole blood samples.

DETAILED DESCRIPTION OF THE DRAWINGS

[0055] FIGS. 1A-1B show in vitro release profiles for immediate release tablets containing 50 mg (FIG. 1A) and 250 mg (FIG. 1B) of Compound 1.

[0056] FIG. 2 shows plasma concentrations of Compound 1 in patients orally administered immediate release tablets containing Compound 1.

[0057] FIG. 3 shows plasma concentrations of Compound 1 resulting from twice daily dosing of an immediate release dosage form. [0058] FIGS. 4A-4C illustrate the effects of treatment with Compound 1 in a fibrosis animal model on plasma levels of ALT (FIG. 4A), AST (FIG. 4B) and total bilirubin (FIG. 4C) on Day 21 of treatment.

[0059] FIGS. 5A to 5C illustrate effects of treatment with Compound 1 in a fibrosis animal model on plasma levels of ALT (FIG. 5A), AST (FIG. 5B) and total bilirubin (FIG. 5C) at the completion of treatment.

[0060] FIGS. 6A-6F show results of the pathological evaluation of liver samples from animals taken at the completion of the study as described in Example 8. Scores were determined for inflammation (FIG. 6A), ballooning degeneration (FIG. 6B), steatosis (FIG. 6C), and fibrosis (FIG. 6D). The total score for each group is illustrated in FIG. 6E. FIG. 6F shows a decrease in a-SMA in serum samples taken from the animals.

[0061] FIG. 7 illustrates pharmacokinetic values determined in subjects treated twice daily with 5 mg per kg (mpk), 15 mpk, or 30 mpk of a P2X7 receptor antagonist.

[0062] FIG. 8 illustrates activity of a P2X7 receptor antagonist to reduced liver fibrosis in a rodent NASH model.

DEFINITIONS

[0063] Throughout this disclosure, various patents, patent applications and publications are referenced. The disclosures of these patents, patent applications and publications in their entireties are incorporated into this disclosure by reference for all purposes in order to more fully describe the state of the art as known to those skilled therein as of the date of this disclosure.

This disclosure will govern in the instance that there is any inconsistency between the patents, patent applications and publications cited and this disclosure.

[0064] For convenience, certain terms employed in the specification, examples and claims are collected here. Unless defined otherwise, all technical and scientific terms used in this disclosure have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure belongs. [0065] Throughout this specification, the word“comprise” or variations such as“comprises” or “comprising” will be understood to imply the inclusion of a stated component, or group of components, but not the exclusion of any other components, or group of components.

[0066] The term“a” or“an” refers to one or more of that entity, i.e. can refer to a plural referents. As such, the terms“a” or“an,”“one or more” and“at least one” are used interchangeably herein. In addition, reference to“an element” by the indefinite article“a” or“an” does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there is one and only one of the elements.

[0067] The term“including” is used to mean“including but not limited to.” “Including” and “including but not limited to” are used interchangeably.

[0068] The term“about” when immediately preceding a numerical value means a range (e.g., plus or minus 10% of that value). For example,“about 50” can mean 45 to 55,“about 25,000” can mean 22,500 to 27,500, etc., unless the context of the disclosure indicates otherwise, or is inconsistent with such an interpretation. For example in a list of numerical values such as“about 49, about 50, about 55, ...”,“about 50” means a range extending to less than half the interval(s) between the preceding and subsequent values, e.g., more than 49.5 to less than 52.5.

Furthermore, the phrases“less than about” a value or“greater than about” a value should be understood in view of the definition of the term“about” provided herein. Similarly, the term “about” when preceding a series of numerical values or a range of values (e.g.,“about 10, 20,

30” or“about 10-30”) refers, respectively to all values in the series, or the endpoints of the range.

[0069] The terms“administer,”“administering” or“administration” as used herein refer to either directly administering a compound or pharmaceutically acceptable salt or ester of the compound or a composition comprising the compound or pharmaceutically acceptable salt or ester of the compound to a patient.

[0070] As used herein, the term "route" of administration is intended to include, but is not limited to oral, intravenous injection, intravenous or subcutaneous infusion, subcutaneous injection, subcutaneous depot, intradermal injection, intramuscular injection, intraperitoneal injection, intratracheal administration, intraadiposal administration, intraarticular administration, intrathecal administration, inhalation, intranasal administration, sublingual administration, buccal administration, rectal administration, vaginal administration, intracisternal administration and topical administration, transdermal administration, or administration via local delivery (for example by catheter or stent).

[0071] The term“salts” as used herein embraces pharmaceutically acceptable salts commonly used to form alkali metal salts of free acids and to form addition salts of free bases. The nature of the salt is not critical, provided that it is pharmaceutically acceptable. The term“salts” also includes solvates of addition salts, such as hydrates, as well as polymorphs of addition salts. Suitable pharmaceutically acceptable acid addition salts can be prepared from an inorganic acid or from an organic acid. Examples of such inorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric, and phosphoric acid. Appropriate organic acids can be selected from aliphatic, cycloaliphatic, aromatic, arylaliphatic, and heterocyclyl containing carboxylic acids and sulfonic acids, for example formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, mesylic, stearic, salicylic, p-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, toluenesulfonic, 2- hydroxyethanesulfonic, sulfanilic, cyclohexylaminosulfonic, algenic, 3-hydroxybutyric, galactaric and galacturonic acid. “Salts” include, without limitation, amino acid salts, inorganic acid salts (such as chloride, sulfate, phosphate, diphosphate, bromide, and nitrate salts) and organic acid salts (such as malate, maleate, fumarate, tartrate, succinate, ethylsuccinate, citrate, acetate, lactate, methanesulfonate, benzoate, ascorbate, para-toluenesulfonate, palmoate, salicylate and stearate, as well as estolate, gluceptate and lactobionate salts). Similarly,“salts” containing pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium (including substituted ammonium).

[0072] "Optional" or "optionally" as used herein means that the subsequently described element, component or circumstance may or may not occur, so that the description includes instances where the element, component, or circumstance occurs and instances where it does not.

[0073] The terms“effective amount” and“therapeutically effective amount” are used

interchangeably in this disclosure and refer to an amount of a compound, or a salt, solvate or ester thereof, that, when administered to a patient, is capable of performing the intended result. For example, an effective amount of Compound 1 is that amount that is required to reduce at least one symptom of a disease associated with P2X7 receptor activation in a patient. The actual amount that comprises the“effective amount” or“therapeutically effective amount” will vary depending on a number of conditions including, but not limited to, the severity of the disorder, the size and health of the patient, and the route of administration. A skilled medical practitioner can readily determine the appropriate amount using methods known in the medical arts.

[0074] The terms "subject," "individual" or "patient" are used interchangeably herein and refer to a vertebrate, preferably a mammal. Mammals include, but are not limited to, humans, non-human primates, and rodents such as mice and rats.

[0075] The term "drug" or "active agent" is used herein to refer to any chemical that elicits a biochemical response when administered to a human or an animal. The drug may act as a substrate or product of a biochemical reaction, or the drug may interact with a cell receptor and elicit a physiological response, or the drug may bind with and block a receptor from eliciting a physiological response.

[0076] "Pharmaceutically acceptable" includes those compounds, materials, compositions, or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[0077] The term“therapeutic effect” as used herein refers to a desired or beneficial effect provided by the method and/or the composition. For example, the method for treating NASH provides a therapeutic effect when the method reduces at least one symptom of NASH in a patient.

[0078] The term“treating” as used herein with regard to a patient, refers to improving at least one symptom of the patient’s disorder. Treating can be curing, improving, or at least partially ameliorating a disorder. The patient may be a mammal, e.g. particularly a human, a non-human primate or a rodent.

DETAILED DESCRIPTION OF THE DISCLOSURE [0079] The P2X7 receptor is a member of the P2X receptor family, a family of ionotropic receptors that are cationic ligand-operated channels that upon ATP binding open the pore permeable to Na+, K+, and Ca++ (Surprenant et al., 1996, Science, 272:735-738; Rassendren et al, 1997, J Biol Chem, 272:5482-5486; Buell et al., 1996, Eur J Neurosci, 8:2221-2228). Activation of the P2X7 receptor has been shown to induce fast release into the cytosol of oxidized mitochondrial DNA that promotes NLRP3 inflammasome assembly (Nakahira et al., 2011, Nat Immunol, 12:222-230; Shimada et al., 2012, Immunity, 36:401-414). Aberrant inflammasome signaling, including chronic NLRP3 inflammasome activation, has been associated with diseases such as age-related macular degeneration (AMD) (Marneros, 2013, Cell Rep, 4:945-958; Tarallo et al., 2012, Cell, 149:847-859) and Alzheimer’s disease (Halle et al, 2008,). Such disease associations include observations of NLRP3 -driven production of IL-1 β, a pro-inflammatory cytokine (Marneros, ibid).

[0080] Inflammation plays a role in the onset and progression of a large variety of diseases and disorders. Both the localization of P2X7 receptors on proinflammatory cells and the demonstration that activation of P2X7 receptors modulates release of IL-1 β is consistent with a role for this receptor in inflammatory diseases (North, 2002, Physiol Rev, 82: 1013-167; Baraldi et al., 2003, J Med Chem, 45: 1318-1329; Honore et al., 2006, J Pharmacol Exp Ther, 319: 1376-1385). Accordingly, the P2X7 receptor has become an attractive therapeutic target for inflammatory disorders and numerous antagonists of the P2X7 receptor have been developed for potential use in the treatment of inflammation-mediated diseases or disorders.

[0081] Inflammation plays a role in various hepatic diseases. Non-alcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease in the United States (Peery et al, 2012, Gastroenterology, 143: 1179-1187) and is associated with obesity, insulin resistance, and type 2 diabetes. Seven to thirty percent of NAFLD patients will develop non-alcoholic steatohepatitis (NASH), and a third of those will develop advanced fibrosis or cirrhosis. Patients suffering from NASH are also more prone to develop hepatocellular carcinoma (Vernon et al., 2011, Aliment Pharmacol Ther, 34:274-285; Cotrim et al., 2016, Clinics (Sao Paulo), 71 :281-284).

[0082] Inflammation also is a critical component in the progression NAFLD to the more serious NASH. NASH is characterized by hepatocellular damage, inflammation and fibrosis. The progression of simple steatosis to NASH is explained in part by a“two-hit” hypothesis (Day and James, 1998, Gastroenterology, 114:842-845) whereby the first hit is the dysregulated hepatic lipid accumulation in hepatocytes and the second hit includes oxidative, metabolic and cytokine stressors, which lead to hepatocyte injury and/or apoptosis, inflammation, and then fibrosis (Chatterjee and Das, 2015, Oxidative Med Cell Longevity, 172493; Syn et al, 2009, Clin Liver Dis, 13:565-580).

[0083] Various studies suggest that inhibition of NLRP3 -containing inflammasome activation can reduce liver inflammation and liver pathology in NASH mouse models. Mridha et al. (2017, J Hepatol, 66: 1037-1046) administered a small molecule NLRP3 inhibitor to a mouse experimental NASH model and showed that this inhibitor partly reversed liver inflammation.

[0084] Accordingly, described herein are uses of a class of P2X7 receptor antagonists for the treatment of subjects suffering from liver diseases associated with inflammation of the liver, such as NASH, cirrhosis and liver fibrosis. The P2X7 receptor is a well-known activator of the NLRP3- containing inflammasome. In response to inflammatory stimuli such as LPS, IL-1 β is synthesized as a biologically inactive 31 kDa pro-cytokine that is retained in the cytosol of the cell. A second stimulus is then required for the efficient proteolytic processing and release of the biologically active 17 kDa IL-1 β. ATP, acting via the P2X7 receptor, can serve as this secondary signal. P2X7 receptor activation leads to disruption of the normal ionic gradients in the cell and sequential recruitment of the proteins ASC and caspase-l to NLRP3. Caspase-l is then activated by autocatalytic processing and subsequently cleaves inactive IL-1 β to the active form. Mature IL-1 β is then released into the extracellular space (Sluyter, 2004; Mariathasan, 2006, Nature, 440:228- 232; Ferrari, 2006, J Immunol, 176:3877-3883; Sharma and Kanneganti, 2016, J Cell Biol, 213:617-629). ATP-activation of the P2X7 receptor and caspase-l also plays a role in processing and secretion of Pro-IL-l8 and Pro-IL-la (Yazdi and Drexler, 2013, Ann Rheum Dis, 72 Suppl 2:ii96-99; Ferrari et al., ibid, Arulkumaran et al., 2011, Expert Opin lnvestig Drugs, 20:8997-915). In addition, the naturally occurring IL-l receptor antagonist (IL-Ra) can be released from cells in a P2X7 receptor dependent fashion (Ferrari, ibid, Wilson, 2004, J Immunol, 173: 1202-1208).

[0085] In some embodiments, the methods of the present disclosure are used to treat a disease or disorder that is mediated by activation of the NLRP3 inflammasome pathway. In some embodiments, the disease or disorder is selected from the group consisting of: renal fibrosis, lung fibrosis, cardiac fibrosis, diabetic kidney disease, chronic kidney disease, heart failure, and metabolic heart failure.

In vitro assay for effects of a P2X7 receptor antagonist on IL-Ib secretion

[0086] IL-1 β, a pro-inflammatory cytokine, is a primary cause of inflammation and a key mediator of host response to infections (Ferrari et al., 2006, J Immunol, 176:3877-3883). IL- l b levels in the blood are increased in several chronic diseases including arthritis, scleroderma, systemic lupus erythematosus, vasculitis, sepsis, septic shock, and in the presence of atherosclerotic lesions leading to myocardial infarction (Dinarello, 2002, Clin Exp Rheumatol, 20:Sl-Sl3). As noted above, activation of the P2X7 receptor, e.g., in the presence of ATP, results in activation of the NLRP3 inflammasome as well as the release of mature IE-1b. Accordingly, there has been development of selective P2X7 receptor antagonists (Letavic et al., 2017, J Medicinal Chem, 60:4559-4572 and U.S. Patent No. 8,093,265, the contents of which are hereby incorporated by reference in their entirety for all purposes.). Preclinical animal studies with several P2X7 receptor antagonists have demonstrated activity of the antagonists (Honore et al, 2006, J Pharmacol Exp Ther, 319: 1376-1385; Carroll et al, 2007, Bioorg Med Chem Lett, 17:4044-4048).

[0087] One means of assessing the efficacy of a P2X7 receptor antagonist is to measure the ability of the antagonist to inhibit or reduce production of IL-1 β by cells that express P2X7 receptor. Such an assay can be performed in vitro, for example, in which cells such as THP-l monocytic cells are stimulated with LPS and incubated with an ATP analogue (e.g., 3’-0-(4-benzoyl)benzoyl- adenosine 5’-triphosphate (BzATP) and with varying concentrations of the P2X7 receptor antagonist to determine the effects of the antagonist on IL-1 β release by the cells (Grahames et al., 1999, Br J Pharmacol, 127: 1915-1921 ; Michel et al, 2006, Br J Pharmacol, 153:737-750). Example 3 describes experiments performed using this assay to show that treatment of LPS- stimulated THP-l cells with Compound 1 resulted in the inhibition of ATP-induced IE-1b release by the cells.

[0088] Alternatively or additionally, an assay can be performed in which IE-1 b production is measured in whole blood wherein whole blood from untreated subjects is collected into tubes coated with sodium heparin, and incubated with 200 ng/ml LPS at about 37°C in 5% CO₂ for about 1.5 hours. A P2X7 receptor antagonist is then added at a range of concentrations (e.g., 1 nM to 10 M) and ATP is added at a concentration of about 2.5 mM. Samples are then incubated at about 37°C in 5% CO₂ for about 45 min. IL-1 β content in the plasma obtained from the treated blood sample is then measured as a function of agonist concentration. As shown in Example 1 below, incubation of whole blood from untreated subjects, with LPS, then with Compound 1 and ATP resulted in an inhibition of IL-1 β production in the whole blood samples.

[0089] An in vitro assay can be used to assess the efficacy of a P2X7 receptor antagonist at blocking ATP-dependent IL-1 β release in LPS-primed whole blood following oral dosing of subjects with the antagonist (Ali et al., Br J Clin Pharmacol, 75: 197-207). In this assay, a whole blood sample is obtained from subjects treated with varying doses of a P2X7 receptor antagonist, wherein the samples are taken pre-dose and at different time points after dosing. This in vitro assay can be used to determine a dose of P2X7 receptor antagonist that is effective in inhibiting IL-1 β production in blood, thereby providing information regarding therapeutically effective dosing amounts for inhibition of P2X7 receptor activation in vivo, inhibition of NLRP3 inflammasome activation, and/or inhibition of in vivo inflammation. Specifically, about 200 μL of whole blood samples are treated with 25 μL of 2 pg LPS in RPMI 1640 + 25 mM HEPES and the mixed samples are incubated for about 1 hour at about 37 °C. To the samples, 25 μL of an ATP solution (ranging from 0 to about 330 mM) is added. Negative control samples are also prepared which lack LPS and/or ATP. The samples are incubated for about 45 min at about 37 °C. The samples are then centrifuged for about 2 minutes at 1000xg and about 60 μL of plasma from each sample is used for analysis of IL-1 β concentrations, e.g., by ELISA.

[0090] Example 5 below demonstrates, e.g., that when whole blood is assayed about 4 hours after administration, doses of Compound 1 ranging from about 80-1280 mg resulted in statistically significant inhibition of ATP-dependent IL-1 β release from whole blood and that at dose levels of about 160 mg to 1280 mg, maximal inhibition was about 81% to 91%.

[0091] In some embodiments, the methods of the present disclosure are used to treat a disease or disorder that is mediated by IL-l . In other embodiments, the disease or disorder is mediated by IL-1 β. P2X7 Receptor Antagonists for Treatment of Liver Disorders

[0092] The Applicants conducted preclinical studies that show that P2X7 receptor antagonists are effective for treating inflammatory liver disorders. Specifically, an animal model was used in which liver fibrosis was induced in cynomolgus monkeys using carbon tetrachloride (CCl₄). These studies are described below in Example 8 and illustrated in part in FIGS. 4-6. The studies show that while CCl₄ induces liver fibrosis in the animals, administration of a P2X7 receptor antagonist (e.g., Compound 1) to these animals results in reduced pathology of liver tissue. For example, administration of a composition comprising Compound 1 resulted in decreased inflammation, liver ballooning (hepatocyte apoptosis, a characteristic of NASH) and fibrosis. Also observed was a decrease in stellate cell activation during liver fibrosis in these animals. a-SMA (a small muscle actin) expression increases upon stellate cell activation or transdifferentiation from healthy quiescent cells to a myofibroblast-like cell characteristic of steatohepatitis.

[0093] Applicants conducted preclinical studies that show that P2X7 receptor antagonists are effective for treating NASH. An experiment was also performed by Applicants to show the effectiveness of the P2X7 receptor antagonist A438079 (3-[[5-(2, 3-Dichlorophenyl)-1H-tetrazol- 1-yl]methyl]pyridine hydrochloride) in reducing liver fibrosis in a mouse NASH model. In this experiment, neonate mice were treated with streptozotocin (STZ), then 4 weeks later put on a high fat diet (HFD). After 2 weeks on the HFD, the mice were treated with A438079 (30 mg/kg, b.i.d.) for 10 weeks while maintained on the HFD. Results, presented in FIG. 8, show that treatment with A438079 was effective in reducing fibrosis in this mouse NASH model (p = 0.11 Mann- Whitney test).

[0094] Accordingly, the present disclosure provides methods of treating P2X7 receptor-mediated inflammation and associated diseases and disorders (including those associated with steatohepatitis and liver fibrosis) by administering a P2X7 receptor antagonist to a patient in need thereof.

[0095] In one aspect, the present disclosure provides methods of treating liver disease associated with P2X7 receptor activation comprising administering to a patient in need thereof a therapeutically effective amount of administering a P2X7 receptor antagonist. In some embodiments, the P2X7 receptor antagonist is a compound of Formula I, Formula II, Formula III, Formula Ilia, Formula IV, or Formula IVa or a pharmaceutically acceptable salt thereof. In some embodiments, the P2X7 receptor antagonist is Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6 or Compound 7 or a pharmaceutically acceptable salt thereof. In certain embodiments, the P2X7 receptor antagonist is a compound of Formula (III). In certain embodiments, the P2X7 receptor antagonist is Compound 1 or a pharmaceutically acceptable salt thereof.

[0096] In some embodiments, the methods of the present disclosure treat a liver disease associated with P2X7 receptor activation, wherein subject has inflammation of the liver. In some embodiments, the methods of the present disclosure reduce inflammation of the liver.

[0097] In some embodiments, the methods of the present disclosure are used to treat a liver disease associated with P2X7 receptor activation, wherein the patient has been diagnosed with or is at risk of developing fibrosis of the liver. In certain embodiments, the patient has been diagnosed with or is at risk of developing advanced liver fibrosis.

[0098] In some embodiments, the methods of the present disclosure are used to treat liver disease associated with P2X7 receptor activation, wherein prior to the administering, the patient’s proton density fat fraction (PDFF) as determined by multiparametric magnetic resonance is≥ 8% steatosis.

[0099] In some embodiments, the methods of the present disclosure are used to treat liver disease associated with P2X7 receptor activation, wherein prior to the administering, the patient’s cT1 score as determined by multiparametric magnetic resonance is≥ 827.5 ms.

[00100] In some embodiments, the methods of the present disclosure are used to treat liver disease associated with P2X7 receptor activation, wherein prior to the administering, the patient has advanced liver fibrosis as determined by 2D MRE score.

[00101] In some embodiments, the methods of the present disclosure are used to treat liver disease associated with P2X7 receptor activation, wherein prior to the administering, the patient has liver cirrhosis as determined by 2D MRE score. [00102] In some embodiments, the methods of the present disclosure are used to treat liver disease associated with P2X7 receptor activation, wherein prior to the administering, the patient’s liver stiffness as determined by MRE is between about 2.5 kPa to about 4.65 kPa.

[00103] In some embodiments, the methods of the present disclosure are used to treat liver disease associated with P2X7 receptor activation, wherein prior to the administering, the patient’s liver stiffness as determined by ultrasound-based transient elastography is > 8.

[00104] In some embodiments, the methods of the present disclosure are used to treat liver disease associated with P2X7 receptor activation, wherein prior to the administering, the patient’s controlled attenuation parameter (CAP) as determined by ultrasound-based transient elastography (Fibroscan) is≥300 dB/m.

[00105] In some embodiments, the methods of the present disclosure are used to treat liver disease associated with P2X7 receptor activation, wherein prior to the administering, the patient’s alanine aminotransferase (ALT) is between about 5 IU/L and 225 IU/L.

[00106] In some embodiments, the methods of the present disclosure are used to treat liver disease associated with P2X7 receptor activation, wherein prior to the administering, the patient’s Steatosis, Activity, and Fibrosis (SAF) activity score is≥ 2.

[00107] In some embodiments, the methods of the present disclosure are used to treat liver disease associated with P2X7 receptor activation, wherein prior to the administering, the patient’s SAF activity score is <2.

[00108] In some embodiments, the methods of the present disclosure are used to treat liver disease associated with P2X7 receptor activation, wherein prior to the administering, the difference in the patient’s spleen and liver Hounsfield units (HUspieen-HUiiver) as determined by CT scan is greater than 0.

[00109] In some embodiments, the methods of the present disclosure are used to treat liver disease associated with P2X7 receptor activation, wherein the patient has had an inadequate response to one or more anti-inflammatories. In certain embodiments, the one or more anti-inflammatories are selected from a 5-aminosalicylate or a corticosteroid. In further embodiments, the 5- aminosalicylate is selected from the group consisting of sulfasalazine, mesalamine, balsalazide, and olsalazine.

[00110] In some embodiments, the methods of the present disclosure are used to treat liver disease associated with P2X7 receptor activation, wherein the patient has had an inadequate response to one or more immunosuppressants. In certain embodiments, the one or more immunosuppressants are selected from the group consisting of azathioprine 6-mercaptopurine (6-MP) and cyclosporine. In some embodiments, the one or more immunosuppressants is selected from the group consisting of infliximab, adalimumab, golimumab, and vedolizumab.

[00111] In some embodiments, the methods of the present disclosure are used to treat liver disease associated with P2X7 receptor activation, wherein the patient was intolerant or refractory to mesalamine treatment. In some embodiments, the methods of the present disclosure are used to treat liver disease associated with P2X7 receptor activation, wherein the patient was non- responsive to mesalamine treatment. In some embodiments, the methods of the present disclosure are used to treat liver disease associated with P2X7 receptor activation, wherein the patient relapsed after effective treatment with mesalamine.

[00112] In some embodiments, the methods of the present disclosure are used to treat liver disease associated with P2X7 receptor activation, wherein the patient has been diagnosed with at least disease or disorder selected from the group consisting of hepatocellular carcinoma, obesity, insulin resistance diabetes and Type 2 diabetes.

[00113] In some embodiments, the methods of the present disclosure are used to treat liver disease associated with P2X7 receptor activation, wherein the patient has been diagnosed with NAFLD, NASH, liver fibrosis, advanced liver fibrosis, cirrhosis, and/or hepatocellular carcinoma.

[00114] In some embodiments, the methods of the present disclosure are used to treat fatty liver disease (FLD).

[00115] In some embodiments, the methods of the present disclosure are used to treat liver disease associated with P2X7 receptor activation, wherein the patient has been diagnosed with or is at risk of developing non-alcoholic fatty liver disease (NAFLD). [00116] In some embodiments, the methods of the present disclosure are used to treat liver disease associated with P2X7 receptor activation, wherein the patient has been diagnosed with or is at risk of developing alcoholic steatohepatitis.

