Methods and compositions for modulating adipor2 expression

Abstract

Provided herein are compositions and methods for increasing expression of ADIPOR2 and for treating diseases and / or conditions characterized by reduced expression of ADIPOR2 (e.g., obesity or liver disease) in subjects in need thereof, by administering compositions comprising compounds described herein.

Description

Attorney Docket No: RRBS-010 / 01WO 353016-2018METHODS AND COMPOSITIONS FOR MODULATING ADIPOR2 EXPRESSIONCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 776,811, filed March 24, 2025, which is incorporated herein in its entirety for all purposes.TECHNICAL FIELD

[0002] The disclosure relates to compositions and methods for increasing expression of ADIPOR2 and for treating diseases and / or conditions characterized by reduced expression of ADIPOR2 (e.g., obesity or liver disease) in subjects in need thereof, by administering compositions comprising compounds described herein.BACKGROUND

[0003] Adiponectin receptor 2 (ADIPOR2) is a key regulator of glucose and lipid metabolism, predominantly expressed in the liver. Upon activation by adiponectin, ADIPOR2stimulates the PPARa pathway, enhancing fatty acid oxidation and reducing lipid accumulation in hepatocytes, which is critical for maintaining hepatic and metabolic health. Enhanced fatty acid oxidation by AdipoR2 also influences the phospholipid composition of cell membranes, creating a balanced lipid environment that supports membrane stability and proper receptor function.

[0004] Structural studies have revealed that ADIPOR2 has intrinsic ceramidase activity, which is further enhanced by adiponectin binding. This activity reduces intracellular ceramide levels, increasing membrane fluidity as well as improving insulin sensitivity and overall metabolic function. Dysregulation of ADIPOR2disrupts these processes, leading to membrane lipid imbalances that contribute to metabolic disorders such as obesity, insulin resistance, and liver disease (e.g., non-alcoholic steatohepatitis (NASH)).

[0005] New therapeutic options for obesity, insulin resistance, and liver disease are needed.SUMMARY

[0006] In some aspects, provided herein is a method of increasing the level of adiponectin receptor 2 (ADIPOR2) transcript and / or protein in a cell, wherein the method comprises contacting a cell with an effective amount of a composition comprising omaveloxolone, or a pharmaceutically acceptable salt thereof. In some aspects, provided herein is a method ofAttorney Docket No: RRBS-OIO / OIWO 353016-2018 decreasing ceramide levels, reducing lipogenesis, improving glucose homeostasis, and / or increasing insulin sensitivity in a cell, wherein the method comprises contacting a cell with an effective amount of a composition comprising omaveloxolone, or a pharmaceutically acceptable salt thereof.

[0007] In some embodiments, the cell has reduced expression or activity of ADIPOR2. In some embodiments, the cell has reduced ceramidase activity. In some embodiments, the cell has increased ceramide levels compared to a control cell. In some embodiments, the cell has increased membrane rigidity, impaired insulin signaling, insulin resistance, and / or lipotoxicity.

[0008] In some embodiments, the cell comprises one or more mutations in one allele of the ADIPOR2 gene. In some embodiments, the one or more mutations comprise a missense mutation, a frameshift mutation, a nonsense mutation, a mutation that introduces a splice site, a mutation that affects an existing splice site, or a deletion. In some embodiments, the one or more mutations result in a mutant gene encoding a protein with reduced function. In some embodiments, the cell has at least one ADIPOR2 allele encoding a protein with residual function.

[0009] In some embodiments, the level of ADIPOR2 transcript and / or protein in the cell is increased by about 1.1 to about 10-fold, compared to a control cell . In some embodiments, the level of ADIPOR2 transcript and / or protein in the cell is increased to a level comparable to that of a control cell that does not comprise reduced expression or activity of ADIPOR2.

[0010] In some embodiments, the cell is a liver cell. In some embodiments, the contact occurs in vivo or ex vivo.