[00117] In some embodiments, the methods of the present disclosure are used to treat liver disease associated with P2X7 receptor activation, wherein the patient has been diagnosed with or is at risk of developing cirrhosis.

[00118] In some embodiments, the methods of the present disclosure are used to treat non alcoholic steatohepatitis (NASH). In certain embodiments, the methods of the present disclosure are used to treat NASH, wherein the patient has Fibrosis stage 1-3 NASH as determined by two- dimensional magnetic resonance elastography (2D MRE) score.

P2X7 receptor antagonists

[00119] P2X7 receptor antagonists as employed in the present methods can form a part of a pharmaceutical composition by combining a P2X7 receptor antagonist, or a pharmaceutically acceptable salt thereof, with a pharmaceutically acceptable carrier. Additionally, the compositions can include an additive selected from the group consisting of adjuvants, excipients, diluents, release-modifying agents and stabilizers. The composition can be an immediate release formulation, a delayed release formulation, a sustained release formulation or an extended release formulation.

[00120] P2X7 receptor antagonists are known to those skilled in the art. For example, P2X7 receptor antagonists have been synthesized and characterized for their ability to inhibit IL-1 β secretion by human cells in vivo and in vitro (for example, in terms of an IC₅₀ value for the inhibition of IL-b secretion by human cells or whole blood).

[00121] In some embodiments, the P2X7 receptor antagonist used in the formulations and methods of the present disclosure is a P2X7 receptor antagonist having an IC₅₀ value of from about 0.1 nM to about 100 nM, including about 1 nM, about 3 nM, about 5 nM, about 10 nM, about 25 nM, about 50 nM, about 75 nM, including all ranges there between, determined by an in vitro assay. In certain embodiments, the P2X7 receptor antagonist used in the formulations and methods of the present disclosure is a P2X7 receptor antagonist having an IC₅₀ value of from about 0.1 nM to about 5 nM, about 0.1 nM to about 3 nM, or 0.1 nM to about 1 nM. In some embodiments, the in vitro assay comprises measuring plasma IL- l b in the sample. In certain embodiments, the in vitro assay comprises incubating human whole blood with LPS (e.g., 200 ng/mL LPS). In still other embodiments, the in vitro assay further comprises incubating the whole blood sample with the antagonist. In certain other embodiments, the in vitro assay further comprises incubating the whole blood sample with ATP (e.g., about 2.5 mM ATP). In still other embodiments, the in vitro assay further comprises incubating the LPS-treated whole blood with the antagonist and ATP at 37°C prior to measuring plasma IL-1 β quantities.

[00122] In some embodiments, the P2X7 receptor antagonist used in the formulations and methods of the present disclosure is a P2X7 receptor antagonist having an IC₅₀ of from about 10 nM to about 100 nM; about 20 nM to about 75 nM; about 30 nM to about 60 nM; about 40 nM to about 60 nM; about 50 nM to about 60 nM, or about 52 nM to about 57 nM as determined by an in vitro assay. In certain embodiments, the in vitro assay comprises the in vitro assay comprises measuring the amount of IL-1 β released by cells in the assay. In some embodiments, the in vitro assay is performed using LPS-primed whole blood from a subject to which the antagonist was administered. In some embodiments, the assay comprises whole blood that is primed with 200 ng/mL LPS. In some embodiments, the in vitro assay comprises treating the whole blood sample with heparin. In some embodiments, the in vitro assay comprises adding ATP to aliquots of the sample wherein the concentration of ATP ranges from about 1 nM to about 10 mM. In still other embodiments, the in vitro assay comprises incubating the whole blood aliquots at 37°C.

[00123] In some embodiments, the P2X7 receptor antagonist used in the formulations and methods of the present disclosure is a P2X7 receptor antagonist having a mean IC₅₀ ranging from about 0.1 nM to about 100 nM, about 0.1 nM to about 75 nM, about 0.1 nM to about 50 nM, about 0.1 nM to about 40 nM, about 0.1 nM to about 30 nM, about 0.1 nM to about 20 nM, about 5 nM to about 20 nM, about 5 nM to about 15 nM, about 1 nM to about 15 nM, about 7.5 nM to about 12.5 nM or about 10 nM, wherein the IC₅₀ is measured in an in vitro assay with LPS-stimulated human monocytic cells.

[00124] The synthesis of P2X7 receptor antagonists is provided in U.S. Patent Nos. 9,464,084; 9,604,982; 9,540,388; 9,102,686; 9,096,596; 9,040,534; 8,431,704; 6,974,812; 8,513,248; 8,501,946; 9,556,117; 9,409,917; 9,388,198; 9,388,197, and 9,221,832, and U.S. Patent

Publication No. 2016/0016962, which are hereby incorporated by reference in their entirety for all purposes.

[00125] In some embodiments, the P2X7 receptor antagonist used in the formulations and methods of the present disclosure is a P2X7 receptor antagonist selected from those described in U.S. Patent

Nos. 9,464,084; 9,604,982; 9,540,388; 9,102,686; 9,096,596; 9,040,534; 8,431,704; 6,974,812; 8,513,248; 8,501,946; 9,556,117; 9,409,917; 9,388,198; 9,388,197, and 9,221,832, and U.S. Patent

Publication No. 2016/0016962.

[00126] In some embodiments, the P2X7 receptor antagonist used in the formulations and methods of the present disclosure is a P2X7 receptor antagonist selected from those described in U.S. Patent No. 8,093,265, which is hereby incorporated by reference in their entirety for all purposes. In certain embodiments, the P2X7 receptor antagonist is a compound of Formula (la) or Formula (lb) as defined in U.S. Patent No. 8,093,265.

[00127] In some embodiments, the P2X7 receptor antagonist used in the formulations and methods of the present disclosure is a compound of Formula (I):

[00128] wherein R¹ is C₂-C₄ alkyl optionally substituted with one or more substituents selected from -CONH₂, -CONHCH₃, hydroxy, amino, aminoalkyl, and substituted or unsubstituted heterocycloalkyl;

[00129] R² is selected from halogen, -CH₃, OCH₃, aminoalkyl, and cyclopropyl;

[00130] R³ is selected from H or CH₃;

[00131] R⁴ is selected from substituted or unsubstituted aryl; [00132] A is selected from -NH(CO)- or -(CO)NH-;

[00133] B and C are CR⁷ and

[00134] R⁷ in B and C are independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, cyano, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cyeioheteroalkyl, halo, and hydroxy.

[00135] In certain embodiments, P2X7 receptor antagonist is a compound of Formula (I), wherein R¹ is a C₂-C₄ alkyl optionally substituted with one or more substituents selected from -CONH₂, hydroxy, and amino; R² is selected from halogen and -CH₃; R³ is H; R⁴ is substituted phenyl; A is -NH(CO)- and R⁷ is H.

[00136] In certain embodiments, P2X7 receptor antagonist is a compound of Formula (I), wherein R¹ is a C₂-C₄ alkyl optionally substituted with one or more substituents selected from -CONH₂, hydroxy, and amino; R² is selected from halogen and -CH₃; R³ is H; R⁴ is substituted phenyl; A is -(CO)NH- and R⁷ is H.

[00137] In some embodiments, the P2X7 receptor antagonist used in the formulations and methods of the present disclosure is a compound of Formula (II)

[00138] wherein R¹; R²; R³, A, B and C are as described for Formula (I) and R⁵ and R⁶ are independently selected from H, -CF₃, and halogen.

[00139] In certain embodiments, P2X7 receptor antagonist is a compound of Formula (II), wherein R¹ is a C₂-C₄ alkyl optionally substituted with one or more substituents selected from -CONH₂, hydroxy, and amino; R² is selected from halogen and --CH₃; R³ is H; A is -NH(CO)- and R⁷ is H. [00140] In certain embodiments, the P2X7 receptor antagonist is a compound of Formula (II), wherein R¹ is a C₂-C₄ alkyl optionally substituted with one or more substituents selected from ---CONH₂., hydroxy, and amino; R² is selected from halogen and -CH₃; R³ is H; A is -(CO)NH- and R⁷ is H.

[00141] In certain further embodiments, R⁵ and R⁶ are independently selected from -CF₃ and halogen. In other further embodiments, R⁵ and R⁶ are independently selected from -CF₃ and F. In other further embodiments, R⁵ is -CF₃ and R⁶ is F.

[00142] In certain further embodiments, the P2X7 receptor antagonist is a compound of Formula (II), wherein R⁵ and R⁶ are independently selected from -CF₃ and H. In other further embodiments, the P2X7 receptor antagonist is a compound of Formula (II), wherein R⁵ is -CF₃ and R⁶ is H.

[00143] In some embodiments, the P2X7 receptor antagonist used in the formulations and methods of the present disclosure is a compound of Formula (ΙII)

[00144] wherein R¹ is C₂-C₄ alkyl optionally substituted with one or more substituents selected from -CONH₂ -CONHCH₃, hydroxy, amino, aminoalkyl, and substituted or unsubstituted heterocy cloal kyl ;

[00145] R² is selected from CH₃ and halogen;

[00146] R³ is selected from H and CH₃; and

[00147] R⁵ andR⁶ are independently selected from H, halogen and CF₃.

[00148] In certain embodiments, the P2X7 receptor antagonist is a compound of Formula (ΙII), wherein R¹ is a C₂-C₄ alkyl optionally substituted with one or more substituents selected from -CONH₂, hydroxy and amino; R² is selected from halogen and CH₃; and R³ is H.

[00149] In certain further embodiments, the P2X7 receptor antagonist is a compound of Formula (III), wherein R⁵ and R⁶ are independently selected from -CF₃ and halogen. In other further embodiments, the P2X7 receptor antagonist is a compound of Formula (III), wherein R⁵ and R⁶ are independently selected from -CF₃ and F. In other further embodiments, the P2X7 receptor antagonist is a compound of Formula (III), wherein R⁵ is -CF₃ and R⁶ is F.

[00150] In certain further embodiments, the P2X7 receptor antagonist is a compound of Formula (III), wherein R⁵ and R⁶ are independently selected from -CF₃ and H. In other further embodiments, the P2X7 receptor antagonist is a compound of Formula (III), wherein R⁵ is -CF₃ and R⁶ is H.

[00151] In certain further embodiments, the P2X7 receptor antagonist is a compound of Formula (III), wherein R¹ is C₂-C₄ alkyl substituted with one or more substituents selected from -OH, -NH₂, and -CONH₂.

[00152] In some embodiments, the P2X7 receptor antagonist used in the formulations and methods of the present disclosure is a compound of Formula (Ilia)

[00153] wherein R¹; R²; R³, R⁵, R⁶ are as defined in Formula (III).

[00154] In some embodiments, the P2X7 receptor antagonist used in the formulations and methods of the present disclosure is a compound of Formula (IV)

[00155] wherein R¹ is C₂-C₄ alkyl optionally substituted with one or more substituents selected from -CONH₂, -CONHCH₃, hydroxy, amino, aminoalkyl, and substituted or unsubstituted heterocy cloal kyl ;

[00156] R² is selected from CH₃ and halogen;

[00157] R³ is selected from H and CH₃; and

[00158] R⁵ andR⁶ are independently selected from H, halogen and CF₃.

[00159] In certain embodiments, the P2X7 receptor antagonist is a compound of Formula (IV), wherein R¹ is a C₂-C₄ alkyl optionally substituted with one or more substituents selected from - CONH₂, hydroxy, and amino; R² is selected from halogen and -CH₃; and R³ is H.

[00160] In certain further embodiments, the P2X7 receptor antagonist is a compound of Formula (IV), wherein R⁵ and R⁶ are independently selected from -CF₃ and halogen. In other further embodiments, the P2X7 receptor antagonist is a compound of Formula (IV), wherein R⁵ and R⁶ are independently selected from -CF₃ and F. In other further embodiments, the P2X7 receptor antagonist is a compound of Formula (IV), wherein R⁵ is -CF₃ and R⁶ is F.

[00161] In certain further embodiments, the P2X7 receptor antagonist is a compound of Formula (IV), wherein R⁵ and R⁶ are independently selected from -CF₃ and H. In other further embodiments, the P2X7 receptor antagonist is a compound of Formula (IV), wherein R⁵ is -CF₃ and R⁶ is H.

[00162] In certain further embodiments, the P2X7 receptor antagonist is a compound of Formula (IV), wherein R¹ is C₂-C₄ alkyl substituted with one or more substituents selected from -OH, -NH₂, and -CONH₂. [00163] In some embodiments, the P2X7 receptor antagonist used in the formulations and methods of the present disclosure is a compound of Formula (IVa)

[00164] wherein R¹; R²; R³, R⁵, R⁶ are as defined in Formula (IV).

[00165] In some embodiments, the P2X7 receptor antagonist used in the formulations and methods of the present disclosure is a compound selected from the group:

[00166] The synthesis of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5 and Compound 6 is described in U.S. Patent No. 8,093,265.

[00167] Compound 1 is also known as (2-(3-Fluoro-4-trifluoromethyl-phenyl)-N-[2-((R)-2- hydroxy-l -methylethyl)-6-methyl-l -oxo-l, 2-dihydro-isoquinolin-5-yl]-acetamide). In some aspects, the P2X7 receptor antagonist used in the formulations and methods of the present disclosure is Compound 1 or a pharmaceutically acceptable salt thereof. In certain embodiments, the P2X7 receptor antagonist used in the formulations and methods of the present disclosure is Compound 1 (i.e., not a pharmaceutically acceptable salt). In vitro studies with Compound 1 (see Examples 1-3 below) showed that Compound 1 inhibits human and rat P2X7 receptor mediated calcium influx expressed in 1321N1 human brain astrocytoma cells. The IC₅₀ for the human P2X7 receptor was l0.0±19.4 nM (n=3) while the IC₅₀ for the rat receptor was 220±2.0 nM (n=4). Compound 1 showed no activity against human P2X1, P2X2, P2X3 or P2X4 receptors at concentrations up to 10 mM (n=l each). Compound 1 has been shown to inhibit ATP dependent IL-1 β release from human LPS stimulated whole blood with a mean IC₅₀ of about 54.5±16.8 nM (n=6) and to cause a concentration-dependent inhibition of ATP induced IL-1 β release from human LPS stimulated THP-l monocytic cell line and mouse J774 cells. As TNF-alpha release is not dependent upon the P2X7 receptor, Compound 1 had no effect on the control cytokine TNF-alpha.

[00168] Furthermore, the Applicants conducted Phase 1 clinical studies to evaluate the safety and pharmacokinetics of Compound 1 administered to healthy subject. The P2X7 receptor antagonist Compound 1 was administered either in capsule form or as an emulsion. These studies are described in Examples 4 and 5. Compound 1 provided dose-dependent reductions in inhibition of ATP-dependent IL-1 β release in whole blood at doses ranging from 80 mg to 1280 mg. The 320 g to 1280 mg doses of Compound 1 showed a statistical difference compared to placebo. The study showed that the studied doses of Compound 1 were safe and well tolerated.

[00169] Compound 2 is also known as (2-(3-Fluoro-4-trifluoromethyl-phenyl)-N-[2-((S)-2- hydroxy-l -methylethyl)-6-methyl-l -oxo-l, 2-dihydro-isoquinolin-5-yl]-acetamide). In some embodiments, the P2X7 receptor antagonist used in the formulations and methods of the present disclosure is Compound 3 or a pharmaceutically acceptable salt thereof.

[00170] In some embodiments, the P2X7 receptor antagonist used in the formulations and methods of the present disclosure is Compound 3 or a pharmaceutically acceptable salt thereof. In some embodiments, the P2X7 receptor antagonist used in the formulations and methods of the present disclosure is Compound 4 or a pharmaceutically acceptable salt thereof. In some embodiments, the P2X7 receptor antagonist used in the formulations and methods of the present disclosure is Compound 5 or a pharmaceutically acceptable salt thereof. In some embodiments, the P2X7 receptor antagonist used in the formulations and methods of the present disclosure is Compound 6 or a pharmaceutically acceptable salt thereof.

[00171] P2X7 receptor antagonist useful in the compositions and methods of the present disclosure include those described herein in any of their pharmaceutically acceptable forms, including isomers such as diastereomers and enantiomers, salts, solvates, and polymorphs, as well as racemic mixtures and pure isomers of the compounds described herein, where applicable.

Formulations

[00172] The methods of the present disclosure can employ various formulations for administration to patients, e.g., humans in unit dosage forms, such as tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions (e.g., intramuscular (IM), subcutaneous (SC) and intravenous (IV)), transdermal patches, and oral solutions or suspensions, and oil-water emulsions containing suitable quantities of a P2X7 receptor antagonist.

[00173] Oral pharmaceutical dosage forms can be either solid or liquid. The solid dosage forms can be tablets, capsules, granules, films (e.g., buccal films) and bulk powders. Types of oral tablets include compressed, chewable lozenges and tablets, which can be enteric-coated, sugar-coated or film-coated. Capsules can be hard or soft gelatin capsules, while granules and powders can be provided in non-effervescent or effervescent form with the combination of other ingredients known to those skilled in the art. In some embodiments, the present oral dosage forms may include orally disintegrating tablets.

[00174] Pharmaceutically acceptable carriers utilized in tablets include binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, and wetting agents.

[00175] Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules.

[00176] Aqueous solutions include, for example, elixirs and syrups. Emulsions can be either oil- in water or water-in-oil. Elixirs are clear, sweetened, hydroalcoholic preparations. Pharmaceutically acceptable carriers used in elixirs include solvents. Syrups can be concentrated aqueous solutions of a sugar, for example, sucrose, and can contain a preservative. An emulsion is a two-phase system in which one liquid is dispersed in the form of small globules throughout another liquid. Pharmaceutically acceptable carriers used in emulsions are non-aqueous liquids, emulsifying agents and preservatives. Suspensions can use pharmaceutically acceptable suspending agents and preservatives. Pharmaceutically acceptable substances used in non- effervescent granules, to be reconstituted into a liquid oral dosage form, include diluents, sweeteners and wetting agents. Pharmaceutically acceptable substance used in effervescent granules, to be reconstituted into a liquid oral dosage form, can include organic acids and a source of carbon dioxide. Coloring and flavoring agents can be used in all of the above dosage forms. [00177] In some embodiments, the present disclosure provides a pharmaceutical composition comprising from about 20 mg to about 550 mg, including about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, about 500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg, and about 550 mg, including all ranges there between, of a P2X7 receptor antagonist.

[00178] In some embodiments, the present disclosure provides a pharmaceutical composition comprising from about 50 mg to about 500 mg, about 50 mg to about 1000 mg, about 50 mg to about 250 mg, about 100 mg to about 1000 mg, about 150 mg to about 500 mg, about 100 mg to about 200 mg, about 100 mg to about 300 mg, about 100 mg to about 400 mg, about 100 mg to about 500 mg, about 250 mg to about 500 mg, about 500 mg to about 1000 mg.

[00179] In some embodiments, the present disclosure provides a pharmaceutical composition comprising about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, about 500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg, or about 550 mg of a P2X7 receptor antagonist.

[00180] In some embodiments, the present disclosure provides a pharmaceutical composition comprising about 100 mg, about 150 mg, about 175 mg, about 200 mg, about 250 mg, about 275 mg, about 300 mg, about 350 mg, about 400 mg, about 500 mg, about 600 mg, about 750 mg, or about 1000 mg of a P2X7 receptor antagonist.

[00181] In some embodiments, the present disclosure provides a pharmaceutical composition comprising wherein the P2X7 receptor antagonist is a compound of Formula I, Formula II, Formula III, Formula Ilia, Formula IV, or Formula IVa, or a pharmaceutically acceptable salt thereof. In some embodiments, the present disclosure provides a pharmaceutical composition comprising wherein the P2X7 receptor antagonist is Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, or Compound 7, or a pharmaceutically acceptable salt thereof.

[00182] In some embodiments, the present disclosure provides a pharmaceutical composition comprising Compound 1 or a pharmaceutically acceptable salt thereof. In some embodiments, the salt of Compound 1 is Compound 1 Hydrochloride.

[00183] In some embodiments, Compound 1 is formulated for oral administration. For example, a pharmaceutically acceptable composition comprising Compound 1 is provided, wherein the composition is a tablet, capsule, caplet, or emulsion. Pharmaceutical compositions of the disclosure suitable for oral administration may thereby be presented as discrete units, each containing a predetermined amount of the active ingredient, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil liquid emulsion. Such compositions may be prepared by any acceptable pharmaceutical process. All such methods include the step of combining the active ingredient(s) with carrier components. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient(s) with a liquid or finely divided solid carrier component, and then, if necessary, manipulating the blend into the desired product form. For example, a tablet may be prepared by compression or molding. Compressed tablets may be prepared by compressing free- flowing powder or granules, containing the active(s) optionally mixed with one or more excipients, e.g., binders, lubricants, diluents, surfactants and disintegrants. Molded tablets may be made by molding a mixture of the powdered compound moistened with an inert liquid. In some embodiments, the active agent may be micronized. Micronization can facilitate solubilization of a poorly soluble active agent. [00184] In some embodiments, the present disclosure provides a dosage form comprising the P2X7 receptor antagonist in an immediate release, sustained release or extended release tablet. An immediate release tablet will release at least 80%, 85%, 90% 95% or 99% of the P2X7 receptor antagonist within 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 45 min or 60 min of oral ingestion of the tablet.

[00185] In some embodiments, the present disclosure provides a dosage form comprising the P2X7 receptor antagonist in an immediate release comprising from about 50 mg to about 300 mg, about 100 mg to about 500 mg, about 100 mg to about 1000 mg, about 100 mg to about 750 mg, about 100 mg to about 200 mg, about 150 mg to about 250 mg, about 500 mg to about 1000 mg, about 500 mg to about 750 mg, about 250 mg to about 500 mg or about 50 mg to about 150 mg of the P2X7 receptor antagonist. In some embodiments, P2X7 receptor antagonist has the structure of Formula I, II, III, Ilia, IV, or IVa or Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7, or a pharmaceutically acceptable salt thereof. In certain embodiments, the compound of Formula I, II, III, Ilia, IV, or IVa or Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7, or a pharmaceutically acceptable salt thereof, in the immediate release tablet is micronized.

[00186] In some embodiments, the present disclosure provides a dosage form comprising the P2X7 receptor antagonist in an extended release tablet wherein at least 80%, 85%, 90% 95% or 99% of the P2X7 receptor antagonist is released with 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, or 12 h of ingestion of the tablet.

[00187] A dosage form, in some embodiments, comprises a tablet with an enteric coating such that the P2X7 receptor antagonist is not released from the tablet until the dosage form has entered the small intestine, e.g., a delayed release dosage form. The dosage form comprising an enteric coating can comprise an immediate release or extended release tablet as described above.

[00188] In some embodiments, the dosage form comprises an extended release or sustained release tablet, capsule or caplet. The extended or sustained release dosage form releases no more than 40% of the P2X7 receptor antagonist within 1 h, 2 h, 3 h, or 4 h of ingestion of the dosage form. In further embodiments, the extended or sustained release dosage form releases no more than about 70% of the P2X7 receptor antagonist over a within about 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, or 12 h after ingestion of the dosage form. In some embodiments, the extended or sustained release dosage form releases not more than about 80% of the P2X7 receptor antagonist over a within about 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, or 12 h after ingestion of the dosage form. In other embodiments, the extended or sustained release dosage form releases not more than about 90% of the P2X7 receptor antagonist over a within about 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, or 12 h after ingestion of the dosage form.

[00189] A dosage form comprising the P2X7 receptor antagonist according to the present disclosure can be one formulated to release a portion or substantially all of the antagonist in the intestine. In some embodiments, a portion or substantially all of the antagonist is released within the colon. In other embodiments, a portion or substantially all of the antagonist is released within the small intestine. In some embodiments, a portion or substantially all of the antagonist can be released within both the small intestine and colon.

[00190] Oral dosage forms formulated to release an active agent into the intestine, e.g., colon and/or small intestine, to achieve an optimal absorption profile for the agent. Such targeted formulations are well-known in the art (for a review, see, e.g., Amidon et al., 2015, AAPS PharmSciTech, 16:731-741) and include a bioadhesive system that allows the formulation to remain in contact with an organ, such as the colon, for an extended period of time. Bioadhesive components for these systems include polycarbophils, polyurethanes, and polyethylene oxide (Chourasia and Jain, 2004, Drug Deliv, 11 :201-207). Timed-release systems are formulations designed to release an active agent after a specified amount of time, i.e., when the dosage form is approximated to have reached the colon. Timed-release systems can include osmotic dosage forms and pulsincap systems, designed to have a lag time of release between when an enteric coating (e.g., acid- insoluble film) dissolves and the drug is released.

[00191] In some embodiments, the P2X7 receptor antagonist is synthesized as a prodrug that is cleaved to release the active agent (e.g., Compound 1 or active derivative thereof), such that cleavage does not happen until after the prodrug has passed through the stomach or through both the stomach and small intestine. [00192] The P2X7 receptor antagonist can be formulated as a solution for injection, wherein the formulation comprises the P2X7 receptor antagonist and one or more pharmaceutically acceptable excipients.

[00193] The pharmaceutical compositions comprising a P2X7 receptor antagonist as described herein can be prepared using conventional methods known to those in the field of pharmaceutical formulation and described in the pertinent texts, e.g., in Gennaro, A. R., editor. "Remington: The Science & Practice of Pharmacy", 2lst ed., Williams & Williams, and in the "Physician's Desk Reference," 2006, Thomson Healthcare.

Co- therapy

[00194] While the compositions can be administered as the sole active pharmaceutical ingredient (i.e., a P2X7 receptor antagonist) or sole active anti-inflammatory ingredient in the methods described herein, in other embodiments they can also be used in combination with one or more ingredients that are known to be therapeutically effective against diseases associated with the P2X7 receptor and/or compliment the anti-inflammatory effect of the P2X7 receptor antagonist ingredient.

[00195] For example, in some embodiments, the present methods can employ a P2X7 receptor antagonist in conjunction with one or more additional anti-inflammatory agents.

[00196] In some embodiments, the P2X7 receptor antagonist is administered in conjunction with one more 5-ASA preparations (such as mesalazine and salazosulfapyridine (sulfasalazine)), corticosteroid preparations (such as prednisolone, betamethasone, and budesonide), or antibiotics agents (such as metronidazole).

[00197] In some embodiments, the P2X7 receptor antagonist is administered in conjunction with one more immunosuppressants. In certain embodiments, the immunosuppressants are selected from the group consisting of azathioprine, 6-mercaptopurine, cyclosporine, and tacrolimus.

[00198] In some embodiments, the P2X7 receptor antagonist is administered in combination with an active agent indicated for treatment of diabetes, obesity, high cholesterol, or hypertension. In some embodiments, the P2X7 receptor antagonist is administered to a patient receiving treatment for diabetes, obesity, high cholesterol, or hypertension

[00199] In some embodiments, the P2X7 receptor antagonist is administered in combination with one or more GLP-l receptor agonist (e.g., GLP-l or functional analog thereof), insulin or functional analog thereof, FGF-21 polypeptide or fragment thereof or modified fragment thereof (e.g., U.S. Patent No. 9,631,004, Examples 2, 3, and 4, the contents of which are incorporated herein by reference in their entirety), and bupropion.

[00200] In some embodiments, the P2X7 receptor antagonist is administered to a subject who has received or is receiving anti-cytokine agents, such as anti-TNF-alpha antibodies (such as infliximab, adalimumab, certolizumabpegol, and golimumab), anti-IL-6 receptor antibodies (such as tocilizumab), anti-IL- 12/23 antibodies (such as ustekinumab and briakinumab), anti-IL-17 receptor antibodies (such as AMG827 and AIN457), IL- 12/23 production inhibitors (such as STA- 5326) as low-molecular-weight agents, PDE-4 inhibitors (such as tetomilast), chemokine inhibitors (such as vercirnon and CCX507), Janus kinase inhibitors (such as tofacitinib and GLPG0634), or SIP agonists (such as KRP203 and RPC1063).

[00201] In some embodiments, the subject administered the P2X7 receptor antagonist is not undergoing treatment with another immunosuppressor, anti-inflammatory, and/or biologic anti cytokine agent. In certain embodiments, the subject has not been administered another immunosuppressor, anti-inflammatory, and/or biologic anti-cytokine agent for at least about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months or about 6 months prior to a first administration with a P2X7 receptor antagonist of the present disclosure.

Dosing and administration:

[00202] The disclosure provides methods for treating diseases associated with P2X7 receptor activation by administering an effective amount of a P2X7 receptor antagonist to a patient in need thereof. An effective amount is an amount sufficient to eliminate or significantly reduce symptoms or to alleviate those symptoms of the disease associated with P2X7 receptor activation (e.g., reduce the symptoms, such as liver fibrosis, compared to the symptoms present prior to treatment). In some embodiments, the P2X7 receptor antagonist is a compound of Formula X.

[00203] According to some embodiments of the present disclosure, administering P2X7 receptor antagonist provides statistically significant therapeutic effect. In one embodiment, the statistically significant therapeutic effect is determined based on one or more standards or criteria provided by one or more regulatory agencies in the United States, e.g., FDA or other countries. In another embodiment, the statistically significant therapeutic effect is determined based on results obtained from regulatory agency approved clinical trial set up and/or procedure.

[00204] In some embodiments, the statistically significant therapeutic effect is determined based on a patient population of at least 20, 50, 60, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or 2000. In some embodiments, the statistically significant therapeutic effect is determined based on data obtained from randomized and double-blinded clinical trial set up. In some embodiments, the statistically significant therapeutic effect is determined based on data with a p value of less than or equal to about 0.05, 0.04, 0.03, 0.02 or 0.01. In some embodiments, the statistically significant therapeutic effect is determined based on data with a confidence interval greater than or equal to 95%, 96%, 97%, 98% or 99%.

[00205] In some embodiments, the statistically significant therapeutic effect is determined by a randomized double-blind clinical trial of patients treated with a P2X7 receptor antagonist and optionally in combination with standard care. In some embodiments, the statistically significant therapeutic effect is determined by a randomized clinical trial and using alanine aminotransferase (ALT) as primary efficacy parameter and optionally in combination with any other commonly accepted criteria for liver disease assessment. In some embodiments, the statistically significant therapeutic effect is determined by a randomized clinical trial and using corrected Tl (cTl) magnetic resonance imaging (MRI) and optionally in combination with any other commonly accepted criteria for liver disease assessment.

[00206] In general, statistical analysis can include any suitable method permitted by a regulatory agency, e.g., FDA in the US or Europe or any other country. In some embodiments, statistical analysis includes non-stratified analysis, log-rank analysis, e.g., from Kaplan-Meier, Jacobson- Truax, Gulliken-Lord-Novick, Edwards-Nunnally, Hageman-Arrindel and Hierarchical Linear Modeling (HLM) and Cox regression analysis.

[00207] According to some embodiments of the present disclosure, the dosing frequency and dose amount per administration of a P2X7 receptor antagonist are selected to inhibit P2X7 receptor activity in the subject. In some embodiments, the P2X7 receptor activity is P2X7 receptor activation. In some embodiments, the P2X7 receptor activity is activation of the NLRP3 inflammasome pathway.

[00208] According to some embodiments of the present disclosure, the dosing frequency and dose amount per administration of a P2X7 receptor antagonist are selected to provide therapeutic effects for the treatment of a disease associated with P2X7 activation.

[00209] In some embodiments, the dosing frequency and dose amount per administration of a P2X7 receptor antagonist are selected to provide a reduction in the IL-1 β secretion by cells in the blood of the subject after administration of the dose of the antagonist to the subject compared to prior to said administration.

[00210] In some embodiments, the dosing frequency and dose amount per administration of a P2X7 receptor antagonist are selected to provide a reduction in the IL-1 β secretion by cells in the whole blood of the subject of at least about 60%, about 70%, about 80%, about 85%, about 90%, or about 95% compared to prior to said administration as determined by an in vitro assay of the whole blood. In some embodiments, the inhibition of IL-1 β secretion in the in vitro assay is determined about 3 hours (h), about 3.5 h, about 4 h, about 4.5 h, about 5 h, about 5.5 h, or about 6 h after administration of the dose to the subject. In some embodiments, the inhibition of IL-1 β secretion in the in vitro assay is determined about 3.5 h to about 4.5 h after administration of the dose to the subject.

[00211] According to some embodiments of the present disclosure, the dosing frequency and dose amount per administration of a P2X7 receptor antagonist are selected to provide therapeutic effects for the treatment of a liver disease associated with P2X7 activation. In some embodiments, a P2X7 receptor antagonist is administered on a once a day, twice a day or three times a day basis to provide effective relief of a liver disease associated with P2X7 receptor activation. In some embodiments, a P2X7 receptor antagonist selected from the group consisting of a compound of Formula I, Formula II, Formula III, Formula Ilia, Formula IV or Formula IVa, or a pharmaceutically acceptable salt thereof, is administered on a once a day, twice a day or three times a day basis to provide effective relief of a liver disease associated with P2X7 receptor activation. In certain embodiments, a P2X7 receptor antagonist selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, and Compound 7, or a pharmaceutically acceptable salt thereof, is administered on a once a day, twice a day or three times a day basis to provide effective relief of a liver disease associated with P2X7 receptor activation. In certain embodiments, Compound 1 or a pharmaceutically acceptable salt thereof is administered on a once a day, twice a day or three times a day basis to provide effective relief of a liver disease associated with P2X7 receptor activation.

[00212] According to some embodiments of the present disclosure, the dosing frequency and dose amount per administration of a P2X7 receptor antagonist are selected to provide therapeutic effects for the treatment of a liver disease associated with P2X7 activation selected from the group consisting of NASH, FLD, and NAFLD. In some embodiments, a P2X7 receptor antagonist is administered on a once a day, twice a day or three times a day basis to provide effective relief of a liver disease associated with P2X7 receptor activation selected from the group consisting of NASH, FLD, and NAFLD. In some embodiments, a P2X7 receptor antagonist selected from the group consisting of a compound of Formula I, Formula II, Formula III, Formula Ilia, Formula IV or Formula IVa, or a pharmaceutically acceptable salt thereof, is administered on a once a day, twice a day or three times a day basis to provide effective relief of a liver disease associated with P2X7 receptor activation selected from the group consisting of NASH, FLD, and NAFLD. In certain embodiments, a P2X7 receptor antagonist selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, and Compound 7, or a pharmaceutically acceptable salt thereof, is administered on a once a day, twice a day or three times a day basis to provide effective relief of a liver disease associated with P2X7 receptor activation selected from the group consisting of NASH, FLD, and NAFLD. In certain embodiments, Compound 1 or a pharmaceutically acceptable salt thereof is administered on a once a day, twice a day or three times a day basis to provide effective relief of a liver disease associated with P2X7 receptor activation selected from the group consisting of NASH, FLD, and NAFLD. [00213] According to some embodiments of the present disclosure, the dosing frequency and dose amount per administration of a P2X7 receptor antagonist are selected to provide therapeutic effects for the treatment of NASH. In some embodiments, a P2X7 receptor antagonist selected from the group consisting of a compound of Formula I, Formula II, Formula III, Formula Ilia, Formula IV or Formula IVa, or a pharmaceutically acceptable salt thereof, is administered on a once a day, twice a day or three times a day basis to provide effective relief of NASH. In certain embodiments, a P2X7 receptor antagonist selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, and Compound 7, or a pharmaceutically acceptable salt thereof, is administered on a once a day, twice a day or three times a day basis to provide effective relief of NASH. In certain embodiments, Compound 1 or a pharmaceutically acceptable salt thereof is administered on a once a day, twice a day or three times a day basis to provide effective relief of NASH.

[00214] In some of the embodiments described herein, reference is made to the dose of a P2X7 receptor antagonist for the treatment of a particular liver disease or disorder associated with P2X7 activation (such as NASH). However, the present disclosure contemplates the disclosed doses for the treatment of other liver diseases associated with P2X7 receptor activation described herein (including fatty liver disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, cirrhosis, liver fibrosis, or hepatocellular carcinoma). Similarly, in some of the embodiments described herein, reference is made to the dose of a P2X7 receptor antagonist for the treatment of a liver disease or disorder associated with P2X7 activation (i.e., without specifying the particular liver disease or disorder). However, the present disclosure contemplates the disclosed doses for the treatment of the specific liver diseases associated with P2X7 receptor activation described herein (including fatty liver disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, cirrhosis, liver fibrosis, or hepatocellular carcinoma).

[00215] Furthermore, in some of the embodiments described herein, reference is made to the dose of a particular P2X7 receptor antagonist (such as Compound 1) for the treatment of a liver disease or disorder associated with P2X7 activation. However, the present disclosure contemplates the disclosed doses using the other P2X7 receptor antagonists (including the compounds of Formula

1, Formula II, Formula III, Formula Ilia, Formula IV, Formula IVa and Compound 1, Compound

2, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7, or a pharmaceutically acceptable salt thereof) described herein for the treatment of other liver diseases associated with P2X7 receptor activation. That is, it is understood that the doses described herein are suitable for a compound of Formula I, Formula II, Formula III, Formula Ilia, Formula IV, or Formula IVa as well as Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, or Compound 7, or a pharmaceutically acceptable salt thereof.

[00216] In some embodiments, the total daily dose of the P2X7 receptor antagonist is about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, about 500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg, and about 550 mg. In certain embodiments, the total daily dose of the P2X7 receptor antagonist is about 100 mg. In certain embodiments, the total daily dose of the P2X7 receptor antagonist is about 200 mg. In certain embodiments, the total daily dose of the P2X7 receptor antagonist is about 500 mg.

[00217] In some embodiments, the total daily dose of the P2X7 receptor antagonist is about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg, about 1000 mg, about 1250 mg, about 1500 mg, about 1750 mg or about 2000 mg.

[00218] In some embodiments, the total daily dose of the P2X7 receptor antagonist is about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 m, about 850 mg, about 900 mg, about 950 mg, about 1000 mg, about 1050 mg, about 1100 mg, about 1150 mg, about 1200 mg, about 1250 mg, about 1300 mg, about 1350 mg, about 1400 mg, about 1450 mg, about 1500 mg, about 1550 mg, about 1600 mg, about 1650 mg, about 1700 mg, about 1750 mg, about 1800 mg, about 1850 mg, about 1900 mg, about 1950 mg, about 2000 mg, about 2100 mg, about 2200 mg, about 2300 mg, about 2400 mg, about 2500 mg, about 2600 mg, about 2700 mg, about 2800 mg, about 2900 mg, or 3000 mg.

[00219] In certain embodiments, the total daily dose of the P2X7 receptor antagonist is from about 20 mg to about 550 mg, including about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, about 500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg. In certain embodiments, the total daily dose of the P2X7 receptor antagonist is from about 50 mg to about 500 mg.

[00220] In some embodiments, the total daily dose of the P2X7 receptor antagonist is about 100 mg to about 2000 mg, about 100 mg to about 1500 mg, about 100 mg to about 1000 mg, about 100 mg to about 750 mg, about 100 mg to about 500 mg, about 100 mg to about 250 mg, about 150 mg to about 250 mg, about 150 mg to about 300 mg, about 150 mg to about 500 mg, about 250 mg to about 500 mg, about 500 mg to about 750 mg, or about 500 mg to about 1000 mg.

[00221] In some embodiments, the total daily dose of the P2X7 receptor antagonist is about 100 mg to about 1500 mg, about 100 mg to about 1000 mg, about 100 mg to about 750 mg, about 100 mg to about 500 mg, about 100 mg to about 400 mg, about 100 mg to about 300 mg, about 100 mg to about 200 mg, about 500 mg to about 3000 mg, about 500 mg to about 2500 mg, about 500 mg to about 2000 mg, about 500 mg to about 1500 mg, about 500 mg to about 1250 mg, about 500 mg to about 1000 mg, about 500 mg to about 900 mg, about 500 mg to about 800 mg, about 500 mg to about 750 mg, about 500 mg to about 700 mg, about 500 mg to about 600 mg, about 250 mg to about 750 mg, about 250 mg to about 500 mg, or about 250 mg to about 350 mg. In certain embodiments, the total daily dose of the P2X7 receptor antagonist is about 50 mg to about 750 mg. [00222] In some embodiments, the methods of the present disclosure comprise administering to a patient in need thereof a dose of from about 100 mg to about 2000 mg, about 100 mg to about 1500 mg, about 100 mg to about 1000 mg, about 100 mg to about 750 mg, about 100 mg to about 500 mg, about 100 mg to about 250 mg, about 150 mg to about 250 mg, about 150 mg to about 300 mg, about 150 mg to about 500 mg, about 250 mg to about 500 mg, about 500 mg to about 750 mg, or about 500 mg to about 1000 mg of a P2X7 receptor antagonist.

[00223] In some embodiments, the methods of the present disclosure comprise administering to a patient in need thereof a dose of from about 100 mg to about 1000 mg, about 150 mg to about 500 mg, about 250 mg to about 750 mg, about 100 mg to about 200 mg, about 400 mg to about 600 mg of a P2X7 receptor antagonist.

[00224] In some embodiments, the methods of the present disclosure comprise administering to a patient in need thereof a dose of from about 80 mg to 1300 mg, 100 mg to 1000 mg, 100 mg to 750 mg, 100 mg to 500 mg, 100 mg to 400 mg, 100 gm to 300 mg, 100 mg to 200 mg, 125 mg to 175 mg, 200 mg to 400 mg or 200 mg to 500 mg of a P2X7 receptor antagonist.

[00225] In some embodiments, the methods of the present disclosure comprise administering to a patient in need thereof a dose of from about 50 mg to about 1500 mg, about 100 mg to about 1500 mg, about 150 mg to about 1000 mg, about 150 mg to about 500 mg, about 150 mg to about 400 mg, about 150 mg to about 300 mg, about 150 mg to about 200 mg, about 500 mg to about 1500 mg, about 500 mg to about 1000 mg, about 500 mg to about 900 mg, about 500 mg to about 800 mg, about 500 mg to about 700 mg, about 500 mg to about 600 mg, about 200 mg to about 500 mg, about 300 mg to about 500 mg or about 400 mg to about 500 mg of a P2X7 receptor antagonist.

[00226] In some embodiments, the methods of the present disclosure comprise administering to a patient in need thereof a dose of from about 100 mg to about 1500 mg per dose, or from about 100 mg to about 1000 mg, about 100 mg to about 750 mg, about 100 mg to about 500 mg, about 100 mg to about 400 mg, about 100 mg to about 300 mg, about 100 mg to about 200 mg, about 500 mg to about 3000 mg, about 500 mg to about 2500 mg, about 500 mg to about 2000 mg, about 500 mg to about 1500 mg, about 500 mg to about 1250 mg, about 500 mg to about 1000 mg, about 500 mg to about 900 mg, about 500 mg to about 800 mg, about 500 mg to about 750 mg, about 500 mg to about 700 mg, about 500 mg to about 600 mg, about 250 mg to about 750 mg, about 250 mg to about 500 mg, or about 250 mg to about 350 mg per dose, or about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, or about 750 mg per dose of a P2X7 receptor antagonist.

[00227] In some embodiments, the methods of the present disclosure comprise administering to a patient in need thereof a dose of about 75 mg, about 100 mg, about 125 mg, about 130 mg, about 135 mg, about 140 mg, about 145 mg, about 150 mg, about 155 mg, about 160 mg, about 165 mg, about 170 mg, about 175 mg, about 180 mg, about 185 mg, about 190 mg, about 195 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg, about 1000 mg, about 1250 mg, about 1500 mg, about 1750 mg or about 2000 mg of the P2X7 receptor antagonist. In certain embodiments, the methods of the present disclosure comprise administering to a patient in need thereof a dose of about 500 mg of the P2X7 receptor antagonist.

[00228] In some embodiments, the methods of the present disclosure comprise administering to a patient in need thereof a dose of about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 900 mg or about 1000 mg of the P2X7 receptor antagonist.

[00229] In some embodiments, the administering comprises orally administering one or more immediate release tablets comprising a P2X7 receptor antagonist. In some embodiments, the administering comprises ingesting one or more dosage forms comprising the P2X7 receptor antagonist. In other embodiments, the P2X7 receptor antagonist is a compound of Formula I, II, III, Ilia, IV, or IVa or Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, or Compound 6, or a pharmaceutically acceptable salt thereof. In some embodiments, the one or more dosage forms is selected from the group consisting of tablets, capsules, and emulsions. In some embodiments, the tablet is an immediate release, extended release, sustained release or delayed release tablet. In other embodiments, the dosage form comprises the tablet and an enteric coating. [00230] In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 50 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 100 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 110 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 120 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 130 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 140 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 150 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 160 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 170 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 180 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 190 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 200 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 210 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 220 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 230 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 240 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 250 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 260 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 270 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 280 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 290 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 300 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 310 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 320 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 330 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 340 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 350 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 360 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 370 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 380 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 390 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 400 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 410 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 420 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 430 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 440 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 450 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 460 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 470 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 480 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 490 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 500 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 510 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 520 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 530 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 540 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is at least about 550 mg a day for the treatment of NASH.

[00231] In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 50 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 100 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 110 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 120 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 130 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 140 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 150 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 160 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 170 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 180 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 190 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 200 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 210 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 220 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 230 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 240 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 250 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 260 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 270 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 280 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 290 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 300 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 310 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 320 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 330 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 340 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 350 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 360 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 370 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 380 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 390 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 400 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 410 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 420 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 430 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 440 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 450 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 460 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 470 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 480 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 490 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 500 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 510 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 520 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 530 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 540 mg a day for the treatment of NASH. In some embodiments, the total daily dose of a P2X7 receptor antagonist is about 550 mg a day for the treatment of NASH.

[00232] In some embodiments, about 50 mg of a P2X7 receptor antagonist once a day (or twice a day) is selected to provide a substantial reduction in NASH. In some embodiments, about 60 mg of a P2X7 receptor antagonist once a day (or twice a day) is selected to provide a substantial reduction in NASH. In some embodiments, about 70 mg of a P2X7 receptor antagonist once a day (or twice a day) is selected to provide a substantial reduction in NASH. In some embodiments, about 80 mg of a P2X7 receptor antagonist once a day (or twice a day) is selected to provide a substantial reduction in NASH. In some embodiments, about 90 mg of a P2X7 receptor antagonist once a day (or twice a day) is selected to provide a substantial reduction in NASH. In some embodiments, about 100 mg of a P2X7 receptor antagonist once a day (or twice a day) is selected to provide a substantial reduction in NASH. In some embodiments, about 110 mg of a P2X7 receptor antagonist once a day (or twice a day) is selected to provide a substantial reduction in NASH. In some embodiments, about 120 mg of a P2X7 receptor antagonist once a day (or twice a day) is selected to provide a substantial reduction in NASH. In some embodiments, about 130 mg of a P2X7 receptor antagonist once a day (or twice a day) is selected to provide a substantial reduction in NASH.In some embodiments, about 140 mg of a P2X7 receptor antagonist once a day (or twice a day) is selected to provide a substantial reduction in NASH.In some embodiments, about 150 mg of a P2X7 receptor antagonist once a day (or twice a day) is selected to provide a substantial reduction in NASH.In some embodiments, about 160 mg of a P2X7 receptor antagonist once a day (or twice a day) is selected to provide a substantial reduction in NASH.In some embodiments, about 170 mg of a P2X7 receptor antagonist once a day (or twice a day) is selected to provide a substantial reduction in NASH. In some embodiments, about 180 mg of a P2X7 receptor antagonist once a day (or twice a day) is selected to provide a substantial reduction in NASH. In some embodiments, about 190 mg of a P2X7 receptor antagonist once a day (or twice a day) is selected to provide a substantial reduction in NASH. In some embodiments, about 200 mg of a P2X7 receptor antagonist once a day (or twice a day) is selected to provide a substantial reduction in NASH. In some embodiments, about 210 mg of a P2X7 receptor antagonist once a day (or twice a day) is selected to provide a substantial reduction in NASH. In some embodiments, about 220 mg of a P2X7 receptor antagonist once a day (or twice a day) is selected to provide a substantial reduction in NASH. In some embodiments, about 230 mg of a P2X7 receptor antagonist once a day (or twice a day) is selected to provide a substantial reduction in NASH. In some embodiments, about 240 mg of a P2X7 receptor antagonist once a day (or twice a day) is selected to provide a substantial reduction in NASH. In some embodiments, about 250 mg of a P2X7 receptor antagonist once a day (or twice a day) is selected to provide a substantial reduction in NASH. In some embodiments, about 260 mg of a P2X7 receptor antagonist once a day (or twice a day) is selected to provide a substantial reduction in NASH. In some embodiments, about 270 mg of a P2X7 receptor antagonist once a day (or twice a day) is selected to provide a substantial reduction in NASH. In some embodiments, about 280 mg of a P2X7 receptor antagonist once a day is selected to provide a substantial reduction in NASH. In some embodiments, about 290 mg of a P2X7 receptor antagonist once a day is selected to provide a substantial reduction in NASH. In some embodiments, about 300 mg of a P2X7 receptor antagonist once a day is selected to provide a substantial reduction in NASH. In some embodiments, about 310 mg of a P2X7 receptor antagonist once a day is selected to provide a substantial reduction in NASH. In some embodiments, about 320 mg of a P2X7 receptor antagonist once a day is selected to provide a substantial reduction in NASH. In some embodiments, about 330 mg of a P2X7 receptor antagonist once a day is selected to provide a substantial reduction in NASH. In some embodiments, about 340 mg of a P2X7 receptor antagonist once a day is selected to provide a substantial reduction in NASH. In some embodiments, about 350 mg of a P2X7 receptor antagonist once a day is selected to provide a substantial reduction in NASH. In some embodiments, about 360 mg of a P2X7 receptor antagonist once a day is selected to provide a substantial reduction in NASH. In some embodiments, about 370 mg of a P2X7 receptor antagonist once a day is selected to provide a substantial reduction in NASH. In some embodiments, about 380 mg of a P2X7 receptor antagonist once a day is selected to provide a substantial reduction in NASH. In some embodiments, about 390 mg of a P2X7 receptor antagonist once a day is selected to provide a substantial reduction in NASH. In some embodiments, about 400 mg of a P2X7 receptor antagonist once a day is selected to provide a substantial reduction in NASH. In some embodiments, about 410 mg of a P2X7 receptor antagonist once a day is selected to provide a substantial reduction in NASH. In some embodiments, about 420 mg of a P2X7 receptor antagonist once a day is selected to provide a substantial reduction in NASH. In some embodiments, about 430 mg of a P2X7 receptor antagonist once a day is selected to provide a substantial reduction in NASH. In some embodiments, about 440 mg of a P2X7 receptor antagonist once a day is selected to provide a substantial reduction in NASH. In some embodiments, about 450 mg of a P2X7 receptor antagonist once a day is selected to provide a substantial reduction in NASH. In some embodiments, about 460 mg of a P2X7 receptor antagonist once a day is selected to provide a substantial reduction in NASH. In some embodiments, about 470 mg of a P2X7 receptor antagonist once a day is selected to provide a substantial reduction in NASH. In some embodiments, about 480 mg of a P2X7 receptor antagonist once a day is selected to provide a substantial reduction in NASH. In some embodiments, about 490 mg of a P2X7 receptor antagonist once a day is selected to provide a substantial reduction in NASH. In some embodiments, about 500 mg of a P2X7 receptor antagonist once a day is selected to provide a substantial reduction in NASH. In some embodiments, about 510 mg of a P2X7 receptor antagonist once a day is selected to provide a substantial reduction in NASH. In some embodiments, about 520 mg of a P2X7 receptor antagonist once a day is selected to provide a substantial reduction in NASH. In some embodiments, about 530 mg of a P2X7 receptor antagonist once a day is selected to provide a substantial reduction in NASH. In some embodiments, about 540 mg of a P2X7 receptor antagonist once a day is selected to provide a substantial reduction in NASH. In some embodiments, about 550 mg of a P2X7 receptor antagonist once a day is selected to provide a substantial reduction in NASH.