[0011] In some aspects, provided herein is a method of decreasing ceramide levels, reducing lipogenesis, and / or improving glucose homeostasis within the liver of a subject in need thereof, comprising administering an effective amount of a composition comprising omaveloxolone, or a pharmaceutically acceptable salt thereof.

[0012] In some aspects, provided herein is a method of reducing adipocyte hypertrophy, reducing plasma levels of triglycerides and free fatty acids, increasing mitochondrial function, and / or increasing expression of thermogenic markers in brown adipose tissue in a subject in need thereof, comprising administering an effective amount of a composition comprising omaveloxolone, or a pharmaceutically acceptable salt thereof.Attorney Docket No: RRBS-OIO / OIWO 353016-2018

[0013] In some aspects, provided herein is a method of reducing liver inflammation, decreasing fibrosis markers, and / or improving insulin sensitivity in a subject in need thereof, comprising administering an effective amount of a composition comprising omaveloxolone, or a pharmaceutically acceptable salt thereof.

[0014] In some aspects, provided herein is a method of treating or preventing a disease or condition characterized by reduced expression and / or function of ADIPOR2 in a subject in need thereof, comprising administering an effective amount of a composition comprising omaveloxolone, or a pharmaceutically acceptable salt thereof. In some embodiments, the subject has reduced ceramidase activity. In some embodiments, the subject has increased ceramide levels compared to a control subject. In some embodiments, the subject has increased membrane rigidity, impaired insulin signaling, insulin resistance, and / or lipotoxicity

[0015] In some embodiments, the subject comprises one or more mutations in one allele of the ADIPOR2 gene. In some embodiments, the one or more mutations comprise a missense mutation, a frameshift mutation, a nonsense mutation, a mutation that introduces a splice site, a mutation that affects an existing splice site, or a deletion. In some embodiments, the one or more mutations result in a mutant gene encoding a protein with reduced function. In some embodiments, the subject is determined to have the one or more mutations by analysis of the subject’s ADIPOR2 genome sequence. In some embodiments, the analysis of the subject’s ADIPOR2 genome sequence is performed using a method selected from the group consisting of: targeted variant analysis, deletion analysis, mutations canning of select exons, mutation scanning of the entire ADIPOR2 coding region, sequence analysis of select exons, sequence analysis of the entire ADIPOR2 coding region, or ADIPOR2 RNA analysis. In some embodiments, the subject has at least one ADIPOR2 allele encoding a protein with residual function.

[0016] In some embodiments, the method results in increased expression and / or activity of ADIPOR2 in the subject. In some embodiments, expression is increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%. In some embodiments, expression is increased within 1 month of beginning administration of omaveloxolone.Attorney Docket No: RRBS-OIO / OIWO 353016-2018

[0017] In some embodiments, the method results in reduced ceramide levels in the subject. In some embodiments, the method results in reduced membrane rigidity, increased insulin signaling, improved insulin sensitivity, and / or reduced lipotoxicity.

[0018] In some embodiments, the method results in decreased levels of ceramide, reduced lipogenesis, and / or improved glucose homeostasis within the liver of the subject. In some embodiments, the method results in reduced adipocyte hypertrophy, decreased plasma levels of triglycerides and free fatty acids, increased mitochondrial function, and / or increased expression of thermogenic markers in brown adipose tissue of the subject. In some embodiments, the method results in reduced liver inflammation, decreased levels of fibrosis markers, and / or improved insulin sensitivity in the subject.

[0019] In some embodiments, the disease or condition is obesity. In some embodiments, the disease or condition is liver disease.

[0020] In some embodiments, the effective amount of the composition is about 1 mg to about 1000 mg per day. In some embodiments, the effective amount of the composition is about 5 mg to about 150 mg per day. In some embodiments, the effective amount of the composition is about 10 mg, about 20 mg, about 30 mg, about 40 mg, 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, or about 150 mg per day.

[0021] In some embodiments, the composition is administered orally or parenterally. In some embodiments, the composition is administered once or twice per day.

[0022] In some embodiments, the method results in reduction or improvement of at least one symptom of the disease or condition. In some embodiments, the method results in prevention of at least one symptom of the disease or condition.