[00233] In some embodiments, the present disclosure provides methods of treating liver disease associated with P2X7 receptor activation by administering an effective amount of a P2X7 receptor antagonist, where the treating provides no worsening in liver disease compared to prior to said administering (i.e., the administering prevents the progression of the liver disease).

[00234] Reduction of liver disease in patients with liver disease associated with P2X7 receptor activation can be determined by various methods. In some embodiments, the effectiveness of a dosage regimen can be determined by evaluation via cTl score as determined by multiparametric magnetic resonance. In some embodiments, the effectiveness of a dosage regimen can be determined by evaluation via Nonalcoholic Fatty Liver Disease Activity Score (NAS) value. In some embodiments, the effectiveness of a dosage regimen can be determined by evaluation of alanine aminotransferase (ALT) levels in the blood or serum. [00235] In some embodiments, the effectiveness of a dosage regimen can be determined by evaluation via cTl score as determined by multiparametric magnetic resonance, NAS value, serum ALT level, serum aspartate aminotransferase (AST) level, liver stiffness as determined by magnetic resonance elastography (MRE), Ultrasonographic Steatosis Score (USS), liver Hounsfield units (HU) as determined by CT scan, hepatic venous pressure gradient (HVPG), and blood IL-1 β concentration, or any combination thereof.

[00236] In some embodiments, the effectiveness of a dosage regimen can be determined by evaluation via cTl score as determined by multiparametric magnetic resonance as a primary efficacy endpoint in association with secondary efficacy endpoints such as the NAS value, serum ALT level, serum AST level, liver stiffness as determined by MRE, USS value, liver Hounsfield units (HU) as determined by CT scan, hepatic venous pressure gradient (HVPG), and blood IL-1 β concentration, or any combination thereof.

[00237] According to some embodiments of the present disclosure, the dosing frequency and dose amount per administration of a P2X7 receptor antagonist are selected to provide therapeutic effects for the treatment of liver disease associated with P2X7 activation selected fatty liver disease, non alcoholic fatty liver disease, non-alcoholic steatohepatitis, cirrhosis, liver fibrosis, or hepatocellular carcinoma.

[00238] According to some embodiments of the present disclosure, the dosing frequency and dose amount per administration of the P2X7 receptor antagonist are selected to provide therapeutic effects for the treatment of NASH. In certain embodiments, the dosing frequency and amount per administration of the P2X7 receptor antagonist are selected to provide for the treatment of Fibrosis stage 1-3 NASH as determined by two-dimensional magnetic resonance elastography (2D MRE) score. In certain embodiments, the dosing frequency and amount per administration of the P2X7 receptor antagonist are selected to provide for the treatment of NASH in a patient having advanced liver fibrosis as determined by 2D MRE score. In certain embodiments, the dosing frequency and amount per administration of the P2X7 receptor antagonist are selected to provide for the treatment of NASH in a patient having liver cirrhosis as determined by 2D MRE score. In certain embodiments, the dosing frequency and amount per administration of the P2X7 receptor antagonist are selected to provide for the treatment of NASH in a patient having liver stiffness as determined by MRE of between about 2.5 kPa to about 4.65 kPa. In certain embodiments, the dosing frequency and amount per administration of the P2X7 receptor antagonist are selected to provide for the treatment of NASH in a patient having proton density fat fraction (PDFF) as determined by multiparametric magnetic resonance of≥ 8% steatosis. In certain embodiments, the dosing frequency and amount per administration of the P2X7 receptor antagonist are selected to provide for the treatment of NASH in a patient having cTl score as determined by multiparametric magnetic resonance of≥ 827.5 ms. In certain embodiments, the dosing frequency and amount per administration of the P2X7 receptor antagonist are selected to provide for the treatment of NASH in a patient having liver stiffness as determined by ultrasound-based transient elastography of > 8. In certain embodiments, the dosing frequency and amount per administration of the P2X7 receptor antagonist are selected to provide for the treatment of NASH in a patient having controlled attenuation parameter (CAP) as determined by ultrasound-based transient elastography (Fibroscan) is >300 dB/m. In certain embodiments, the dosing frequency and amount per administration of the P2X7 receptor antagonist are selected to provide for the treatment of NASH in a patient having serum alanine aminotransferase (ALT) of between about 5 IU/L and 225 IU/L. In certain embodiments, the dosing frequency and amount per administration of the P2X7 receptor antagonist are selected to provide for the treatment of a liver disease associated with P2X7 activation in a patient having Steatosis, Activity, and Fibrosis (SAF) activity score of≥ 2. In certain embodiments, the dosing frequency and amount per administration of the P2X7 receptor antagonist are selected to provide for the treatment of a liver disease associated with P2X7 activation in a patient having SAF activity score is <2. In certain embodiments, the dosing frequency and amount per administration of the P2X7 receptor antagonist are selected to provide for the treatment of NASH in a patient having a difference of spleen and liver Hounsfield units (HU_spleen-HU_liver) as determined by CT scan of greater than 0.

[00239] In certain embodiments, the P2X7 receptor antagonist is administered on a once a day or twice a day basis for at least a week, for example, about a week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 12 weeks, about 18 weeks, about 24 weeks, and about 50 weeks.

[00240] In certain embodiments, the P2X7 receptor antagonist is administered on a once a day or twice a day basis for at least a month, for example, about a month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, about 24 months, about 30 months, and about 36 months.

[00241] In certain embodiments, at least about 50 mg or about 50 mg of the P2X7 receptor antagonist is administered on a once a day or twice a day basis for at least a week. In certain embodiments, at least about 60 mg or about 60 mg of the P2X7 receptor antagonist is administered on a once a day or twice a day basis for at least a week. In certain embodiments, at least about 70 mg or about 70 mg of the P2X7 receptor antagonist is administered on a once a day or twice a day basis for at least a week. In certain embodiments, at least about 80 mg or about 80 mg of the P2X7 receptor antagonist is administered on a once a day or twice a day basis for at least a week. In certain embodiments, at least about 90 mg or about 90 mg of the P2X7 receptor antagonist is administered on a once a day or twice a day basis for at least a week. In certain embodiments, at least about 100 mg or about 100 mg of the P2X7 receptor antagonist is administered on a once a day or twice a day basis for at least a week. In certain embodiments, at least about 110 mg or about 110 mg of the P2X7 receptor antagonist is administered on a once a day or twice a day basis for at least a week. In certain embodiments, at least about 120 mg or about 120 mg of the P2X7 receptor antagonist is administered on a once a day or twice a day basis for at least a week. In certain embodiments, at least about 130 mg or about 130 mg of the P2X7 receptor antagonist is administered on a once a day or twice a day basis for at least a week. In certain embodiments, at least about 140 mg or about 140 mg of the P2X7 receptor antagonist is administered on a once a day or twice a day basis for at least a week. In certain embodiments, at least about 150 mg or about 150 mg of the P2X7 receptor antagonist is administered on a once a day or twice a day basis for at least a week. In certain embodiments, at least about 160 mg or about 160 mg of the P2X7 receptor antagonist is administered on a once a day or twice a day basis for at least a week. In certain embodiments, at least about 170 mg or about 170 mg of the P2X7 receptor antagonist is administered on a once a day basis for at least a week. In certain embodiments, at least about 180 mg or about 180 mg of the P2X7 receptor antagonist is administered on a once a day basis for at least a week. In certain embodiments, at least about 190 mg or about 190 mg of the P2X7 receptor antagonist is administered on a once a day basis for at least a week. In certain embodiments, at least about 200 mg or about 200 mg of the P2X7 receptor antagonist is administered on a once a day basis for at least a week. In certain embodiments, at least about 210 mg or about 210 mg of the P2X7 receptor antagonist is administered on a once a day or twice a day basis for at least a week. In certain embodiments, at least about 220 mg or about 220 mg of the P2X7 receptor antagonist is administered on a once a day or twice a day basis for at least a week. In certain embodiments, at least about 230 mg or about 230 mg of the P2X7 receptor antagonist is administered on a once a day or twice a day basis for at least a week. In certain embodiments, at least about 240 mg or about 240 mg of the P2X7 receptor antagonist is administered on a once a day or twice a day basis for at least a week. In certain embodiments, at least about 250 mg or about 250 mg of the P2X7 receptor antagonist is administered on a once a day or twice a day basis for at least a week. In certain embodiments, at least about 260 mg or about 260 mg of the P2X7 receptor antagonist is administered on a once a day or twice a day basis for at least a week. In certain embodiments, at least about 270 mg or about 270 mg of the P2X7 receptor antagonist is administered on a once a day basis for at least a week. In certain embodiments, at least about 280 mg or about 280 mg of the P2X7 receptor antagonist is administered on a once a day basis for at least a week. In certain embodiments, at least about 290 mg or about 290 mg of the P2X7 receptor antagonist is administered on a once a day basis for at least a week. In certain embodiments, at least about 300 mg or about 300 mg of the P2X7 receptor antagonist is administered on a once a day basis for at least a week. In certain embodiments, at least about 310 mg or about 310 mg of the P2X7 receptor antagonist is administered on a once a day or twice a day basis for at least a week. In certain embodiments, at least about 320 mg or about 320 mg of the P2X7 receptor antagonist is administered on a once a day or twice a day basis for at least a week. In certain embodiments, at least about 330 mg or about 330 mg of the P2X7 receptor antagonist is administered on a once a day or twice a day basis for at least a week. In certain embodiments, at least about 340 mg or about 340 mg of the P2X7 receptor antagonist is administered on a once a day or twice a day basis for at least a week. In certain embodiments, at least about 350 mg or about 350 mg of the P2X7 receptor antagonist is administered on a once a day or twice a day basis for at least a week. In certain embodiments, at least about 360 mg or about 360 mg of the P2X7 receptor antagonist is administered on a once a day or twice a day basis for at least a week. In certain embodiments, at least about 370 mg or about 370 mg of the P2X7 receptor antagonist is administered on a once a day basis for at least a week. In certain embodiments, at least about 380 mg or about 380 mg of the P2X7 receptor antagonist is administered on a once a day basis for at least a week. In certain embodiments, at least about 390 mg or about 390 mg of the P2X7 receptor antagonist is administered on a once a day basis for at least a week. In certain embodiments, at least about 400 mg or about 400 mg of the P2X7 receptor antagonist is administered on a once a day basis for at least a week. In certain embodiments, at least about 410 mg or about 410 mg of the P2X7 receptor antagonist is administered on a once a day or twice a day basis for at least a week. In certain embodiments, at least about 420 mg or about 420 mg of the P2X7 receptor antagonist is administered on a once a day or twice a day basis for at least a week. In certain embodiments, at least about 430 mg or about 430 mg of the P2X7 receptor antagonist is administered on a once a day or twice a day basis for at least a week. In certain embodiments, at least about 440 mg or about 440 mg of the P2X7 receptor antagonist is administered on a once a day or twice a day basis for at least a week. In certain embodiments, at least about 450 mg or about 450 mg of the P2X7 receptor antagonist is administered on a once a day or twice a day basis for at least a week. In certain embodiments, at least about 460 mg or about 460 mg of the P2X7 receptor antagonist is administered on a once a day or twice a day basis for at least a week. In certain embodiments, at least about 470 mg or about 470 mg of the P2X7 receptor antagonist is administered on a once a day basis for at least a week. In certain embodiments, at least about 480 mg or about 480 mg of the P2X7 receptor antagonist is administered on a once a day basis for at least a week. In certain embodiments, at least about 490 mg or about 490 mg of the P2X7 receptor antagonist is administered on a once a day basis for at least a week. In certain embodiments, at least about 500 mg or about 500 mg of the P2X7 receptor antagonist is administered on a once a day basis for at least a week. In certain embodiments, at least about 510 mg or about 510 mg of the P2X7 receptor antagonist is administered on a once a day or twice a day basis for at least a week. In certain embodiments, at least about 520 mg or about 520 mg of the P2X7 receptor antagonist is administered on a once a day or twice a day basis for at least a week. In certain embodiments, at least about 530 mg or about 530 mg of the P2X7 receptor antagonist is administered on a once a day or twice a day basis for at least a week. In certain embodiments, at least about 540 mg or about 540 mg of the P2X7 receptor antagonist is administered on a once a day or twice a day basis for at least a week. In certain embodiments, at least about 550 mg or about 550 mg of the P2X7 receptor antagonist is administered on a once a day or twice a day basis for at least a week. [00242] Applicants discovered that there is a food effect associated with certain formulations of P2X7 receptor antagonists of the present disclosure (that is, in some cases, the bioavailability of the P2X7 receptor antagonists depends at least in part on the patient’s food consumption prior to, during or after administration of the P2X7 receptor antagonist). Specifically, a Phase 1 clinical study was conducted (as described in Example 6, below) in which human subjects were dosed with an immediate release tablet containing micronized Compound 1 P2X7 receptor antagonist. Oral dosing was conducted in a fed and fasted state, with dosing once per day. These data suggest that absorption of Compound 1 in the fasted state nears saturation at approximately 1500 mg while the 3000 mg dose showed a near dose proportional increase vs. 1500 g in the fasted state. Twice a day (b.i.d.) dosing of 150 mg or 500 mg was also studied, each with a standard meal. The data (see, Example 6), show that the dosing gave a projected therapeutic plasma exposure after 14 days (336 hours) of dosing (see FIG. 3). A dose of 150 mg resulted in an AUC_0-inf of about 4230 ng*hr mL, a dose of 500 mg resulted in an AUC_0-inf of about 9970 ng*hr mL, and a dose of 1500 mg resulted in an AUC_0-inf of about 6040 ng*hr mL. These data show a nonlinear pharmacokinetic profile. Importantly, however, administration of the P2X7 receptor antagonist with a meal significantly increased the AUC_0-inf of Compound 1. Specifically, a dose of about 500 mg as immediate release tablets resulted in an AUC_0-inf of about 13700 ng*hr mL when the tablet was administered to a healthy subject with food, whereas administration of the same dose to the subject in a fasted mode resulted in an AUC_0-inf of about 11700 ng*hr mL, demonstrating that oral administration of Compound 1 with food can significantly enhance plasma availability of the agent. The study showed that the studied doses of Compound 1 were safe and well tolerated.

[00243] In some embodiments, the methods of the present disclosure require that the P2X7 receptor antagonist is administered without food (i.e., patient is in a fasted state). In certain embodiments, the P2X7 receptor antagonist administered without food is Compound 1 or a pharmaceutically acceptable salt thereof.

[00244] In some embodiments, the methods of the present disclosure require that the P2X7 receptor antagonist is administered with food. In alternative embodiments, it is recommended that the P2X7 receptor antagonist is taken with a meal such as a morning meal and/or an evening meal. In certain embodiments, the P2X7 receptor antagonist administered with food is Compound 1 or a pharmaceutically acceptable salt thereof. In certain further embodiments, the methods of the present disclosure comprise administering a therapeutically effective amount of Compound 1 or a pharmaceutically acceptable salt thereof with food. In certain further embodiments, the therapeutically effective amount of Compound 1 is about 250 mg or 500 mg provided twice a day.

[00245] In some embodiments, the methods of the present disclosure require that the P2X7 receptor antagonist is administered with food in the stomach. In such embodiments, the presence of food provides two signals, one that is said to stem from stomach distension and the other a chemical signal based on food in the stomach.

[00246] In some embodiments, the methods of the present disclosure require the patient in need thereof to ingest food at a time from about 120 minutes before, to about 60 minutes after administration of the P2X7 receptor antagonist described herein.

[00247] In other embodiments, the food is ingested about 15 minutes before administration, about 30 minutes before administration, about 45 minutes before administration, about 60 minutes before administration, about 75 minutes before administration, about 90 minutes before administration, about 105 minutes before administration, or about 120 minutes before administration, or within a time range between any of the aforementioned times.

[00248] In some embodiments, the P2X7 receptor antagonist is administered from about 1 to about 90 minutes, about 1 to about 75 minutes, about 1 to about 60 minutes, about 1 to 30 minutes, about 1 to about 15 minutes, about 1 to about 10 minutes, after food is ingested. In some embodiments, the P2X7 receptor antagonist is administered about 1, about 2, about 3, about 4, about 5, about 10, about 15, about 20, about 25, about 30, about 45, about 60, about 75, or about 90 minutes after food is ingested.

[00249] In some embodiments, the P2X7 receptor antagonist is administered when food is ingested.

[00250] In other embodiments, the food is ingested about 15 minutes after administration, about 30 minutes after administration, about 45 minutes after administration, or about 60 minutes after administration or within a time range between any of the aforementioned times. [00251] In some embodiments, the P2X7 receptor antagonist is administered from about 1 to about 90 minutes, about 1 to about 75 minutes, about 1 to about 60 minutes, about 1 to 30 minutes, about 1 to about 15 minutes, about 1 to about 10 minutes, before food is ingested. In some embodiments, the P2X7 receptor antagonist is administered about 1, about 2, about 3, about 4, about 5, about 10, about 15, about 20, about 25, about 30, about 45, about 60, about 75, or about 90 minutes before food is ingested.

[00252] In some embodiments, the food ingested is a high fat and high calorie food. In certain embodiments, the caloric content of the high fat and high calorie food is at least about 700 kilocalories (kcal), and at least about 40 percent of the caloric content of the food is from fat. For example, the fat can contribute to about 50 percent of the caloric content of the food of high fat and high calorie. In particular embodiments, the caloric content of the high fat and high calorie food is about 900 kilocalories.

[00253] In some embodiments, a high fat food is a food that provides about or at least about 500 calories to the patient of which about 25%, about 35%, about 40% or about 50% of the caloric content of the food is from fat.

[00254] In some embodiments, the food ingested is a medium fat and medium calorie food. In certain embodiments, the caloric content of the medium fat and medium calorie food is about 300 kcal to about 700 kcal, and between about 20 percent to about 40 percent of the caloric content of the food is from fat. In particular embodiments, the caloric content of the medium fat and medium calorie food is about 400 kcal.

[00255] In some embodiments, the food ingested is a low fat and low calorie food. In certain embodiments, the caloric content of the low fat and low calorie food is between about 100 kcal to about 300 kcal, and the fat content is approximately 3 grams or less, or about 20 percent or less of the caloric content of the food are from fat In particular embodiments, the caloric content of the food of low fat and low calorie is about 100 kilocalories.

[00256] According to some embodiments, the substantial reduction in liver disease provided by the methods of the present disclosure requires treatment for a specified time interval (e.g., at least one week) before the patient experiences substantial reduction of liver. In some embodiments, after treatment for at least one week, at least two weeks, at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least eleven weeks or at least twelve weeks the patient experiences a substantial reduction of liver disease compared to prior to the treatment. In certain embodiments, after treatment for at least one week the patient experiences a substantial reduction of liver disease compared to prior to the treatment. In certain embodiments, after treatment for at least twelve weeks the patient experiences a substantial reduction of liver disease compared to prior to the treatment. In certain embodiments, after treatment for at least twenty-four weeks the patient experiences a substantial reduction of liver disease compared to prior to the treatment. In certain embodiments, after treatment for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, about least 9 months, at least 10 months, at least 11 months, or at least 12 months, the patient experiences a substantial reduction of liver disease compared to prior to the treatment. According to this embodiment, the substantial reduction in liver disease may be expressed using any of the methods described herein (for example, decline in ALT or ASP value compared to prior to the treatment, improvement in the Nonalcoholic Fatty Liver Disease Activity Score value compared to prior to the treatment, etc.).

[00257] According to methods of the present disclosure, the therapeutic effectiveness (e.g., a substantial reduction in liver disease) of the P2X7 receptor antagonists of the present disclosure in the treatment of liver disease associated with P2X7 receptor activation may be expressed using any suitable method known to those skilled in the art (see, e.g., Hannah et al, 2016, Gasteroenterology Hepatol, 12:756-763). In some embodiments, the therapeutic effectiveness of the P2X7 receptor antagonists of the present disclosure in the treatment of liver disease associated with P2X7 receptor activation is determined using a liver biopsy performed for a pathologic evaluation of lobular and portal inflammation as well as hepatocyte ballooning, each of which can be used to distinguish non-NASH NAFLD from NASH. In other embodiments, a liver biopsy is performed to evaluate the presence and/or extent of liver fibrosis.

[00258] As known to those skilled in the art, liver biopsy reveals damage similar to that seen in alcoholic hepatitis, usually including large fat droplets (macrovesicular fatty infiltration). Indications for biopsy include unexplained signs of portal hypertension (e.g., splenomegaly, cytopenia) and unexplained elevations in aminotransferase levels that persist for greater than 6 months in a patient with diabetes, obesity, or dyslipidemia.

[00259] According to some embodiments, the therapeutic effectiveness of the P2X7 receptor antagonists of the present disclosure in the treatment of liver disease associated with P2X7 receptor activation is determined using imaging tests known to those skilled in the art, including ultrasonography, CT, and particularly magnetic resonance imaging (MRI), magnetic resonance elastography, and body composition by dual-energy X-ray absorptiometry (DXA), are used to identify hepatic steatosis. In some embodiments, magnetic resonance is used to quantify hepatic steatosis by measuring the proton density fat fraction (PDFF) which is the fraction of MRI-visible protons bound to fat divided by all protons in the liver (bound to fat and water) (see, e.g., Jayakumar et al, 2016, Curr Hepatol Rep, 15:86-95; Dubai et al., 2016, J Hepatol, 65: 1006-1016).

[00260] According to some embodiments, after the treatment the patient experiences a substantial reduction of liver disease that is characterized by decline in the patient’s extent of fibrosis in the liver compared to prior to the treatment. In certain embodiments, the reduction of liver disease is characterized by a decline in the patient’s extent of fibrosis in the liver ranging from about 2% to about 60%, for example, about 2%, about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, and about 60%, compared to prior to the treatment. In certain further embodiments, the decline is the in the patient’s extent of fibrosis in the liver is provided by administration of a P2X7 receptor antagonist of the present disclosure for between about 1 to about 5 years.

[00261] According to some embodiments, after the treatment the patient experiences a substantial reduction of liver disease that is characterized by decline in the patient’s rate of liver fibrosis compared to prior to the treatment.

[00262] Since liver biopsy are painful and inconvenient, non-invasive methods have been developed as reliable methods of liver fibrosis assessment and diagnosis. In some embodiments, the accumulation of fat in the liver can be indicated by several non-invasive means, for example, by ultrasonography, computed tomography (CT), magnetic resonance imaging (MRI), measurement of serum alanine transaminase and aspartate transaminase, measurement of liver size or weight, and/or biopsy (Pavlides et al., 2016, J Hepatol, 64:308-315). In other embodiments, hepatic fibrosis is detected using body composition by dual-energy X-ray absorptiometry (DXA).

[00263] Multiparametric magnetic resonance (e.g., Liver MultiScan, Perspectum Diagnostics LTD) is one reliable method for in vivo diagnosis of liver disease (Banerjee et al., 2014, J Hepatol, 60:69-77). This technology enables post-processing of MRI data into parametric maps of proton density fat fraction (PDFF), T2* and corrected T1 (cT1). Effective treatment of fibrosis or steatohepatitis can be determined using this MR-PDFF technology wherein, e.g., a decrease in cT1 in a NASH patient treated with, e.g., a P2X7 receptor antagonist, is observed relative to the cT1 value for a NASH patient treated with a placebo or not treated.

[00264] In some embodiments, after the treatment the patient experiences a substantial reduction of liver disease that is characterized by at least about a 2% decline in in the patient’s proton density fat fraction (PDFF) as determined by multiparametric magnetic resonance compared to prior to the treatment. In some embodiments, the reduction of liver disease is characterized by a decline in patient’s PDFF ranging from about 2% to about 100%, for example, about 2%, about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, and about 100%, compared to prior to the treatment.

[00265] In some embodiments, after the treatment the patient experiences a substantial reduction of liver disease that is characterized by at least about a 2% decline in the cTl score as determined by multiparametric magnetic resonance compared to prior to the treatment. In some

embodiments, the reduction of liver disease is characterized by a decline in patient’s cTl score ranging from about 2% to about 100%, for example, about 2%, about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, and about 100%, compared to prior to the treatment.

[00266] In some embodiments, after the treatment the patient experiences a substantial reduction of liver disease that is characterized a 35 ms reduction in cTl compared to placebo. In some embodiments, after the treatment the patient experiences a substantial reduction of liver disease that is characterized a 35 ms reduction in cTl relative compared to prior to the treatment.

[00267] Liver stiffness measurement using magnetic resonance elastography (MRE), ultrasound- based transient elastography (Fibroscan™), or acoustic radiation force impulse have been used to assess fibrosis and predicting clinical outcomes (Bohte et al., 2014, Eur Radiol, 24:638-648; Singh et al., 2015, Clin Gastroenterol Hepatol, 13:440-451). MRE can detect and stage liver fibrosis and can differentiate isolated fatty liver disease from steatohepatitis with or without fibrosis (Venkatesh et al, 2013, J Comput Assist Tomogr, 37:887-896). Magnetic resonance can quantify hepatic steatosis by measuring the proton density fat fraction (PDFF) which is the fraction of MRI- visible protons bound to fat divided by all protons in the liver (bound to fat and water) (see, e.g., Jayakumar et al, 2016, Curr Hepatol Rep, 15:86-95; Dubai et al., 2016, J Hepatol, 65: 1006-1016).

[00268] In some embodiments, after the treatment the patient experiences a substantial reduction of liver disease that is characterized by at least a 10% decline in liver stiffness as determined by magnetic resonance elastography (MRE) compared to prior to administering. In some embodiments, the reduction of liver disease is characterized by a decline in liver stiffness as determined by MRE ranging from about 10% to about 100%, for example, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, and about 100%, compared to prior to the treatment.

[00269] In some embodiments, after the treatment the patient experiences a substantial reduction of liver disease that is characterized by at least a 10% decline in MRI-PDFF compared to prior to administering. In some embodiments, the reduction of liver disease is characterized by a decline in MRI-PDFF ranging from about 10% to about 100%, for example, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, and about 100%, compared to prior to the treatment.

[00270] Ultrasonography methods for assessing liver are known to those of skill in the art.

Ultrasonography assessment of hepatic steatosis (e.g., fat accumulation in liver) can comprise use of conventional B-mode ultrasonography. Assessment of various hepatic ultrasonography parameters can be used for the assessment of hepatic steatosis. Exemplary ultrasonography parameters for the assessment of hepatic steatosis include but are not limited to (1) parenchymal brightness, (2) liver-to-kidney contrast, (3) deep beam attenuation, (4) bright vessel walls, and (5) gallbladder wall definition. Assessment of such ultrasonography parameters can be used to calculate an ultrasonographic steatosis score (USS). USS can be calculated, e.g., as follows: absent (score 0) steatosis was defined as normal liver echotexture; mild (score 1) steatosis as slight and diffuse increase in fine parenchymal echoes with normal visualization of diaphragm and portal vein borders; moderate (score 2) steatosis as moderate and diffuse increase in fine echoes with slightly impaired visualization of portal vein borders and diaphragm; and severe (score 3) steatosis as fine echoes with poor or no visualization of portal vein borders, diaphragm, and posterior portion of the right lobe.

[00271] In some embodiments, after the treatment the patient experiences a substantial reduction of liver disease that is characterized by at least a one point decline in ultrasonographic steatosis score (USS) compared to prior to the treatment. In some embodiments, the reduction of liver disease is characterized by a decline in USS ranging from about one point to about three points compared to prior to the treatment. In some embodiments, the reduction of liver disease is characterized by a decline USS of about two points. In some embodiments, the reduction of liver disease is characterized by a decline in USS of about three points. In some embodiments, the reduction of liver disease is characterized by a decline in USS to less than 3. In some embodiments, the reduction of liver disease is characterized by a decline in USS to less than 2. In some embodiments, the reduction of liver disease is characterized by a decline in USS to less than 1.

[00272] In some embodiments, after the treatment the patient experiences a substantial reduction of liver disease that is characterized by at least about a 50% decline in USS compared to prior to the treatment. In some embodiments, the reduction of liver disease is characterized by a decline in USS ranging from about 50% to about 100%, for example, about 50%, about 60%, about 70%, about 80%, about 90%, and about 100%, compared to prior to the treatment.

[00273] A variety of biomarkers present in the serum are becoming recognized as reliable markers for diagnosis and assessment of hepatic fibrosis and related disorders such as NASH, NAFLD, and liver fibrosis, as well as other metabolic diseases and disorders such as obesity, type 2 diabetes mellitus, or dyslipidemia. Diagnosis of NASH is often suspected in patients with risk factors such as obesity, type 2 diabetes mellitus, or dyslipidemia and in patients with unexplained laboratory abnormalities suggesting liver disease (see, e.g., www.merckmanuals.com/professional/hepatic- and-biliary-disorders/approach-to-the-patient-with-liver-disease/nonalcoholic-steatohepatitis- nash). The most common laboratory abnormalities are elevations in aminotransferase levels. Unlike in alcoholic liver disease, the ratio of AST/ALT in NASH is usually < 1. Alkaline phosphatase and gamma-glutamyl transpeptidase (GGT) occasionally increase. Hyperbilirubinemia, prolongation of PT, and hypoalbuminemia are uncommon.

[00274] For diagnosis, strong evidence (such as a history corroborated by friends and relatives) that alcohol intake is not excessive (e.g., is < 20 g/day) can be considered. Serologic tests can be done to show the absence of hepatitis B and C.

[00275] Various serum biomarkers have been shown to correlate with inflammation and can be used in the diagnosis of, e.g., NASH or NAFLD. These include inflammation markers such as high-sensitivity C-reactive protein (hs-CRP), IL-1 β, IL-6, TNF-a, cytokeratin 18 (CK-18) cleavage products CK-18-M30 and CK-18-M65, FGF-21, adiponectin (inversely correlated), leptin, and resistin. Hepatocyte fibrosis biomarkers include those measured in the Enhanced Liver Fibrosis (ELF) blood test (hyaluronic acid (HA), procollagen III amino terminal peptide (PIIINP) and tissue inhibitor of metalloproteinase 1 (TIMP-l), those measured in the Fibrosis-4 (FIB-4) Index (aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and N-terminal type III collagen propeptide (pro-C3) (see, e.g., Hansen et al., 2018, Scan J Gastroenterol, 53:83-87).

[00276] Liver fibrosis can also result from or be associated with metabolic disorders such as metabolic syndrome. Variables commonly associated with increased risk of fibrosis in NASH include the presence of diabetes, increasing age, increased AST/ALT ratio, decreased platelets, hyaluronic acid, increased body mass index (BMI), and increased homeostasis model assessment for insulin resistance (HOMA-IR).

[00277] In some embodiments, hepatic steatosis can be evidenced by measurement of serum alanine transaminase (ALT) and/or aspartate transaminase (AST) levels. Methods of measuring serum alanine transaminase and/or aspartate transaminase levels can be any known to those of skill in the art or otherwise described herein. Hepatic steatosis can be indicated by an increase in serum ALT levels as compared to a control subject without hepatic steatosis. In some cases, hepatic steatosis can be indicated by an increase in serum ALT levels and serum aspartate transaminase levels as compared to a control subject without hepatic steatosis. In some cases, hepatic steatosis can be indicated by an aspartate transaminase to alanine transaminase ratio that is greater than one. [00278] In some embodiments, after the treatment the patient experiences a substantial reduction of liver disease that is characterized by at least a 10% decline in alanine aminotransferase (ALT) compared to prior to the treatment. In some embodiments, the reduction of liver disease is characterized by a decline in ALT ranging from about 10% to about 100%, for example, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about

50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about

90%, about 95%, and about 100%, compared to prior to the treatment.

[00279] In some embodiments, after the treatment the patient experiences a substantial reduction of liver disease that is characterized by at least a 10% decline in aspartate aminotransferase (AST) compared to prior to the treatment. In some embodiments, the reduction of liver disease is characterized by a decline in AST ranging from about 10% to about 100%, for example, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about

90%, about 95%, and about 100%, compared to prior to the treatment.

[00280] In some embodiment, the Steatosis, Activity, and Fibrosis (SAF) scoring system is used, which has been shown to differentiate between NASH and NAFLD without NASH (Bedossa et al, 2012, Hepatology, 56: 1751-1759). In some embodiments, after the treatment the patient experiences a substantial reduction of liver disease that is characterized by a SAF activity score less than 2.

[00281] CT methods for assessing liver are also known to those of skill in the art. CT images can be assessed by, e.g., a radiologist. Assessment of CT images can include, e.g., measuring density of regions of interest in the images. Regions of interest within images can be selected so as not to contain blood vessels or other artifacts (e.g., motion artifacts). Density of regions of interest in a CT image can be measured in Hounsfield units (HU). Normal liver tissue can have a HU measurement of 40-60 HU. By contrast, fat typically has a lower density. For example, fat can have an HU measurement of, e.g., about -100 to about -500. Hepatic steatosis can be evidenced by an HU measurement less than 40 HU. Hepatic steatosis can be evidenced by an HU measurement that is between -500 and 40 HU, for example, an HU measurement that is -500-1 HU, -100-10 HU, 0-20 HU, 5-30 HU, or 20-39.9 HU. Hepatic steatosis can be evidenced by a difference in HU measurement between spleen and liver (e.g., HUspieen-HUiiver). For example, hepatic steatosis can be evidenced if HUspieen-HUiiver is greater than 0, for example, if HUspieen- HUiiver is between 1 - 10, 10-20, or more than 20. In some embodiments, a difference in HU measurement between spleen and liver of 18.5 is used to diagnose hepatic steatosis. In some embodiments, the present disclosure provides a method of treating liver disease associated with P2X7 receptor activation, wherein the difference in the patient’s spleen and liver Hounsfield units (HUspieen-HUiiver) as determined by CT scan is greater than 0.

[00282] In some embodiments, after the treatment the patient experiences a substantial reduction of liver disease that is characterized by an increase of the patient’s liver Hounsfield units (HU) to at least about 20 HU as determined by CT scan. In some embodiments, after the treatment the patient experiences a substantial reduction of liver disease that is characterized by an increase of the patient’s liver HU to at least about 30 HU as determined by CT scan.

[00283] Hepatic steatosis can be assessed by a variety of methods for the purpose of diagnosis and/or monitoring therapeutic efficacy of treatment with a P2X7 receptor antagonist according to the embodiments described herein. Regression of hepatic steatosis and the associated inflammatory process are features that can be used to assess response to therapy. Currently, repeat biopsy is one reliable means to assess such changes. For example, hepatic steatosis in a subject can be evidenced, by an accumulation of fat in the liver of the subject (e.g., by an accumulation of fat in hepatic cells of the subject).

[00284] Hepatic steatosis can be evidenced by tissue biopsy. A liver biopsy sample can be obtained by any means known to those of skill in the art, for example, by needle biopsy. The sample can be processed by any means known to those of skill in the art or otherwise described herein. The sample can be fixed (e.g., with formalin) or may be unfixed. The sample may be snap-frozen. For example, the sample may be sectioned into thin sections. The sections may be stained, e.g., with hematoxylin and eosin. Accumulation of fat in the liver can be evidenced by appearance of vacuoles which are filled with lipids such as, by way of example only, triglycerides. Such vacuoles can appear to be optically "empty," since fats can dissolve during histological tissue processing. Accordingly, levels of hepatic steatosis can be determined by measuring the number, size, or density of hepatic lipid vacuoles. In some embodiments, a therapeutically effective dosing regimen with a P2X7 receptor antagonist as described herein is one that results in a decrease in the size, number, and/or density of hepatic lipid vacuoles in a subject treated according to the dosing regimen, as determined by a liver biopsy

[00285] In some embodiments, after the treatment the patient experiences a substantial reduction of liver disease that is characterized by a decrease in the size, number, and/or density of hepatic lipid vacuoles of at least about 5% compared to prior to the administering. In certain embodiments, after the treatment the patient experiences a substantial reduction of liver disease that is characterized by a decrease in the size, number, and/or density of hepatic lipid vacuoles of at least about 5%, 10%, 20%, 30%, 40%, 50% or 60% compared to prior to the administering.

[00286] In an alternative embodiment, the patient treated according to the present disclosure, e.g., treated with Compound 1, experiences no decrease in the size, number, and/or density of hepatic lipid vacuoles as compared to an untreated patient or a patient treated with a placebo.

[00287] The Nonalcoholic Fatty Liver Disease Activity Score (NAS) was developed to provide a numerical pathologic score that differentiates between necroinflammatory activity and fibrosis (Bedossa et al., 2016, Gastroenterology, 150: 1811-1822). Total NAS score is the sum of three subscale scores: severity of steatosis (Score: 0, 1, 2 or 3), inflammation (Score: 0, 1, 2, or 3) and hepatocellular ballooning (Score: 0, 1 or 2). A score of 5 or greater correlates with the histologic diagnosis of NASH.

[00288] In some embodiments, after the treatment the patient experiences a substantial reduction of liver disease that is characterized by at least a 1 point decline in total NAS score compared to prior to the treatment. Decline in NAS score is synonymous with improvement. In some embodiments, the reduction of liver disease is characterized by a decline in total NAS score ranging from about 1 point to about 8 points compared to prior to the treatment. In some embodiments, the reduction of liver disease is characterized by a decline total NAS score of about 2 points. In some embodiments, the reduction of liver disease is characterized by a decline in Total NAS score of about 3 points. In some embodiments, the reduction of liver disease is characterized by a decline in Total NAS score of about 4 points. In some embodiments, the reduction of liver disease is characterized by a decline in Total NAS score of about 5 points. In some embodiments, the reduction of liver disease is characterized by a decline in Total NAS score of about 6 points. In some embodiments, the reduction of liver disease is characterized by a decline in Total NAS score of about 7 points. In some embodiments, the reduction of liver disease is characterized by a decline in Total NAS score of about 8 points. In some embodiments, the reduction of liver disease is characterized by a decline total NAS score of at least 2 points. In some embodiments, the reduction of liver disease is characterized by a decline in Total NAS score of at least 3 points. In some embodiments, the reduction of liver disease is characterized by a decline in Total NAS score of at least 4 points. In some embodiments, the reduction of liver disease is characterized by a decline in Total NAS score of at least 5 points. In some embodiments, the reduction of liver disease is characterized by a decline in Total NAS score of at least 6 points. In some embodiments, the reduction of liver disease is characterized by a decline in Total NAS score of at least 7 points. In some embodiments, the reduction of liver disease is characterized by a decline in Total NAS score of at least 8 points.

[00289] In some embodiments, the reduction of liver disease is characterized by a decline in total NAS score to less than 5. In some embodiments, the reduction of liver disease is characterized by a decline in total NAS score to less than 4. In some embodiments, the reduction of liver disease is characterized by a decline in total NAS score to less than 3. In some embodiments, after the treatment the patient experiences a substantial reduction of liver disease that is characterized by at least a 1 point decline in at least one subscale value selected from steatosis, inflammation, and hepatocellular ballooning compared to prior to the treatment.

[00290] In some embodiments, after the treatment the patient experiences a substantial reduction of liver disease that is characterized by at least about a 50% decline in total NAS score compared to prior to the treatment. In some embodiments, the reduction of liver disease is characterized by a decline in total NAS score ranging from about 50% to about 100%, for example, about 50%, about 60%, about 70%, about 80%, about 90%, and about 100%, compared to prior to the treatment.

[00291] The hepatic venous pressure gradient (HVPG) can be measured wherein a reduction in the HVPG after administration of a therapeutic agent indicates efficacy of the therapeutic agent in treating a liver disorder such as NASH, cirrhosis or liver fibrosis. [00292] In some embodiments, after the treatment the patient experiences a substantial reduction of liver disease that is characterized by a decrease in HVPG to less than 12 mm Hg. In some embodiments, after the treatment the patient experiences a substantial reduction of liver disease that is characterized by a decrease in HVPG to less than 10 mm Hg.

[00293] As shown in Example 5 below, an in vitro assay was performed to show that oral dosing of a subject with a P2X7 receptor antagonist according to the present disclosure resulted in plasma amounts of the antagonist that were effective in blocking ATP-dependent IL-1 β release in LPS- primed whole blood of the subject. These data showed statistically significant inhibition of ATP- dependent IL-1 β at oral dose amounts ranging from about 80 mg to 1280 mg when measured about 4 hours after dosing. Specifically, the estimated IC₅₀ of Compound 1 in the IL-1 β release assay, at 4 hours post dose, was 130 ng/mL to 143 ng/mL. The estimated 95% inhibitory concentration (IC95) of Compound 1 at 4 hours post dose was 205 ng/mL to 206 ng/mL. Accordingly, it is contemplated that a dosing regimen which maintains a plasma concentration of at least about 150 ng/mL, 175 ng/mL, 200 ng/mL, 205 ng/mL or 225 ng/mL will be therapeutically effective in treating a P2X7 receptor-mediated disorder.

[00294] In some embodiments, the present methods provide steady state plasma levels of Compound 1 that correlate to one or more statistically significant therapeutic effects. In certain embodiments, the therapeutically effective a mean steady state plasma levels of Compound 1 provided by the methods of the present disclosure range from about 50 ng/mL to about 700 ng/mL, including about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, about 100 ng/mL, about 110 ng/mL, about 120 ng/mL, about 130 ng/mL, about 140 ng/mL, about 150 ng/mL, about 160 ng/mL, about 170 ng/mL, about 180 ng/mL, about 190 ng/mL, about 200 ng/ml, about 210 ng/mL, about 220 ng/mL, about 230 ng/mL, about 240 ng/mL, about 250 ng/mL, about 260 ng/mL, about 270 ng/mL, about 280 ng/mL, about 290 ng/mL, about 300 ng/mL, about 310 ng/mL, about 320 ng/mL, about 330 ng/mL, about 340 ng/mL, about 350 ng/mL, about 360 ng/mL, about 370 ng/mL, about 380 ng/mL, about 390 ng/mL, about 400 ng/mL, about 410 ng/mL, about 420 ng/mL, about 430 ng/mL, about 440 ng/mL, about 450 ng/mL, about 460 ng/mL, about 470 ng/mL, about 480 ng/mL, about 490 ng/mL, about 500 ng/mL, about 510 ng/mL, about 520 ng/mL, about 530 ng/mL, about 540 ng/mL, about 550 ng/mL, about 560 ng/mL, about 570 ng/mL, about 580 ng/mL, about 590 ng/mL, about 600 ng/mL, about 610 ng/mL, about 620 ng/mL, about 630 ng/mL, about 640 ng/mL, about 650 ng/mL, about 660 ng/mL, about 670 ng/mL, about 680 ng/mL, about 690 ng/mL, about 700 ng/mL, including all ranges there between. In certain embodiments, the therapeutically effective steady state plasma levels of Compound 1 provided by the methods of the present disclosure range from about 150 ng/mL to about 550 ng/ml, from about 150 ng/mL to about 300 ng/mL, or from about 150 ng/mL to about 250 ng/mL.

[00295] In certain embodiments, the therapeutically effective a mean steady state plasma levels of Compound 1 provided by the methods of the present disclosure is greater than about 100 ng/mL, about 125 ng/mL, about 150 ng/mL, about 175 ng/mL, about 190 ng/mL, about 200 ng/mL, about 210 ng/mL, about 225 ng/mL, about 240 ng/mL, about 250 ng/mL, about 275 ng/mL or about 300 ng/mL. In certain embodiments, the therapeutically effective a mean steady state plasma levels of Compound 1 provided by the methods of the present disclosure are greater than about 150 ng/mL, about 175 ng/mL, about 180 ng/mL, about 190 ng/mL, about 200 ng/mL, about 205 ng/mL, about 210 ng/mL, about 215 ng/mL or about 225 ng/mL.

[00296] In certain embodiments, the therapeutically effective a mean steady state plasma levels of Compound 1 provided by the methods of the present disclosure are greater than about 100 ng/mL, about 125 ng/mL, about 150 ng/mL, about 175 ng/mL, about 190 ng/mL, about 200 ng/mL, about 210 ng/mL, about 225 ng/mL, about 240 ng/mL, about 250 ng/mL, about 275 ng/mL or about 300 ng/mL over a time period of about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, or 24 hours.

[00297] In certain embodiments, the therapeutically effective mean steady state plasma levels of Compound 1 is provided by administering a daily dose of Compound 1 or a pharmaceutically acceptable salt thereof of about 100 mg. In further embodiments, the therapeutically effective steady state plasma levels of Compound 1 is provided by administering about 50 mg of Compound 1 or a pharmaceutically acceptable salt thereof twice a day.

[00298] In certain embodiments, the therapeutically effective mean steady state plasma levels of Compound 1 is provided by administering a daily dose of Compound 1 or a pharmaceutically acceptable salt thereof of about 200 mg. In further embodiments, the therapeutically effective steady state plasma levels of Compound 1 is provided by administering about 100 mg of Compound 1 or a pharmaceutically acceptable salt thereof twice a day.

[00299] In certain embodiments, the therapeutically effective steady state plasma levels of Compound 1 is provided by administering a daily dose of Compound 1 or a pharmaceutically acceptable salt thereof of about 500 mg. In further embodiments, the therapeutically effective steady state plasma levels of Compound 1 is provided by administering about 250 mg of Compound 1 or a pharmaceutically acceptable salt thereof twice a day.

[00300] In some embodiments, the present methods provide steady state plasma Cmin levels of Compound 1 that correlate to one or more statistically significant therapeutic effects. In certain embodiments, the therapeutically effective a mean steady state plasma Cmin levels of Compound 1 provided by the methods of the present disclosure range from about 50 ng/mL to about 700 ng/mL, including about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, about 100 ng/mL, about 110 ng/mL, about 120 ng/mL, about 130 ng/mL, about 140 ng/mL, about 150 ng/mL, about 160 ng/mL, about 170 ng/mL, about 180 ng/mL, about 190 ng/mL, about 200 ng/ml, about 210 ng/mL, about 220 ng/mL, about 230 ng/mL, about 240 ng/mL, about 250 ng/mL, about 260 ng/mL, about 270 ng/mL, about 280 ng/mL, about 290 ng/mL, about 300 ng/mL, about 310 ng/mL, about 320 ng/mL, about 330 ng/mL, about 340 ng/mL, about 350 ng/mL, about 360 ng/mL, about 370 ng/mL, about 380 ng/mL, about 390 ng/mL, about 400 ng/mL, about 410 ng/mL, about 420 ng/mL, about 430 ng/mL, about 440 ng/mL, about 450 ng/mL, about 460 ng/mL, about 470 ng/mL, about 480 ng/mL, about 490 ng/mL, about 500 ng/mL, about 510 ng/mL, about 520 ng/mL, about 530 ng/mL, about 540 ng/mL, about 550 ng/mL, about 560 ng/mL, about 570 ng/mL, about 580 ng/mL, about 590 ng/mL, about 600 ng/mL, about 610 ng/mL, about 620 ng/mL, about 630 ng/mL, about 640 ng/mL, about 650 ng/mL, about 660 ng/mL, about 670 ng/mL, about 680 ng/mL, about 690 ng/mL, about 700 ng/mL, including all ranges there between. In certain embodiments, the therapeutically effective steady state plasma Cmin levels of Compound 1 provided by the methods of the present disclosure range from about 150 ng/mL to about 550 ng/ml.

[00301] In certain embodiments, the therapeutically effective mean steady state plasma Cmin levels of Compound 1 is provided by administering a daily dose of Compound 1 or a pharmaceutically acceptable salt thereof of about 100 mg. In further embodiments, the therapeutically effective steady state plasma Cmin levels of Compound 1 is provided by administering about 50 mg of Compound 1 or a pharmaceutically acceptable salt thereof twice a day.

[00302] In certain embodiments, the therapeutically effective mean steady state plasma Cmin levels of Compound 1 is provided by administering a daily dose of Compound 1 or a pharmaceutically acceptable salt thereof of about 200 mg. In further embodiments, the therapeutically effective steady state plasma Cmin levels of Compound 1 is provided by administering about 100 mg of Compound 1 or a pharmaceutically acceptable salt thereof twice a day.

[00303] In certain embodiments, the therapeutically effective steady state plasma Cmin levels of Compound 1 is provided by administering a daily dose of Compound 1 or a pharmaceutically acceptable salt thereof of about 500 mg. In further embodiments, the therapeutically effective steady state plasma levels of Compound 1 is provided by administering about 250 mg of Compound 1 or a pharmaceutically acceptable salt thereof twice a day.