[0023] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0024] In some embodiments, the composition comprises omaveloxolone. In some embodiments, the composition additionally comprises a pharmaceutically acceptable carrier.

[0025] In one aspect, the present disclosure provides a method of identifying a subject in need of treatment with an effective amount of a composition comprising omaveloxolone, comprising: analyzing a genome sequence of the subject; and determining that the subject comprises a mutation in the ADIPOR2 gene.Attorney Docket No: RRBS-OIO / OIWO 353016-2018

[0026] In some embodiments, the genome sequence comprises a sequence of the ADIPOR2 gene, or a portion thereof. In some embodiments, the subject comprises one or more mutations in one or both alleles of the ADIPOR2 gene.

[0027] The method of identifying a subject in need of treatment with an effective amount of a composition comprising omaveloxolone may further comprise administering to the subject an effective amount of a composition comprising omaveloxolone, or a pharmaceutically acceptable salt thereof.

[0028] In one aspect, the present disclosure provides a composition comprising omaveloxolone for use in a method of increasing the level of adiponectin receptor 2 (ADIPOR2) transcript and / or protein in a cell.

[0029] In one aspect, the present disclosure provides a composition comprising omaveloxolone for use in decreasing ceramide levels, reducing lipogenesis, improving glucose homeostasis, and / or increasing insulin sensitivity in a cell.

[0030] In one aspect, the present disclosure provides a composition comprising omaveloxolone for use in treating or preventing a disease or disorder.

[0031] In one aspect, the present disclosure provides a composition comprising omaveloxolone in the manufacture of a medicament for increasing the level of adiponectin receptor 2 (ADIPOR2) transcript and / or protein in a cell.

[0032] In one aspect, the present disclosure provides use of a composition comprising omaveloxolone in the manufacture of a medicament for decreasing ceramide levels, reducing lipogenesis, improving glucose homeostasis, and / or increasing insulin sensitivity in a cell.

[0033] In one aspect, the present disclosure provides use of a composition comprising omaveloxolone in the manufacture of a medicament for treating or preventing a disease or disorder.FIGURE DESCRIPTIONS

[0034] FIG. 1 provides a concentration-response curve (CRC) for adiponectin receptor 2 (ADIPOR2) expression in omaveloxolone-treated motor neurons.

[0035] FIGS.2A-2B provide quantification of immunocytochemistry data showing ADIPOR2 protein abundance in HEPG2 cells treated with 125 nM, 250 nM, and 500 nM omaveloxolone.Attorney Docket No: RRBS-OIO / OIWO 353016-2018

[0036] FIGS.3A-3B provide visualization (FIG.3A) and quantification (FIG.3B) of western blot data showing ADIPOR2 protein expression in motor neurons treated with 125 nM, 250 nM, and 500 nM omaveloxolone.DETAILED DESCRIPTION

[0037] The present disclosure provides compositions and methods for increasing expression of ADIPOR2. The present disclosure further provides compositions and methods for decreasing ceramide levels, reducing lipogenesis, and / or improving glucose homeostasis within the liver of a subject in need thereof; for reducing plasma levels of triglycerides and free fatty acids, increasing mitochondrial function, and / or increasing expression of thermogenic markers in brown adipose tissue; and for reducing liver inflammation, decreasing fibrosis markers, and / or improving insulin sensitivity in a subject in need thereof, comprising administering an effective amount of a composition comprising omaveloxolone, or a pharmaceutically acceptable salt thereof. The present disclosure further provides compositions and methods for treating diseases and / or conditions characterized by reduced expression of ADIPOR2 (e.g., obesity or liver disease) in subjects in need thereof, by administering compositions comprising compounds described herein.Definitions

[0038] Unless otherwise defined, all terms of art, notations, and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this application pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art. All publications, patent applications, patents, GenBank or other accession numbers and other references mentioned herein are incorporated by reference in their entirety for all purposes.