[00304] In some embodiments, the present methods provide mean steady state AUC_{0- 12} (expressed in terms of ng*hr/mL) levels of Compound 1 that correlate to one or more statistically significant therapeutic effects. In certain embodiments, the therapeutically effective mean steady state AUCo- 12 levels of Compound 1 provided by the methods of the present disclosure range from about 500 ng*hr/mL to about 11500 ng*hr/mL, about 500 ng*hr/mL, about 600 ng*hr/mL, about 700 ng*hr/mL, about 800 ng*hr/mL, about 900 ng*hr/mL, about 1000 ng*hr/mL, about 1100 ng*hr/mL, about 1200 ng*hr/mL, about 1300 ng*hr/mL, about 1400 ng*hr/mL, about 1500 ng*hr/mL, about 1600 ng*hr/mL, about 1700 ng*hr/mL, about 1800 ng*hr/mL, about 1900 ng*hr/mL, about 2000 ng*hr/mL, about 2100 ng*hr/mL, about 2200 ng*hr/mL, about 2300 ng*hr/mL, about 2400 ng*hr/mL, about 2500 ng*hr/mL, about 2600 ng*hr/mL, about 2700 ng*hr/mL, about 2800 ng*hr/mL, about 2900 ng*hr/mL, about 3000 ng*hr/mL, about 3100 ng*hr/mL, about 3200 ng*hr/mL, about 3300 ng*hr/mL, about 3400 ng*hr/mL, about 3500 ng*hr/mL, about 3600 ng*hr/mL, about 3700 ng*hr/mL, about 3800 ng*hr/mL, about 3900 ng*hr/mL, about 4000 ng*hr/mL, about 4100 ng*hr/mL, about 4200 ng*hr/mL, about 4300 ng*hr/mL, about 4400 ng*hr/mL, about 4500 ng*hr/mL, about 4600 ng*hr/mL, about 4700 ng*hr/mL, about 4800 ng*hr/mL, about 4900 ng*hr/mL, about 5000 ng*hr/mL, about 5100 ng*hr/mL, about 5200 ng*hr/mL, about 5300 ng*hr/mL, about 5400 ng*hr/mL, about 5500 ng*hr/mL, about 5600 ng*hr/mL, about 5700 ng*hr/mL, about 5800 ng*hr/mL, about 5900 ng*hr/mL, about 6000 ng*hr/mL, about 6100 ng*hr/mL, about 6200 ng*hr/mL, about 6300 ng*hr/mL, about 6400 ng*hr/mL, about 6500 ng*hr/mL, about 6600 ng*hr/mL, about 6700 ng*hr/mL, about 6800 ng*hr/mL, about 6900 ng*hr/mL, about 7000 ng*hr/mL, about 7100 ng*hr/mL, about 7200 ng*hr/mL, about 7300 ng*hr/mL, about 7400 ng*hr/mL, about 7500 ng*hr/mL, about 7600 ng*hr/mL, about 7700 ng*hr/mL, about 7800 ng*hr/mL, about 7900 ng*hr/mL, about 8000 ng*hr/mL, about 8100 ng*hr/mL, about 8200 ng*hr/mL, about 8300 ng*hr/mL, about 8400 ng*hr/mL, about 8500 ng*hr/mL, about 8600 ng*hr/mL, about 8700 ng*hr/mL, about 8800 ng*hr/mL, about 8900 ng*hr/mL, about 9000 ng*hr/mL, about 9100 ng*hr/mL, about 9200 ng*hr/mL, about 9300 ng*hr/mL, about 9400 ng*hr/mL, about 9500 ng*hr/mL, about 9600 ng*hr/mL, about 9700 ng*hr/mL, about 9800 ng*hr/mL, about 9900 ng*hr/mL, about 10000 ng*hr/mL, about 10100 ng*hr/mL, about 10200 ng*hr/mL, about 10300 ng*hr/mL, about 10400 ng*hr/mL, about 10500 ng*hr/mL, about 10600 ng*hr/mL, about 10700 ng*hr/mL, about 10800 ng*hr/mL, about 10900 ng*hr/mL, about 11000 ng*hr/mL, about 11100 ng*hr/mL, about 11200 ng*hr/mL, about 11300 ng*hr/mL, about 11400 ng*hr/mL, and about 11500 ng*hr/mL, including all ranges there between. In certain embodiments, the therapeutically effective mean steady state AUC_{0- 12} levels of Compound 1 provided by the methods of the present disclosure range from about 1500 ng*h/ml to about 10500 ng*h/ml.

[00305] In certain embodiments, the therapeutically effective mean steady state AUC_{0- 12} levels of Compound 1 is provided by administering a daily dose of Compound 1 or a pharmaceutically acceptable salt thereof of about 100 mg. In further embodiments, the therapeutically effective mean steady state AUC_{0- 12} levels of Compound 1 is provided by administering about 50 mg of Compound 1 or a pharmaceutically acceptable salt thereof twice a day.

[00306] In certain embodiments, the therapeutically effective mean steady state AUC_{0- 12} levels of Compound 1 is provided by administering a daily dose of Compound 1 or a pharmaceutically acceptable salt thereof of about 200 mg. In further embodiments, the therapeutically effective mean steady state AUC_{0- 12} levels of Compound 1 is provided by administering about 100 mg of Compound 1 or a pharmaceutically acceptable salt thereof twice a day. [00307] In certain embodiments, the therapeutically effective mean steady state AUC_{0- 12} levels of Compound 1 is provided by administering a daily dose of Compound 1 or a pharmaceutically acceptable salt thereof of about 500 mg. In further embodiments, the therapeutically effective mean steady state AUC_{0- 12} plasma levels of Compound 1 is provided by administering about 250 mg of Compound 1 or a pharmaceutically acceptable salt thereof twice a day.

[00308] In some embodiments, the present methods provide mean steady state AUC_0-inf (expressed in terms of ng*hr/mL) levels of Compound 1 that correlate to one or more statistically significant therapeutic effects. In certain embodiments, the therapeutically effective mean steady state AUCo- inf levels of Compound 1 provided by the methods of the present disclosure range from about 500 ng*hr/mL to about 12000 ng*hr/mL, about 500 ng*hr/mL, about 600 ng*hr/mL, about 700 ng*hr/mL, about 800 ng*hr/mL, about 900 ng*hr/mL, about 1000 ng*hr/mL, about 1100 ng*hr/mL, about 1200 ng*hr/mL, about 1300 ng*hr/mL, about 1400 ng*hr/mL, about 1500 ng*hr/mL, about 1600 ng*hr/mL, about 1700 ng*hr/mL, about 1800 ng*hr/mL, about 1900 ng*hr/mL, about 2000 ng*hr/mL, about 2100 ng*hr/mL, about 2200 ng*hr/mL, about 2300 ng*hr/mL, about 2400 ng*hr/mL, about 2500 ng*hr/mL, about 2600 ng*hr/mL, about 2700 ng*hr/mL, about 2800 ng*hr/mL, about 2900 ng*hr/mL, about 3000 ng*hr/mL, about 3100 ng*hr/mL, about 3200 ng*hr/mL, about 3300 ng*hr/mL, about 3400 ng*hr/mL, about 3500 ng*hr/mL, about 3600 ng*hr/mL, about 3700 ng*hr/mL, about 3800 ng*hr/mL, about 3900 ng*hr/mL, about 4000 ng*hr/mL, about 4100 ng*hr/mL, about 4200 ng*hr/mL, about 4300 ng*hr/mL, about 4400 ng*hr/mL, about 4500 ng*hr/mL, about 4600 ng*hr/mL, about 4700 ng*hr/mL, about 4800 ng*hr/mL, about 4900 ng*hr/mL, about 5000 ng*hr/mL, about 5100 ng*hr/mL, about 5200 ng*hr/mL, about 5300 ng*hr/mL, about 5400 ng*hr/mL, about 5500 ng*hr/mL, about 5600 ng*hr/mL, about 5700 ng*hr/mL, about 5800 ng*hr/mL, about 5900 ng*hr/mL, about 6000 ng*hr/mL, about 6100 ng*hr/mL, about 6200 ng*hr/mL, about 6300 ng*hr/mL, about 6400 ng*hr/mL, about 6500 ng*hr/mL, about 6600 ng*hr/mL, about 6700 ng*hr/mL, about 6800 ng*hr/mL, about 6900 ng*hr/mL, about 7000 ng*hr/mL, about 7100 ng*hr/mL, about 7200 ng*hr/mL, about 7300 ng*hr/mL, about 7400 ng*hr/mL, about 7500 ng*hr/mL, about 7600 ng*hr/mL, about 7700 ng*hr/mL, about 7800 ng*hr/mL, about 7900 ng*hr/mL, about 8000 ng*hr/mL, about 8100 ng*hr/mL, about 8200 ng*hr/mL, about 8300 ng*hr/mL, about 8400 ng*hr/mL, about 8500 ng*hr/mL, about 8600 ng*hr/mL, about 8700 ng*hr/mL, about 8800 ng*hr/mL, about 8900 ng*hr/mL, about 9000 ng*hr/mL, about 9100 ng*hr/mL, about 9200 ng*hr/mL, about 9300 ng*hr/mL, about 9400 ng*hr/mL, about 9500 ng*hr/mL, about 9600 ng*hr/mL, about 9700 ng*hr/mL, about 9800 ng*hr/mL, about 9900 ng*hr/mL, about 10000 ng*hr/mL, about 10100 ng*hr/mL, about 10200 ng*hr/mL, about 10300 ng*hr/mL, about 10400 ng*hr/mL, about 10500 ng*hr/mL, about 10600 ng*hr/mL, about 10700 ng*hr/mL, about 10800 ng*hr/mL, about 10900 ng*hr/mL, about 11000 ng*hr/mL, about 11100 ng*hr/mL, about 11200 ng*hr/mL, about 11300 ng*hr/mL, about 11400 ng*hr/mL, about 11500 ng*hr/mL, about 11600 ng*hr/mL, about 11700 ng*hr/mL, about 11800 ng*hr/mL, about 11900 ng*hr/mL, and about 12000 ng*hr/mL, including all ranges there between. In certain embodiments, the therapeutically effective mean steady state AUC_0-inf levels of Compound 1 provided by the methods of the present disclosure range from about 1500 ng*h/ml to about 10500 ng*h/ml.

[00309] In certain embodiments, the therapeutically effective mean steady stateAUC_0-inf levels of Compound 1 provided by the methods of the present disclosure range from about 1000 ng·h/mL to about 12000 ng·h/mL, about 4000 ng·h/mL to about 12000 ng·h/mL, about 4000 ng·h/mL to about 10000 ng·h/mL, or about 5000 ng·h/mL to about 10000 ng·h/mL.

[00310] In certain embodiments, the therapeutically effective mean steady stateAUC_0-inf levels of Compound 1 provided by the methods of the present disclosure range from about 4000 to about 9000 ng·h/mL, about 4000 to about 8000 ng·h/mL, about 4000 to about 7000 ng·h/mL, about 4000 to about 6000 ng·h/mL, about 4000 to about 5000 ng·h/mL, or about 10000 to about 15000 ng·h/mL, about 10000 to about 14000 ng·h/mL, about 10000 to about 13000 ng·h/mL, or about 12000 to about 15000 ng·h/mL.

[00311] In certain embodiments, the therapeutically effective mean steady stateAUC_0-inf levels of Compound 1 provided by the methods of the present disclosure range from about 4000 to 9000 ng·h/mL, 4000 to 8000 ng·h/mL, 4000 to 7000 ng·h/mL, 4000 to 6000 ng·h/mL, 4000 to 5000 ng·h/mL, or 10000 to 15000 ng·h/mL, 10000 to 14000 ng·h/mL, 10000 to 13000 ng·h/mL, or 12000 to 15000 ng·h/mL.

[00312] In certain embodiments, the therapeutically effective mean steady stateAUC_0-inf levels of Compound 1 is provided by administering a daily dose of Compound 1 or a pharmaceutically acceptable salt thereof of about 100 mg. In further embodiments, the therapeutically effective mean steady state AUC_0-inf levels of Compound 1 is provided by administering about 50 mg of Compound 1 or a pharmaceutically acceptable salt thereof twice a day.

[00313] In certain embodiments, the therapeutically effective mean steady state AUC_0-inf levels of Compound 1 is provided by administering a daily dose of Compound 1 or a pharmaceutically acceptable salt thereof of about 200 mg. In further embodiments, the therapeutically effective mean steady state AUC_0-inf levels of Compound 1 is provided by administering about 100 mg of Compound 1 or a pharmaceutically acceptable salt thereof twice a day.

[00314] In certain embodiments, the therapeutically effective mean steady state AUC_0-inf levels of Compound 1 is provided by administering a daily dose of Compound 1 or a pharmaceutically acceptable salt thereof of about 500 mg. In further embodiments, the therapeutically effective mean steady state AUC_0-inf plasma levels of Compound 1 is provided by administering about 250 mg of Compound 1 or a pharmaceutically acceptable salt thereof twice a day.

EXAMPLES

[00315] The following examples are offered to illustrate but not to limit the compounds and methods described herein. Various modifications may be made by the skilled person without departing from the true spirit and scope of the subject matter described herein.

Example 1: Inhibition of IL-Ib Release in Whole Blood Samples

[00316] Studies were performed to show that Compound 1 inhibits the secretion of IL-1 β from cells in whole blood and to determine the IC₅₀ of Compound 1. P2X7 receptor has been shown to mediate the stimulatory action of ATP as a stimulator of caspase-l activation and IL-1 β secretion (Solle et al, 2001, J Biol Chem, 276: 125-132). For this experiment, whole blood from untreated human subjects was collected in vacutainer tubes spray-coated with sodium heparin. The whole blood was diluted 1 : 1 with RPMI 1640 media containing 25 mM HEPES and 150 uL added to wells in a 96 well plate. LPS was added to each well of whole blood to a final concentration of 200 ng/mL and the blood was incubated for 1.5 hr at 37°C/5% CO₂. Compound 1 in RPMI 1640 media containing 25 mM HEPES was added to wells to give a final concentrations ranging from 1 nM to 10 mM and the blood incubated for an additional 30 min 37°C/5% CO₂. ATP was then added to each well the blood to give a final concentration of 2.5 mM and the plates incubated for an additional 45 minutes at 37°C/5% CO₂. Each condition was run in quadruplicate.

[00317] Plates containing the samples were centrifuged at lOOOxg for 2 min and plasma collected and analyzed for IL-1 β content using a human IL-1 β ELISA.

[00318] OD450 values for each well were converted to pg/mL values of IL-1 β from standard curves run on each ELISA plate. Values are expressed in pg/mL. Compound 1 IC50 and ATP EC₅₀ were determined with a four parameter logistic curve fitting algorithm using GraphPad Prism (GraphPad Software, Inc., San Diego, CA). Y=Bottom + (Top - Bottom)/(1+10^((LogEC5o-X)*HillSlope)). Maximum inhibition was determined by comparing background corrected values of the highest concentration of drug used to background corrected values for no drug added.

[00319] The mean IC50 calculated using blood from 6 donors was 54.5 nM±16.8 nM.

Example 2: Inhibition of Calcium Influx in 1321N1 Cells Expression P2X7 Receptor

[00320] An experiment was performed to measure the ability of Compound 1 to inhibit calcium influx in P2X7 receptor-expressing cells upon activation by ATP of BzATP. To perform this experiment, the 1321N1 human astrocytoma cell line was used because it does not give an endogenous calcium response to P2X7 receptor agonists and thereby serves as a useful background cell line for expressing P2X7 receptor to assess function.

[00321] The 1321N1 cells were transfected to establish stable cell lines. Cells were transfected with expression vectors harboring sequences encoding human P2X7, human P2X1, human P2X2, human P2X3, or human P2X4

[00322] Twenty-four hours prior to the assay, the cells stably expressing human P2X7 receptor were plated at a density of 50,000 cells/well and incubated at 37°C/5% CO₂. The media was then removed and replace with 100 μL dye loading mix (10 mL Hanks Balanced Salt Solution supplemented with 20 mM HEPES and containing 100 μL of 250 mM probenecid, 500 μL Signal Enhancer and 5 μL dye from the BD calcium kit) per well. The cells were incubated for 60 min at 37°C/5% CO₂ and an additional 10 min at room temperature. [00323] The cells were pretreated with Compound 1 to give a final concentration ranging from about 300 pM to 3 mM Compound 1) for 30 min at room temperature. The P2X7 BzATP then added and the fluorescence was read from 1 to 90 sec with the FLIPR^tetra (Molecular Devices, Sunnyvale, CA). The final concentration of BzATP used was at the EC₈₀ for each cell line/receptor.

[00324] The max-min relative fluorescent units were determined for each well over the first 90 seconds post agonist addition. To analyze the data, the background (derived from wells where no agonist was added) was subtracted from each well and values normalized to the maximum possible effect (derived from wells where agonist, but no REN-2708 was added). Values are expressed as the effect observed divided by the maximum possible effect (E/Emax). IC₅₀ values were determined with a four parameter logistic curve fitting algorithm using GraphPad Prism (GraphPad Software, Inc., San Diego, CA). Y=Bottom + (Top - Bottom)/(1+10^((LogEC5o-X)*HillSlope)). For each IC₅₀ calculation, the“Top” parameter was constrained to 1.00. For the BzATP response curves, the“Bottom” parameter was constrained to 0.0.

[00325] Compound 1 inhibited human P2X7 receptor-mediated calcium influx in 1321N1 cells stably transfected with human P2X7 receptor. The IC₅₀ for the human P2X7 was 10.0 ± 2.0 nM (N=3).

[00326] The same experiment was performed using each of the 1321N1 cells stably transformed with human P2X1, P2X2, P2X3 and P2X4, except that the P2X2 buffer was supplemented with 5 mM calcium rather than the 1.3 mM present in Hanks balanced Salt Solution. Compound 1 and agonist additions were also the same as for the P2X7 experiment except that ATP was used as the agonist at the EC80 concentration for each cell line/receptor.

[00327] Compound 1 showed no activity against human P2X1, P2X2, P2X3 or P2X4 receptors at concentrations up to 10 mM (N=l for each cell line).

Example 3: Inhibition of ATP Induced IL-Ib Release from LPS Stimulated THP-1 Cells

[00328] THP-1 cells, a human acute monocytic leukemia cell line, have been shown to be responsive to P2X7 receptor- mediated IL-1 β release and are thus useful as a tool to study P2X7 receptor antagonists (Grahames et al, 1999, Br J Pharmacol, 127: 1915-1921). Accordingly, experiments were performed using these cells to study the inhibitory activity of Compound 1 with respect to IE-1b release in the presence of the P2X7 receptor agonists BzATP, ATP and LL-37.

[00329] Prior to the assay, THP-l cells were plated at a density of 200,000 cells/well and grown for 16 hr at 37°C/5% CO₂ in RPMI media containing 10% FBS, 100 ng/mL LPS and 100 ng/mL IFN-g.

[00330] The media was then replaced with RPMI 1640 media containing 100 ng/mL fresh LPS for an additional 2 hr at 37°C/5% CO₂.

[00331] After the 2 hr incubation with fresh LPS, the cells were pre-incubated with Compound 1 (amounts ranging from 30 pM to 300 nM) for 30 min. followed by addition of ATP, BzATP or LL-37 for 45 minutes and cell culture supernatant analyzed for IL-1 β content using a human IL- 1b ELISA BzATP as the agonist, the final concentration was either 275 mM (EC₅₀), or 1.5 mM (>EC99), for ATP as the agonist, the final concentration was 10 mM (>EC99), and for LL-37 as the agonist, the final concentration was 22 mM (EC₅₀).

[00332] To analyze the data, OD450 values for each well were converted to pg/mL values of IL-1 β from standard curves run on each ELISA plate. The background (derived from wells where no agonist was added) was subtracted from each well and values normalized to the maximum possible effect (derived from wells where agonist, but no Compound 1 was added). Values are expressed as the effect observed divided by the maximum possible effect (E/Emax). IC₅₀ were determined with a four parameter logistic curve fitting algorithm using GraphPad Prism (GraphPad Software, Inc., San Diego, CA). Y=Bottom + (Top - Bottom)/(1+10^((LogEC₅₀-X)*HillSlope)). For each calculation, the“Top” parameter was constrained to 1.00.

[00333] Compound 1 inhibited IL-1 β release from THP-l cells treated with each of the three P2X7 agonists tested. At EC₅₀ of BzATP (275 mM), IC₅₀ = 0.62±0.09 nM (N=l5). At 1.5 mM BzATP, IC₅₀ = 10 mM (N=1). At 10 mM ATP, IC₅₀ = 19.08 ± 8.01 (N=2). At 22 mM LL-37, IC₅₀ = 1.07 nM (N=l). Compound 1 did not completely block IE-1b release (89% and 78% maximum inhibition vs. 10 mM ATP and 1.5 mM BzATP, respectively), but each Compound 1 did have inhibitory activity.

Example 4: Clinical Study for Safety and Pharmacokinetics ( PK) [00334] A double-blind, placebo-controlled, ascending single oral dose, sequential group study, incorporating a 2-period crossover bioavailability study to compare capsule and suspension formulations of Compound 1 was completed in healthy male subjects.

[00335] Ninety-six fasted subjects aged 18 to 55 years received a single dose of 1 mg, 3 mg, 10 mg, 20 mg, 40 mg, 80 mg, 160 mg, 320 mg, 640 mg, and 1280 mg Compound 1 or placebo as an oral suspension (8 subjects per dose group). An additional dose group received a single dose of 160 mg Compound 1 as a capsule, and a further dose group received 40 mg Compound 1 both as a suspension and as a capsule. Compound 1 was well tolerated at all dose levels and formulations. There were no severe or serious adverse events reported during the study, and no subjects withdrew due to adverse events.

[00336] Pharmacokinetic data indicated that absorption was rapid with individual tmax estimates ranging from 0.5 hours to 1.5 hours post dose. The disposition kinetics of Compound 1 occurred in a generally bi-phasic manner and the mean t½ was similar for the 10 mg to 1280 mg dose levels (14 hours to 23 hours). Exposure increased in a dose proportional manner across the 10 mg to 160 mg dose levels based on area under the plasma concentration time curve from time zero to infinity (AUC_0-inf) and Cmax, and appeared to plateau beyond 160 mg to 1280 mg, with mean Cmax values of 7l2 ng/mL to 1191 ng/mL and mean AUCo-inf of 5312 ng h/mL to 8965 ng h/mL.

[00337] The disposition kinetics of Compound 1 were generally similar between capsule and suspension formulations at 40 mg and 160 mg dose levels. However, systemic exposure to Compound 1 was reduced following administration of the hand filled capsule compared with suspension, with the difference in exposure being most prominent at the 160 mg dose level.

Example 5: Pharmacodynamic Endpoint Analysis of IL-Ib Release

[00338] An in vitro assay was used to assess the efficacy of Compound 1 at blocking ATP dependent IL-1 β release in LPS primed whole blood following oral dosing as described in Example 4 above. Blood samples were taken pre-dose and at 4 hours, 24 hours, and 7 days after a single oral dose of Compound 1 suspension. Whole human blood primed with LPS for 1.5 hours was pre incubated with Compound 1 for 30 min prior to addition of ATP (final concentration 2.5 mM). After a 45 -min incubation, the supernatant was analyzed for IL-1 β content using a commercially available human IL-1 β ELISA.

[00339] At 4 hours post-dose, a statistically significant inhibition of ATP dependent IL-1 β release from whole blood occurred at dose levels of Compound 1 ranging from 80 mg to 1280 mg. At dose levels of 160 mg to 1280 mg, maximal inhibition ranged from 81% to 91% compared with 7% to 9% for placebo.

[00340] At 24 hours post-dose, dose-dependent trends in inhibition of ATP dependent IL-1 β release from whole blood were evident from 80 mg to 1280 mg, although only the 320 mg to 1280 mg doses showed a statistical difference compared with placebo. Mean inhibition values ranged from 35% to 66% at dose levels of 320 mg to 1280 mg compared with 0% to 1% for placebo. On Day 7, there was no statistically significant difference between treatments and placebo, nor was there any apparent trend for inhibition to increase with increasing dose.

[00341] The estimated IC₅₀ of Compound 1 in the IE-1b release assay, at 4 hours post dose, was 130 ng/mL to 143 ng/mL. The estimated 95% inhibitory concentration (IC95) of Compound 1 at 4 hours post dose was 205 ng/mL to 206 ng/mL.

Example 6: Clinical Study for Safety and Pharmacokinetics ( PK)

[00342] A safety study was performed to determine the safety and tolerability of single and multiple oral (immediate release tablet) doses of Compound 1 in healthy subjects as well as to determine the pharmacokinetic (PK) profiles of Compound 1. Additionally, the effect of PK of dosing subjects who were fasted vs. fed a high-fat meal was determined. The study comprised two parts: Part 1 was a single ascending dose (SAD), placebo-controlled, sequential-group study incorporating the food effect evaluation. Part 2 was a multiple ascending (MAD), placebo- controlled sequential-group study.

[00343] Subjects were orally administered immediate release tablets containing 50 mg or 250 mg Compound 1. The tablets were formulated with micronized Compound 1 due to the poor aqueous solubility of Compound 1. The tablets comprised about 33 wt% Compound 1 with a diluent, binder, disintegrant, surfactant and lubricant. Dissolution studies were performed using a USP II apparatus, 75 RPM at 37°C in 500 ml (for 50 mg tablet) or 900 ml (for 250 mg tablet) aqueous 0.5% sodium lauryl sulfate. Results are presented in FIG. 1A (50 mg Compound 1) and FIG. 1B (250 mg Compound 1). As seen in FIGS. 1A and 1B, the dissolution profile appears unaffected by the presence of the coating and both formulations (50 mg Compound 1 and 260 mg Compound 1), released more than 80% of the active agent in less than 20 minutes, and substantially of the active agent within about 30 minutes.

[00344] The subjects in this study were divided into 4 groups and administered: A) a single dose of 150 mg on Day 1 in a fasted state; B) a single dose of 500 mg in a fasted state; C) a single dose of 1500 mg in a fasted state; and D) a single dose of 3000 mg in a fasted state. At least 14 days after completion of this SAD study, subjects in Group B were administered a single dose of 500 mg in a fed state— about 30 minutes after a high fat breakfast. Plasma and urine concentrations of Compound 1 and its glucuronide metabolite (Compound 1-M3) were measured at various time points over a period of about 5 days (120 hr) after dosing. Results for Groups A, B and C are presented in Table 1 below and FIG. 2.

[00345] Notably, the Cmax values for the 500 mg and 1500 mg doses administered in the fasted mode differed only slightly, suggesting saturation. In contrast, the 500 mg dose administered with food resulted in a Cmax that was about 3 times greater than the Cmax for the 500 mg dose administered in the fasted state. Administration with food also significantly increased both the AUC and Tmax values. No severe adverse events were observed in any of the subjects during the course of the study.

Table 1

*0-24 hr PK data only

[00346] In part 2 of the phase 1 study, the MAD study, 36 subjects are divided into 3 groups (Groups E, F and G), with each group consisting of 12 subjects (9 active; 3 placebo). Each subject in the groups receives a dose (or placebo) every 12 hours (b.i.d.) for 13 days and once on Day 14. The dose for Group E: 150 mg, Group F: 500 mg, Group G: 1500 mg (fed or fasted mode). Plasma and urine concentrations of Compound 1 and its glucuronide metabolite (Compound 1-M3) are measured at various time points over a period of about 5 days (120 hr) after dosing.

[00347] Completion of the dosing of part 2 of the phase 1 study (150 mg and 500 mg b.i.d. with standard meals) showed that both dosing regimens provided the projected therapeutic plasma exposure after 14 days of dosing (see FIG. 3 and Tables 2A, 2B, and 2C below with geometric mean (%CV) in parentheses).

Table 2A: 150 mg b.i.d. (n=9)

Table 2B: 500 mg b.i.d. (n=8)

Table 2C: 1500 mg b.i.d. (n=9)

[00348] Again, the study showed that Compound 1 administration is safe and well tolerated at least up to 500 mg b.i.d. with standard meals. The data also showed that steady state is generally achieved within 7 days for all dosing regimens.

[00349] In Part 1 of this phase 1 study, this phase 1 study, an ex vivo assay was used to assess the efficacy of Compound 1 at blocking ATP-dependent IL-1 β release in LPS-primed whole blood following oral dosing. Blood samples were taken pre-dose and at 1, 6, 24 hrs and at study follow up. Blood from each participant was tested for ATP-induced IL-1 β release by running an 8-point response curve to ATP at each time point in a 96- well format. IL-1 β concentrations were determined by Quantikine® ELISA (R&D Systems, Minneapolis, MN).

[00350] In fasted subjects at 1 hours post-dose, the mean IAUC_IL-lβ at doses of 150 to 3000 mg was 92.7 to 98.1% versus 11.8% placebo. Mean IEmax (% inhibition of Emax relative to baseline) was generally similar at 88.7-96.4% compared with 14.2% for placebo. All doses were statistically different from placebo for both IAUC_IL-lβ and IEmax Mean inhibition in fed vs. fasted subjects receiving 500 mg Compound 1 for IAUC_IL-lβ was 83.5% (fed) and 97.8% (fasted). For IEmax inhibition was 82.7% (fed) vs. 96.9% (fasted). The difference between fasted and fed was not significant for either IAUC_IL-lβ or IEmax.

[00351] In fasted subjects at 6 hours post-dose, the mean IAUC_IL-lβ at doses of 150 to 3000 mg was 71.1% to 96.5% versus -3.73% for placebo. Mean IEmax was generally similar at 56.6% to 93.8% compared with -4.9%% for placebo. All doses were statistically different from placebo for both IAUC_IL-lβ and IEmax. Mean inhibition in fed vs. fasted subjects receiving 500 mg Compound 1 for IAUC_IL-lβ was 95.5% (fed) and 96.2% (fasted). For IE_max inhibition was 93.2% (fed) vs. 94.3% (fasted). The difference between fasted and fed was not significant for either IAUC_IL-lβ or IEmax .

[00352] In fasted subjects at 24 hours post-dose the mean IAUC_IL-lβ across doses of 150 to 3000 mg was 56.5% to 91.0% versus 17.6% for placebo. Mean IEmax was generally similar at 48.5% to 85.4% compared with 17.6% for placebo. All doses were statistically different from placebo for IAUC_IL-lβ, but only the 500 and 3000 mg dose group for IEmax. Mean inhibition in fed vs. fasted subjects receiving 500 mg Compound 1 for IAUC_IL-lβ was 94.5% (fed) and 88.7% (fasted). For IEmax inhibition was 92.0% (fed) vs. 84.6% (fasted). The difference between fasted and fed was not significant for either IAUC_IL-lβ or IEmax.

[00353] At post-study follow-up, IAUC_IL-lβ and IEmax decreased from the 2-, 6- and 24-hour values for all dose groups. The mean IAUC_IL-lβ for doses of 150, 1500 and 3000 mg were 9.31%, 33.6% -15.5% compared with 20.8% for placebo. Mean IEm_ax was generally similar with inhibition values of 12.8%, 27.4% and -16.9% for doses of 150, 1500 and 3000 mg compared with 16.9% for placebo. No significant differences from placebo for either IAUC_IL-lβ or IEmax. were observed. In the 500-mg Compound 1 (fed/fasted) group, arithmetic means for IAUC_IL-lβ and IEmax at the post-study visit were 24.3% and 18.6% respectively.

[00354] The same whole blood ex vivo assays were also performed for samples obtained in Part 2 of this phase 1 study, blood samples were taken on Day 1 pre-dose and 2, 6 hours post- AM dose, Day 7 pre-AM dose, Day 14 pre-AM dose and at post-study follow-up.

[00355] On Day 1, 2 hours post- AM dose the mean IAUC_IL-lβ at doses of 150 to 1500 mg was 71.4 to 94.6% versus -31.9% for placebo (Figure 15). Mean IEmax was generally similar at 63.3% to 93.7% compared with -28.5% for placebo. All doses were statistically different from placebo for both IAUC_IL-lβ and IE max.

[00356] On day 1, 6 hours post- AM dose the IAUC_IL-lβ at doses of 150 to 1500 mg was 75.1 to 94.6% versus -47.9% for placebo (Figure 15). Mean IEmax was generally similar at 64.0% to 94.0% compared with -42.7% for placebo. All doses were statistically different from placebo for both IAUC_IL-lβ and IE max.

[00357] On Day 7 pre-AM dose the IAUC_IL-lβ at doses of 150 to 1500 mg was 89.0% to 94.9% versus -15.0% for placebo (Figure 15). Mean IEmax was generally similar at 88.7 to 92.6% compared with -8.71% for placebo. All doses were statistically different from placebo for both IAUC_IL-lβand IE max.

[00358] On Day 14 pre-AM dose the IAUC_IL-lβ at doses of 150 to 1500 mg was 82.2% to 93% versus -11.9% for placebo (Figure 15). Mean IEmax was generally similar at 88.2% to 91.0% compared with -3.58% for placebo. All doses were statistically different from placebo for both IAUC_IL-lβand IE max. [00359] At post-study follow-up IAUC_IL-lβ and IEmax decreased from the Day 14 pre-AM values for all dose groups. IAUC_IL-lβ at doses of 150 to 1500 mg was 20.0% to -89.7% versus -76.6% for placebo. Mean IEmax was generally similar at 10% to -86.6% compared with -66.7% for placebo.

[00360] In summary, at steady state, all multiple doses of Compound 1 evaluated gave maximal inhibition of 88.2% to 94.9% for IAUC_IL-lβ and 88.2% to 92.6% for IEmax at all time points assessed, including trough. This indicates that maximal inhibition was achieved for multiple doses ≥150 mg throughout the dosing cycle.

Example 7: Phase 2 Study

[00361] Patients with fibrosis stage 1-3 (F1-F3) NASH will be treated with oral tablets of Compound 1 to study the safety, tolerability, pharmacokinetics, and efficacy of Compound 1 oral tablets in the treatment of NASH. Two doses of Compound 1 will be studied: 250 mg B.I.D. and 50 mg B.I.D.

[00362] Study Design

[00363] The study will be a multicenter, randomized, double-blind, placebo-controlled study evaluating the safety, PK, and PD of 12 weeks’ administration of Compound 1 in subjects with fibrosis stage 1-3 (F1-F3) NASH.

[00364] About one hundred (100) subjects will be randomized 2: 1 : 1 into one of three treatment arms: 250 mg Compound 1 B.I.D, 50 mg Compound 1 B.I.D., and matching placebo BID.

[00365] If necessary, the top dose may be lowered from 250 mg to 100 mg and additional subjects enrolled. Additionally, after at least 33% of the subjects have been enrolled and been in the study for at least 4 weeks, an interim analysis may be performed to determine if the sample size is adequate. The total sample size may then be increased if it is determined that the study is not large enough to show a statistically significant difference in either of the two secondary efficacy endpoints (i.e., ALT, cTl).

[00366] Screening Period: [00367] The Screening Period will be up to 42 days in duration (Day -42 to Day -1). Prior to any clinical trial procedures, participants will provide written informed consent to participate in the clinical trial. Screening assessments will include: Measure height. Measure body weight and calculate BMI. Ascertain medical history. Assess inclusion/exclusion criteria. Conduct complete physical examination. Obtain 12-lead ECG (after at least 5 minutes of resting in supine or semi- recumbent position). Measure vital signs: temperature, blood pressure, respiratory rate, pulse (after at least 5 minutes of resting in seated, supine or semi-recumbent position). Record prior and concomitant medications. Record AEs. Obtain clinical laboratory samples for the following: Drug and alcohol screen, Hepatitis and HIV screen (HAV IgM, HBsAg, HBsAb, HBcAb (total), HCVAb (including HCV viral load if HCVAb positive), HIVAb), Serum pregnancy test, women of childbearing potential only, FSH, postmenopausal women only, HbAlc, Hematology, Prothrombin Time/INR, Chemistry, Urinalysis, Glucose (fasting), Insulin (fasting)/HOMA-IR, Lipids (fasting), Fibrinogen, hs-CRP, Cytokines, Cytokeratin 18, ELF score, Pro-C3, sCDl4 and sCDl63, LBP, Other relevant biomarkers. Obtain Fibroscan, after all the above assessments have met inclusion/exclusion criteria. Obtain MRI (PDFF, cTl, 2D MRE), after all the above assessments (including Fibroscan) have met inclusion/exclusion criteria. Obtain a second set of clinical laboratory samples for ALT, AST, alkaline phosphatase and total/direct/indirect bilirubin no less than 28 days after the first chemistry (ALT) assessment.

[00368] Treatment Period:

[00369] The treatment period will be 12 weeks in duration. Subjects will be instructed to dose BID, once in the morning and once in the evening. Study drug will be administered within 30 minutes after a meal (i.e., breakfast and dinner).

[00370] Subjects will take one tablet BID for the Compound 1 50 mg or 250 mg or matching placebo doses. If the 100 mg dose is evaluated, subjects will take two tablets of 50 mg or matching placebo BID within 30 minutes after a meal.

[00371] Day 1 Procedures:

[00372] Subjects will report to this visit 8-hours fasted. The following procedures will be performed at the Day 1 Visit: Measure body weight and calculate BMI. Ascertain interim medical history. Conduct physical examination (symptom- directed). Obtain l2-lead ECG (after at least 5 minutes of resting in supine or semi-recumbent position). Measure vital signs: temperature, blood pressure, respiratory rate, and pulse (after at least 5 minutes of resting in seated, supine or semi- recumbent position). Record prior and concomitant medications. Record AEs. Conduct urine pregnancy test, women of child bearing potential only. Reassess inclusion/exclusion criteria. Randomization. Study drug dispensing. Obtain predose PK sample. Obtain predose clinical laboratory samples for the following: Hematology, Prothrombin Time/INR, Chemistry, Urinalysis, Glucose (fasting), Insulin (fasting)/HOMA-IR, Lipids (fasting), Fibrinogen, hs-CRP, Cytokines, Cytokeratin 18, ELF score, Pro-C3, sCDl4 and sCDl63, LBP, Other relevant biomarkers and P2X₇ polymorphism assessment. Intensive PK Sampling Cohort Only: Provide breakfast to subject. Within 30 minutes after meal, take first dose in clinic. Obtain postdose PK samples at 0.5, 1, and 2 hours (±5 minutes) and at 4, 6, and 8 hours (±15 minutes).

[00373] Week 2 Procedures (±3 days):

[00374] Subjects will report to this visit 8-hours fasted and undosed. The following procedures will be performed at the Week 2 Visit: Conduct physical examination (symptom-directed). Obtain 12-lead ECG (after at least 5 minutes of resting in supine or semi-recumbent position). Measure vital signs: temperature, blood pressure, respiratory rate, and pulse (after at least 5 minutes of resting in seated, supine or semi-recumbent position). Record prior and concomitant medications. Record AEs. Obtain predose/trough PK sample. Obtain predose clinical laboratory samples for the following: Chine pregnancy test, women of child bearing potential only; Hematology, Prothrombin Time/INR, Chemistry, Urinalysis, Glucose (fasting), Insulin (fasting)/HOMA-IR, Lipids (fasting), Fibrinogen, hs-CRP, Cytokines, Cytokeratin 18, ELF score, Pro-C3, sCDl4 and sCDl63, LBP and Other relevant biomarkers. Collect old study drug and conduct reconciliation. Dispense new study drug. Intensive PK Sampling Cohort Only: Provide breakfast to subject. Within 30 minutes after meal, take dose in clinic. Obtain postdose PK samples at 0.5, 1, and 2 hours (±5 minutes) and at 4, 6, and 8 hours (±15 minutes).

[00375] Week 4 Procedures (±3 days):

[00376] Subjects will report to this visit 8-hours fasted and undosed. The following procedures will be performed at the Week 4 Visit: Measure body weight and calculate BMC Conduct physical examination (symptom-directed). Obtain 12-lead ECG (after at least 5 minutes of resting in supine or semi-recumbent position). Measure vital signs: temperature, blood pressure, respiratory rate, and pulse (after at least 5 minutes of resting in seated, supine or semi-recumbent position). Record prior and concomitant medications. Record AEs. Obtain predose/trough PK sample. Obtain predose clinical laboratory samples for the following: Urine pregnancy test, women of child bearing potential only; Hematology, Prothrombin Time/INR, Chemistry, Urinalysis, Glucose (fasting), Insulin (fasting)/HOMA-IR, Lipids (fasting), Fibrinogen, hs-CRP, Cytokines, Cytokeratin 18, ELF score, Pro-C3, sCDl4 and sCDl63, LBP and Other relevant biomarkers. Collect old study drug and conduct reconciliation. Dispense new study drug.

[00377] Week 8 Procedures (±3 days):

[00378] Subjects will report to this visit 8-hours fasted and undosed. The following procedures will be performed at the Week 8 Visit: Measure body weight and calculate BMI. Conduct physical examination (symptom-directed). Obtain 12-lead ECG (after at least 5 minutes of resting in supine or semi-recumbent position). Measure vital signs: temperature, blood pressure, respiratory rate, and pulse (after at least 5 minutes of resting in seated, supine or semi-recumbent position). Record prior and concomitant medications. Record AEs. Obtain predose/trough PK sample. Obtain predose clinical laboratory samples for the following: Urine pregnancy test, women of child bearing potential only; Hematology, Prothrombin Time/INR, Chemistry, Urinalysis, Glucose (fasting), Insulin (fasting)/HOMA-IR, Lipids (fasting), Fibrinogen, hs-CRP, Cytokines, Cytokeratin 18, ELF score, Pro-C3, sCDl4 and sCDl63, LBP and Other relevant biomarkers. Collect old study drug and conduct reconciliation. Dispense new study drug.

[00379] Week 12 Procedures (±3 days):

[00380] Subjects will report to this visit 8-hours fasted and undosed. The following procedures will be performed at the Week 12 EOT/ED Visit: Measure body weight and calculate BMI. Conduct physical examination (symptom-directed). Obtain 12-lead ECG (after at least 5 minutes of resting in supine or semi-recumbent position). Measure vital signs: temperature, blood pressure, respiratory rate, and pulse (after at least 5 minutes of resting in seated, supine or semi-recumbent position). Record prior and concomitant medications. Record AEs. Obtain predose/trough PK sample. Obtain predose clinical laboratory samples for the following: Urine pregnancy test, women of child bearing potential only; Hematology, Prothrombin Time/INR, Chemistry, Urinalysis, Glucose (fasting), Insulin (fasting)/HOMA-IR, Lipids (fasting), Fibrinogen, hs-CRP, Cytokines, Cytokeratin 18, ELF score, Pro-C3, sCDl4 and sCDl63, LBP and Other relevant biomarkers. Obtain MRI (PDFF, cTl). Collect old study drug and conduct reconciliation.

[00381] Safety Follow-up Period:

[00382] This visit will be performed 4 weeks after the last dose of study medication. Subjects will report to this visit 8-hours fasted. The following procedures will be performed at the Week 16 Visit: Measure body weight and calculate BMI. Conduct physical examination (symptom- directed). Obtain 12-lead ECG (after at least 5 minutes of resting in supine or semi-recumbent position). Measure vital signs: temperature, blood pressure, respiratory rate, and pulse (after at least 5 minutes of resting in seated, supine or semi-recumbent position). Record prior and concomitant medications. Record AEs. Obtain predose/trough PK sample. Obtain predose clinical laboratory samples for the following: Urine pregnancy test, women of child bearing potential only; Hematology, Prothrombin Time/INR, Chemistry, Urinalysis, Glucose (fasting), Insulin (fasting)/HOMA-IR, Lipids (fasting), Fibrinogen, hs-CRP, Cytokines, Cytokeratin 18, ELF score, Pro-C3, sCDl4 and sCDl63, LBP and Other relevant biomarkers.

[00383] Patient population

[00384] Number of Participants: The clinical trial is planned to include one hundred (100) subjects are planned to be enrolled. In the event the 250 mg dose is lowered to 100 mg, or a sample size adjustment is made after an optional IA, the maximum number of subjects to be enrolled will not exceed 160. Subjects will be randomized 2: 1 : 1 into one of three treatment arms: 250 mg Compound 1 B.I.D, 50 mg Compound 1 B.I.D., and matching placebo BID.

[00385] Inclusion Criteria: A participant must meet the following criteria at Screening to be eligible to participate in this clinical trial:

• Must have given written informed consent (signed and dated) and any authorizations required by local law

Age 18 to 75 years (inclusive) • ALT≥45 IU/L and≤225 IU/L.

• F1-F3 NASH as defined by a two-stage workup: o Stage 1 - Subjects must have a Fibroscan CAP≥300 dB/m and kPa>8.5. o Stage 2 - Subjects meeting Stage 1 criteria (and all other inclusion and exclusion criteria) must then undergo MRI and demonstrate all of the following:

■ MRI-protein density fat fraction (PDFF) with≥8% steatosis

■ MRE with liver stiffness 2.5-4.65 kPa

■ cTl score≥827.5 ms

• Body mass index (BMI)≥25 kg/m²

• Female subjects are eligible for the study if they meet the following criteria:

• Are not pregnant or nursing o Not heterosexually active or of non-childbearing potential defined as women who have had a hysterectomy, bilateral oophorectomy, other sterilization procedure, medically documented ovarian failure, or are documented postmenopausal (follicle stimulating hormone (FSH) within the postmenopausal range by central laboratory assessment)

OR o If heterosexually active and of child bearing potential, defined as women <55 years of age with <2 years of amenorrhea, must meet both the following criteria:

• Both a negative serum pregnancy test at Screening and urine pregnancy test at the Day 1 visit prior to randomization • Agrees to correct and consistent use of one of the following methods of birth control in addition to a male partner using a condom (unless surgically sterile) from Screening to 90 days after the last dose of study medication:

• hormone-containing contraceptive

• intrauterine device

• cervical cap or diaphragm with spermicidal agent

• Heterosexually active male subjects of child bearing potential (i.e., not known to be

surgically sterile) must agree to consistently and correctly use a condom in combination with one of the methods of birth control from Screening to 90 days after the last dose of study medication.

• Subjects must be able to comply with the dosing instructions for study drug administration and able to complete the study schedule of assessments.

[00386] Exclusion Criteria: A participant who meets any of the following criteria at Screening (unless otherwise specified) will be excluded from this clinical trial:

• Use of any of the following medications within 4 weeks of randomization: a) Amiodarone b) Methotrexate c) Oral glucocorticoids at doses greater than the equivalent of prednisone 5 mg/day d) Tamoxifen e) Estrogens at doses greater than those used for hormone replacement or contraception f) Anabolic steroids g) Valproic acid h) Tetracyclines i) Known hepatotoxins j) Medications administered to treat NASH k) Medications, including over the counter and herbal remedies, which are moderate or strong inhibitors/inducers of P-gp or CYP3A4 or otherwise prohibited. 2) High-dose vitamin E (>400 IU/day) or milk thistle (silymarin) use within the 6 and 3 months, respectively, prior to randomization. 3) Subjects taking doses of sodium/glucose cotransporter (SGLT) 2 inhibitors or GLP-l

agonists or pioglitazone which are not stable over the 3 -month period prior to randomization. 4) In the 6 months prior to screening, significant alcohol consumption, defined as more than

2 drink units per day (equivalent to 20 g) in women and 3 drink units/day (equivalent to 30 g) in men, or inability to reliably quantify alcohol intake 5) Planned (during the 22-week study conduct period) or prior bariatric surgery 6) Poorly controlled type 2 diabetes (hemoglobin Ale [HbAlc]≥9.5%) or type 1 diabetes 7) Significant weight loss (i.e., >10%) within the 6 months prior to randomization 8) Histologic evidence of cirrhosis on prior biopsy or suspected cirrhosis based on clinical suspicion 9) Hepatic decompensation defined as the presence of any of the following: esophageal varices, ascites, hepatorenal syndrome, or hepatic encephalopathy 10) Other chronic liver diseases including, but not limited to: a) Active hepatitis B infection as defined by presence of hepatitis B surface antigen

(HBsAg), or presence of HBV DNA b) Hepatitis C as defined by presence of HCV antibody and positive HCV RNA.

Documented cured HCV infection is acceptable if >2 years from time of randomization c) History or evidence of autoimmune hepatitis, primary biliary cirrhosis, primary

sclerosing cholangitis, Wilson’s disease, alpha- 1 -antitrypsin (A1AT) deficiency, hemochromatosis, or drug-induced liver disease (as defined on the basis of typical exposure and history) d) Known bile duct obstruction e) Suspected or proven liver cancer (even if successfully treated) f) History of liver transplantation 11) Any of the following laboratory abnormalities a) Creatine kinase above 3 xULN b) Serum creatinine >1.5 xULN c) Platelet count <140, 000/mm³ d) Serum albumin <3.5 g/dL e) International normalized ratio (INR) >1.3, in the absence of warfarin or other

anticoagulant therapy f) Total bilirubin >1 0x ULN EXCEPT: i) For subjects with previously diagnosed Gilbert’s disease, a total bilirubin >1.0x ULN is permitted only if the following laboratory parameters are in the normal range: direct bilirubin, reticulocyte count, hemoglobin ii) For subjects with suspected, but not previously diagnosed, Gilbert’s disease, a total bilirubin >l.0x ULN is permitted only if genetic testing confirms the diagnosis and the following laboratory parameters are in the normal range: direct bilirubin, reticulocyte count, hemoglobin g) All other abnormal laboratory results are excluded only if they are deemed to be clinically significant and to put subjects at unacceptable risk, as determined by the Investigator or the Medical Monitor

12) History of biliary diversion

13) Seropositive for human immunodeficiency virus (HIV)

14) Active infection at time of randomization

15) Prior history of tuberculosis infection

16) Unstable and/or serious medical disease which is likely to impair the subject’s ability to

participate in all aspects of the study or result in substantially shortened life expectancy (e.g., malignancy, end-stage heart failure).

17) Any other condition which, in the opinion of the Investigator, would impede compliance with study drug/assessments or which may interfere with the interpretation of study assessments or put the subject at unacceptable risk by participating

18) Substance abuse, including inhaled or injected drugs, or a positive drugs of abuse screen at the time of screening or randomization. Cannabis use is not exclusionary.

19) Has any contraindications to undergoing MRI procedures

20) Known hypersensitivity to the study drug or its excipients

21) Subjects who are investigational site staff members directly involved in the conduct of the trial and their family members, site staff members otherwise supervised by the Investigator, or subjects who are employees of the sponsor or any contractors directly involved in the conduct of the trial.

[00387] Dosing:

[00388] Patients will be treated with Compound 1 for 12 consecutive weeks, unless dosing is halted by the Safety Review Committee (SRC). Adult patients with NASH will self-administer Compound 1. All patients are dosed with Compound 1, or placebo, on a b.i.d. basis in the fed state for a l2-week duration (e.g., ingestion within 0-30 min of meals twice daily such as at around times of breakfast and dinner). Patients in the high dose cohort receive 500 mg Compound l/placebo (i.e., one 250 mg tablet/b.i.d.) while patients in the low dose are dosed with 100 mg Formula II/placebo (i.e., one 50 mg tablet/b.i.d.).

[00389] Formulation

[00390] The Compound 1 drug product will be supplied as oral tablets, of different shapes and sizes, containing 50 mg or 250 mg Compound 1. Matching placebos will also be provided.

[00391] Pharmacokinetic (PK) Assessments

[00392] PK parameters for NASH patients in each cohort will be compared to assess the suitability of Compound 1 tablets for the treatment of NASH. Data will be obtained from the blood plasma samples collected from each cohort according to the schedule provided.

[00393] Plasma samples will be analyzed to determine Compound 1 concentrations using a validated assay method. Pharmacokinetic variables will be calculated using non-compartmental analysis.

[00394] Protocol:

[00395] Blood: Blood samples will be analyzed to determine plasma concentrations of Compound 1 and Compound 1-M3. Samples will be collected predose (all subjects) and up to 8-hours postdose on Day 1 and Week 2 (intensive PK subset only). For all other visits, samples will be collected at trough timepoints.

[00396] In the subjects who participate in the Intensive PK Sampling Cohort, PK parameters to be assessed will include Cmax, Tmax, Ciast, Tiast, AUCo-iast, AUCo-tau, and t½.

[00397] Pharmacodynamic Assessment

[00398] For ALT and cTl, the absolute change from baseline at 12 weeks will be assessed to determine the effects of Compound 1 on these PD markers. For purposes of PD analysis, baseline for ALT is defined as the Day 1 value. An ANCOVA model with terms for treatment and baseline values will be used for statistical inference. Comparisons of both active groups to the placebo group will be performed using two-sided testing and evaluated at a significance level of 0.05.

[00399] In addition, samples for the following exploratory PD parameters will be collected and may be assessed: Additional metabolic and liver function test markers: fasting glucose/insulin/lipid panel, homeostasis model assessment-estimated insulin resistance (HOMA-IR), fibrinogen, sCDl4, sCDl63; Inflammation markers: high-sensitivity C-reactive protein (hs-CRP), interleukin (IL)-6, IL-lb, IL-1RA, CK-18-M30, CK-18-M65, IL-18, lipopolysaccharide binding protein (LBP); and Fibrosis markers: pro-C3, ELF, FIB-4.

Example 8: Pre-Clinical Study in a Monkey Liver Fibrosis Model

[00400] A pre-clinical study was performed in cynomolgus monkeys using a carbon tetrachloride (CCl₄)-induced monkey liver fibrosis model. CCL is used to induce liver lesion and liver fibrosis. CCL was administered intraperitoneally (i.p.) at 1.0 ml/kg (dissolved in olive oil 1 : 1), twice per week for 4 weeks.

[00401] The study included 6 groups: Group 1 : 8 animals treated with vehicle only (1% HPMC dosed orally (p.o.), twice per day (b.i.d.; every 12 hours); Group 2: 8 animals treated with CCL + vehicle p.o., b.i.d.; Group 3: 8 animals treated with CCL + obetacholic acid (OCA) (15 mg/kg, p.o., once per day (q.d.); Group 4: 8 animals treated with CCL + 5 mg/kg Compound 1, p.o., b.i.d.; Group 5: 8 animals treated with CCL + 15 mg/kg Compound 1, p.o., b.i.d.; Group 6: 8 animals treated with CCL + 30 mg/kg Compound 1, p.o., b.i.d.

[00402] The study was performed over a period of 6 weeks wherein for the first two weeks, Group 2-6 animals were treated only with CCL. Dosing with vehicle, OCA or Compound 1 was initiated after the 2 week CCL-only treatment period. Animals were treated and evaluated over a period of 4 weeks, during which time measurements were taken for body weight (weekly) and levels of the markers alanine aminotransferase (ALT), aspartate aminotransferase (AST), and bilirubin, each of which is increased in damaged or diseased liver tissue.

[00403] Animals in Groups 2-6 experienced a small decrease in body weight over the 4 week period that was not observed in Group 1 control animals, likely due to the toxicity of the CCL treatment. Liver weight of the animals, measured at the end of the study, did not significantly change with any of the treatments (data not shown). Similarly, minimal effects on liver weight at the end of the study were observed (data not shown). The effects of treatment with OCA or with varying doses of the P2X7 receptor antagonist showed decreases in ALT levels both at Day 21 and at the end of the study (FIGS. 4A-4C and FIGS. 5A-5C).

[00404] A pathological evaluation was also performed after completion of the study, in which evaluation criteria were scored as shown in Table 3 below. For these studies, medium lobe liver tissue was collected in SNAP frozen tubes and left liver lobe tissue was collected in a tube containing 10% NBS. Graphical representations of the histology analyses are provided in FIGS. 6A- 6E, showing clear improvement in the disease phenotypes.

Table 3

[00405] Immuno-histochemistry was performed to quantitate a-smooth muscle actin (α-SMA) in tissue samples from the subjects in this study. A rabbit polyclonal anti-a-SMA antibody was used to detect a-SMA. Positive pixel counts were determined using Aperio Image Scope Software. (Statistical analysis: ##P≤0.01 vs. normal; unpaired TTEST;** P≤0.01 vehicle one-way ANOVA, Dunnett’s post hoc.) [00406] Results are summarized in FIG. 6F. Pharmacokinetic (PK) analysis of the subjects was performed and results are summarized in Table 4 below and in FIG. 7. At day 41, 200 μL blood was collected and plasma prepared for Compound 1 and OCA treated animals only. The PK time- points were: 5 min, 15 minutes 30 minutes, 1, 2, 4, 8, 12 hours post AM dose.

[00407] The methods for quantitative analysis of Compound 1 were developed on UPLC (Waters) chromatographic system equipped with an API5500QTrap mass spectrometer (Applied Biosystems, Concord, Ontario, Canada). Analyst 1.5 software packages (Applied Biosystems) were used to control the LC-MS/MS system, as well as for data acquisition and processing. For Compound 1, an aliquot of 50 μL plasma was spiked into a 96- well plate, and 200 μL of acetonitrile containing internal standard were added for protein precipitation. The mixture was vortexed, centrifuged at 4000 rpm for 20 min. 200 μL of supernatant were injected for LC-MS/MS analysis.

[00408] Results were expressed as mean ± SEM. Statistical analysis was performed using student’s t-test, one way ANOVA or two ways ANOVA followed if significant by Tukey's multiple comparison test or post-hoc Dunnett s test. Non parametric test like Mann- Whitney were used when N was too small or data did not follow Gaussian distribution. The difference was considered significant when p < 0.05.

[00409] As illustrated in FIG. 7, plasma concentrations of the administered P2X7 receptor antagonist, measured on Day 41 over a period of about 12 hours, remained above the IC95.

INCORPORATION BY REFERENCE

[00410] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entireties for all purposes. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

FURTHER EMBODIMENTS OF THE DISCLOSURE

[00411] Other subject matter contemplated by the present disclosure is set out in the following numbered embodiments:

1. A method of treating liver disease associated with P2X7 receptor activation comprising administering for at least a week to a patient in need thereof a therapeutically effective amount of compound of Formula (III):

or a pharmaceutically acceptable salt thereof, wherein

R¹ is selected from C₂-C₄ alkyl optionally substituted with -OH, -NH₂, and -CONH₂; R² is selected from CH₃ and halogen;

R³ is selected from H and CH₃; and

R⁵ andR⁶ are independently selected from H, halogen and CF₃.

2. The method of embodiment 1, wherein the compound of Formula (III) is selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.

3. The method of embodiment 1, wherein the compound of Formula (III) is:

or a pharmaceutically acceptable salt thereof. 4. The method of any one of the embodiments 1-3, wherein the liver disease associated with P2X7 receptor activation is nonalcoholic steatohepatitis (NASH).

5. The method of embodiment 4, wherein the NASH is Fibrosis stage 1-3 NASH as determined by two-dimensional magnetic resonance elastography (2D MRE) score.

6. The method of any one of the embodiments 1-3, wherein the liver disease associated with P2X7 receptor activation is Fatty Liver Disease (FLD).

7. The method of any one of the embodiments 1-3, wherein the liver disease associated with P2X7 receptor activation is Nonalcoholic Fatty Liver Disease (NAFLD).

8. The method of any one of the embodiments 1-7, wherein prior to the administering, the patient has advanced liver fibrosis as determined by 2D MRE score.

9. The method of any one of the embodiments 1-8, wherein prior to the administering, the patient has liver cirrhosis as determined by 2D MRE score.

10. The method of any one of the embodiments 1 -9, wherein prior to the administering, the patient’s liver stiffness as determined by MRE is between about 2.5 kPa to about 4.65 kPa.

11. The method of any one of the embodiments 1-10, wherein prior to the administering, the patient’s proton density fat fraction (PDFF) as determined by multiparametric magnetic resonance is≥ 8% steatosis.

12. The method of any one of the embodiments 1-11, wherein prior to the administering, the patient’s cTl score as determined by multiparametric magnetic resonance is≥ 827.5 ms.

13. The method of any one of the embodiments 1-12, wherein prior to the administering, the patient’s liver stiffness as determined by ultrasound-based transient elastography is > 8.

14. The method of any one of the embodiments 1-13, wherein prior to the administering, the patient’s controlled attenuation parameter (CAP) as determined by ultrasound-based transient elastography (Fibroscan) is≥300 dB/m. 15. The method of any one of the embodiments 1-14, wherein prior to the administering, the patient’s alanine aminotransferase (ALT) is between about 5 IU/L and 225 IU/L.

16. The method of any one of the embodiments 1-15, wherein prior to the administering, the patient’s Steatosis, Activity, and Fibrosis (SAF) activity score is≥ 2.

17. The method of any one of the embodiments 1-16, wherein prior to the administering, the patient’s SAF activity score is <2.

18. The method of any one of the embodiments 1-17, wherein prior to the administering, the difference in the patient’s spleen and liver Hounsfield units (HUspieen-HUiiver) as determined by CT scan is greater than 0.

19. The method of any one of the embodiments 1-18, wherein the patient has been diagnosed with at least disease or disorder selected from the group consisting of hepatocellular carcinoma, obesity, insulin resistance diabetes and Type 2 diabetes.

20. The method of any one of the embodiments 1-19, wherein about 50 mg to about 750 mg of the compound of Formula (III) or a pharmaceutically acceptable salt thereof is administered daily.

21. The method of any one of the embodiments 1-20, wherein the administering provides a mean blood plasma of about 10,000 to about 50,000 ng h/mL of the compound of

Formula (III).

22. The method of any one of the embodiments 1-21, wherein the administering provides a mean steady state blood plasma concentration of greater than about 125 ng/mL of the compound of Formula (III).

23. The method of any one of the embodiments 1-22, wherein the compound of Formula (III) is Compound 1 or a pharmaceutically acceptable salt thereof.

24. The method of embodiment 23, wherein about 50 mg to about 500 mg of Compound 1 is administered daily. 25. The method of embodiment 23, wherein about 500 mg of Compound 1 is administered daily.

26. The method of embodiment 23, wherein about 100 mg of Compound 1 is administered daily.

27. The method of embodiment 23, wherein about 250 mg of Compound 1 is administered twice daily.

28. The method of embodiment 23, wherein about 50 mg of Compound 1 is administered twice daily.

29. The method of embodiment 23, wherein the administering provides a mean steady state blood plasma AUC (0-12) hours from about 1500 ng*h/ml to about 10500 ng*h/ml of

Compound 1.

30. The method of embodiment 23, wherein the administering provides a mean steady state blood plasma Cmin from about 150 ng/mL to about 550 ng/ml of Compound 1.

31. The method of embodiment 23, wherein the administering provides a mean steady state blood plasma from about 150 ng/mL to about 550 ng/ml of Compound 1.

32. The method of any one of the embodiments 1-31, wherein the compound of Formula (III) is administered with food.

33. The method of any one of the embodiments 1-32, wherein the compound of Formula (III) is administered in an immediate release tablet.

34. The method of any one of the embodiments 1-33, wherein the compound of Formula (III) is administered for at least about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 12 weeks, about 18 weeks, about 24 weeks, or about 50 weeks. 35. The method of any one of the embodiments 1-34, wherein after administering for a period of at least 1 week, the patient experiences no worsening in liver disease compared to prior to said administering.

36. The method of any one of the embodiments 1-35, wherein after administering for a period of at least 1 week, the patient experiences a substantial reduction in liver disease compared to prior to said administering.

37. The method of any one of the embodiments 1-36, wherein after administering for a period of at least 1 week, the patient experiences a reduction in liver disease that is characterized by an at least 2% reduction in the patient’s cT1 score as determined by multiparametric magnetic resonance.

38. The method of any one of the embodiments 1-37, wherein after administering for a period of at least 1 week, the patient experiences a reduction in liver disease that is characterized by an at least two point improvement in Nonalcoholic Fatty Liver Disease Activity Score (NAS) value.

39. The method of any one of the embodiments 1-38, wherein after administering for a period of at least 1 week, the patient experiences a reduction in liver disease that is characterized by an at least 50% reduction in NAS value.

40. The method of any one of the embodiments 1-39, wherein after administering for a period of at least 1 week, the patient experiences a reduction in liver disease that is characterized by an at least 10% reduction in alanine aminotransferase (ALT) compared to prior to

administering.

41. The method of any one of the embodiments 1-40, wherein after administering for a period of at least 1 week, the patient experiences a reduction in liver disease that is characterized by an at least 10% reduction in aspartate aminotransferase (AST) compared to prior to administering.

42. The method of any one of the embodiments 1-41, wherein after administering for a period of at least 1 week, the patient experiences a reduction in liver disease that is characterized by an at least 10% reduction in liver stiffness as determined by magnetic resonance elastography (MRE) compared to prior to administering.

43. The method of any one of the embodiments 1-42, wherein after administering for a period of at least 1 week, the patient experiences a reduction in liver disease that is characterized by an at least one point reduction in Ultrasonographic Steatosis Score (USS).

44. The method of any one of the embodiments 1-43, wherein after administering for a period of at least 1 week, the patient experiences a reduction of liver disease that is characterized by an increase of the patient’s liver Hounsfield units (HU) to at least about 20 HU as determined by CT scan.

45. The method of any one of the embodiments 1-44, wherein after administering for a period of at least 1 week, the patient experiences a reduction of liver disease that is characterized by a hepatic venous pressure gradient (HVPG) of less than about 12 mm Hg.

46. The method of any one of the embodiments 1-45, wherein after administering for a period of at least 1 week, the patient experiences a reduction of liver disease that is characterized by a decrease in blood IL-1 β concentration of at least about 30% compared to prior to the administering.

47. The method of any one of the embodiments 1-46, wherein the compound of Formula (III) is administered with drug selected from the group consisting of a 5-aminosalicyate agent, a corticosteroid, and an antibiotic, or a combination thereof.

48. The method of any one of the embodiments 1-47, wherein after administering for a period of at least 1 week, 2 weeks, 3 weeks or 4 weeks, the patient experiences a decrease in the blood concentration of IL-1 β compared to prior to the administering.

49. The method according to embodiment 48, wherein the decrease in the patient’s blood concentration of IL-1 β is at least about 30%, about 40%, about 50%, about 60%, about 70% or about 80% compared to prior to the administering. 50. The method of any one of the embodiments 1-49, wherein the compound of Formula (III) has an IC₅₀ against the P2X7 receptor of about 0.1 nM to about 10 nM as determined by an in vitro assay that measures the concentration of IL-1 β in whole blood samples.

51. The method of any one of the embodiments 1-50, wherein the compound of Formula (III) has an IC₅₀ against the P2X7 receptor of about 10 nM to about 100 nM as determined by an in vitro assay that measures the concentration of IL-1 β in whole blood samples.

52. A method of treating liver disease associated with P2X7 receptor activation comprising administering for at least a week to a patient in need thereof a therapeutically effective amount of compound of Formula:

or a pharmaceutically acceptable salt thereof.

53. A method of treating liver disease associated with P2X7 receptor activation comprising administering for at least a week to a patient in need thereof a therapeutically effective amount of compound of Formula:

or a pharmaceutically acceptable salt thereof, wherein the administering provides a mean steady state blood plasma AUC (0-12) hours from about 1500 ng*h/ml to about 10500 ng*h/ml of the compound.

54. The method of embodiment 53, wherein the liver disease associated with P2X7 receptor activation is NASH.

55. A method of treating liver disease associated with P2X7 receptor activation comprising administering for at least a week to a patient in need thereof a therapeutically effective amount of compound of Formula:

or a pharmaceutically acceptable salt thereof, wherein the administering provides a mean steady state blood plasma from about 150 ng/mL to about 550 ng/ml of the compound.

56. The method of embodiment 55, wherein the liver disease associated with P2X7 receptor activation is NASH.

57. A method of treating liver disease associated with P2X7 receptor activation comprising administering for at least a week to a patient in need thereof a therapeutically effective amount of compound of Formula:

or a pharmaceutically acceptable salt thereof, wherein about 50 mg to about 500 mg of the compound is administered daily.

58. The method of embodiment 57, wherein the liver disease associated with P2X7 receptor activation is NASH.

59. A method for inhibiting P2X7 receptor activity in the cells of a subject in need thereof comprising administering to the subject a dose of about 100 to about 2000 mg of a P2X7 receptor antagonist, wherein the P2X7 receptor antagonist has an IC₅₀ of about 0.1 nM to about 10 nM as determined by an in vitro assay that measures the concentration of IL-1 β in whole blood samples.

Claims

1. A method of treating a liver disease associated with P2X7 receptor activation comprising administering for at least 1 week to a patient in need thereof a therapeutically effective amount of compound of Formula (III):

or a pharmaceutically acceptable salt thereof, wherein

R³ is selected from H and CH₃; and

R⁵ andR⁶ are independently selected from H, halogen and CF₃.

2. The method of claim 1, wherein the compound of Formula (III) is selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.

3. The method of claim 1, wherein the compound of Formula (III) is:

or a pharmaceutically acceptable salt thereof.

4. The method of claim 1, wherein the liver disease associated with P2X7 receptor activation is nonalcoholic steatohepatitis (NASH).

5. The method of claim 4, wherein the NASH is Fibrosis stage 1-3 NASH as determined by two-dimensional magnetic resonance elastography (2D MRE) score.

6. The method of claim 1, wherein the liver disease associated with P2X7 receptor activation is Fatty Liver Disease (FLD).

7. The method of claim 1, wherein the liver disease associated with P2X7 receptor activation is Nonalcoholic Fatty Liver Disease (NAFLD).

8. The method of claim 1, wherein prior to the administering, the patient has advanced liver fibrosis as determined by 2D MRE score.

9. The method of claim 1, wherein prior to the administering, the patient has liver cirrhosis as determined by 2D MRE score.

10. The method of claim 1, wherein prior to the administering, the patient’s liver stiffness as determined by MRE is between about 2.5 kPa to about 4.65 kPa.

11. The method of claim 1, wherein prior to the administering, the patient’s proton density fat fraction (PDFF) as determined by multiparametric magnetic resonance is≥ 8% steatosis.

12. The method of claim 1, wherein prior to the administering, the patient’s cTl score as determined by multiparametric magnetic resonance is≥ 827.5 ms.

13. The method of claim 1, wherein prior to the administering, the patient’s liver stiffness as determined by ultrasound-based transient elastography is > 8.

14. The method of claim 1, wherein prior to the administering, the patient’s controlled attenuation parameter (CAP) as determined by ultrasound-based transient elastography

(Fibroscan) is≥300 dB/m.

15. The method of claim 1, wherein prior to the administering, the patient’s alanine aminotransferase (ALT) is between about 5 IU/L and 225 IU/L.

16. The method of claim 1, wherein prior to the administering, the patient’s Steatosis, Activity, and Fibrosis (SAF) activity score is≥ 2.

17. The method of claim 1, wherein prior to the administering, the patient’s SAF activity score is <2.

18. The method of claim 1, wherein prior to the administering, the difference in the patient’s spleen and liver Hounsfield units (HUspieen-HUiiver) as determined by CT scan is greater than 0.

19. The method of claim 1, wherein the patient has been diagnosed with at least 1 disease or disorder selected from the group consisting of hepatocellular carcinoma, obesity, insulin resistance diabetes and Type 2 diabetes.

20. The method of claim 1, wherein about 50 mg to about 750 mg of the compound of Formula (III) or a pharmaceutically acceptable salt thereof is administered daily.

21. The method of claim 1, wherein the administering provides a mean blood plasma

of about 10,000 to about 50,000 ng h/mL of the compound of Formula (III).

22. The method of claim 1, wherein the administering provides a mean steady state blood plasma concentration of greater than about 125 ng/mL of the compound of Formula (III).

23. The method of claim 1, wherein the compound of Formula (III) is Compound 1 or a pharmaceutically acceptable salt thereof.

24. The method of claim 23, wherein about 50 mg to about 500 mg of Compound 1 or a pharmaceutically acceptable salt thereof is administered daily.

25. The method of claim 23, wherein about 500 mg of Compound 1 or a pharmaceutically acceptable salt thereof is administered daily.

26. The method of claim 23, wherein about 100 mg of Compound 1 or a pharmaceutically acceptable salt thereof is administered daily.

27. The method of claim 23, wherein about 250 mg of Compound 1 or a pharmaceutically acceptable salt thereof is administered twice daily.

28. The method of claim 23, wherein about 50 mg of Compound 1 or a pharmaceutically acceptable salt thereof is administered twice daily.

29. The method of claim 23, wherein the administering provides a mean steady state blood plasma AUC (0-12) hours from about 1500 ng*h/ml to about 10500 ng*h/ml of Compound 1.

30. The method of claim 23, wherein the administering provides a mean steady state blood plasma Cmin from about 150 ng/mL to about 550 ng/ml of Compound 1.

31. The method of claim 23, wherein the administering provides a mean steady state blood plasma from about 150 ng/mL to about 550 ng/ml of Compound 1.

32. The method of claim 1, wherein the compound of Formula (III) is administered with food.

33. The method of claim 1, wherein the compound of Formula (III) is administered in an immediate release tablet.

34. The method of claim 1, wherein the compound of Formula (III) is administered for at least about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 12 weeks, about 18 weeks, about 24 weeks, or about 50 weeks.

35. The method of claim 1, wherein after administering for a period of at least 1 week, the patient experiences no worsening in liver disease compared to prior to said administering.

36. The method of claim 1, wherein after administering for a period of at least 1 week, the patient experiences a substantial reduction in liver disease compared to prior to said

administering.

37. The method of claim 1, wherein after administering for a period of at least 1 week, the patient experiences a reduction in liver disease that is characterized by an at least 2% reduction in the patient’s cT1 score as determined by multiparametric magnetic resonance.

38. The method of claim 1, wherein after administering for a period of at least 1 week, the patient experiences a reduction in liver disease that is characterized by an at least 2 point improvement in Nonalcoholic Fatty Liver Disease Activity Score (NAS) value.

39. The method of claim 1, wherein after administering for a period of at least 1 week, the patient experiences a reduction in liver disease that is characterized by an at least 50% reduction in NAS value.

40. The method of claim 1, wherein after administering for a period of at least 1 week, the patient experiences a reduction in liver disease that is characterized by an at least 10% reduction in alanine aminotransferase (ALT) compared to prior to administering.

41. The method of claim 1, wherein after administering for a period of at least 1 week, the patient experiences a reduction in liver disease that is characterized by an at least 10% reduction in aspartate aminotransferase (AST) compared to prior to administering.

42. The method of claim 1, wherein after administering for a period of at least 1 week, the patient experiences a reduction in liver disease that is characterized by an at least 10% reduction in liver stiffness as determined by magnetic resonance elastography (MRE) compared to prior to administering.

43. The method of claim 1, wherein after administering for a period of at least 1 week, the patient experiences a reduction in liver disease that is characterized by an at least 1 point reduction in Ultrasonographic Steatosis Score (USS).

44. The method of claim 1, wherein after administering for a period of at least 1 week, the patient experiences a reduction of liver disease that is characterized by an increase of the patient’s liver Hounsfield units (HU) to at least about 20 HU as determined by CT scan.

45. The method of claim 1, wherein after administering for a period of at least 1 week, the patient experiences a reduction of liver disease that is characterized by a hepatic venous pressure gradient (HVPG) of less than about 12 mm Hg.

46. The method of claim 1, wherein after administering for a period of at least 1 week, the patient experiences a reduction of liver disease that is characterized by a decrease in blood IL-1 β concentration of at least about 30% compared to prior to the administering.

47. The method of claim 1, wherein the compound of Formula (III) is administered with drug selected from the group consisting of a 5-aminosalicyate agent, a corticosteroid, and an antibiotic, or a combination thereof.

48. The method of claim 1, wherein after administering for a period of at least 1 week, 2 weeks, 3 weeks or 4 weeks, the patient experiences a decrease in the blood concentration of IL- 1 b compared to prior to the administering.

49. The method according to claim 48, wherein the decrease in the patient’s blood concentration of IL-1 β is at least about 30%, about 40%, about 50%, about 60%, about 70% or about 80% compared to prior to the administering.

50. The method of claim 1, wherein the compound of Formula (III) has an IC₅₀ against the P2X7 receptor of about 0.1 nM to about 10 nM as determined by an in vitro assay that measures the concentration of IL-1 β in whole blood samples.

51. The method of claim 1, wherein the compound of Formula (III) has an IC₅₀ against the P2X7 receptor of about 10 nM to about 100 nM as determined by an in vitro assay that measures the concentration of IL-1 β in whole blood samples.

or a pharmaceutically acceptable salt thereof.

53. A method of treating a liver disease associated with P2X7 receptor activation comprising administering for at least 1 week to a patient in need thereof a therapeutically effective amount of compound of Formula:

54. The method of claim 53, wherein the liver disease associated with P2X7 receptor activation is NASH.

55. A method of treating a liver disease associated with P2X7 receptor activation comprising administering for at least a week to a patient in need thereof a therapeutically effective amount of compound of Formula:

56. The method of claim 55, wherein the liver disease associated with P2X7 receptor activation is NASH.

57. A method of treating a liver disease associated with P2X7 receptor activation comprising administering for at least a week to a patient in need thereof a therapeutically effective amount of compound of Formula:

58. The method of claim 57, wherein the liver disease associated with P2X7 receptor activation is NASH.