[0039] The term “a” or “an” refers to one or more of that entity, z.e., can refer to a plural referent. As such, the terms “a” or “an”, and “one or more” 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.Attorney Docket No: RRBS-OIO / OIWO 353016-2018

[0040] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, z.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, z.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0041] It is understood that aspects and embodiments of the disclosure described herein include “comprising,” “consisting,” and “consisting essentially of’ aspects and embodiments. As used herein, “comprising” is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, “consisting of’ excludes any elements, steps, or ingredients not specified in the claimed composition or method. As used herein, “consisting essentially of’ does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claimed composition or method. Any recitation herein of the term “comprising”, particularly in a description of components of a composition or in a description of steps of a method, is understood to encompass those compositions and methods consisting essentially of and consisting of the recited components or step.

[0042] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0043] Certain ranges are presented herein with numerical values being preceded by the term “approximately” or “about.” The terms “approximately” and “about” are used interchangeablyAttorney Docket No: RRBS-OIO / OIWO 353016-2018 and mean a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1%, of a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length, inclusive of the endpoints. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth, unless otherwise apparent from context that it is impossible to extend the boundary beyond certain points (e.g., below 0% or above 100% in some cases).

[0044] “Treating” or “treatment” as used herein covers the treatment of the disease, injury, or condition of interest, e.g., a disease or condition characterized by reduced expression and / or function of adiponectin receptor 2 (ADIPOR2), in a biological material, e.g., mammal, preferably a human, having the disease or condition of interest, and includes: (i) reducing the severity or duration of the disease, injury or condition, e.g., when it occurs, e.g., in a mammal predisposed to the condition; (ii) inhibiting the disease, injury, or condition, i.e., arresting its development; (iii) relieving the disease, injury, or condition, i.e., causing regression of the disease or condition; or (iv) relieving the symptoms resulting from the disease, injury, or condition.

[0045] In certain embodiments, as used herein, the term “prevention” includes inhibiting or impeding the onset or progression of a disease, or reducing the amount of damage caused by a disease. As used herein, the terms “disease,” “disorder,” and “condition” may be used interchangeably.

[0046] The terms “increase” and “increased” are all used herein generally to mean an increase by a statistically significant amount. However, for avoidance of doubt, “increase” and “increased” mean an increase by at least 10% as compared to a reference level, for example an increase by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least 1-fold, at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 500-fold, or at least 1000-fold, or any increase between 10% and 1000-fold as compared to a reference level.

[0047] As used herein, the term “comparable level” refers to a level that is in between 50% and 200%, inclusive of end points, of the reference level in the control set of conditions or circumstances, unless otherwise apparent from context that it is impossible to extend theAttorney Docket No: RRBS-OIO / OIWO 353016-2018 boundary beyond certain points. In some embodiments, a comparable level may be preferably between 60% and 170%, more preferably between 70% and 140%, even more preferably between 80% and 125%, or more preferably still between 90% and 110%, of the reference level. Those of ordinary skill in the art will appreciate the appropriate control set in obtaining the reference level. In general, the appropriate control set shares identical features with the test set except the feature(s) of interest, so that the observed results warrant a reasonable conclusion that any changes in the level observed are caused by or indicative of the variation in the feature(s) that are varied.

[0048] As used herein, the term “control cell” refers to a cell treated with vehicle or control instead of omaveloxolone, unless otherwise indicated. A “control cell” may comprises one or more mutations in one or both alleles of the ADIPOR2 gene. Alternatively, a “control cell” may not comprise a mutation in either allele of the ADIPOR2 gene.

[0049] As used herein, a “subject” or an “individual” includes animals, such as human (e.g., human subjects) and non-human animals. In some embodiments, a “subject” or “individual” is a patient under the care of a physician. Thus, the subject can be a human patient or an individual who has, is at risk of having, or is suspected of having a disease of interest (e.g., cancer) and / or one or more symptoms of the disease. The subject can also be an individual who is diagnosed with a risk of the condition of interest at the time of diagnosis or later. The term “non-human animals” includes all vertebrates, e.g., mammals, e.g., rodents, e.g., mice, e.g. non-human primates, and non- mammals, e.g., sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0050] “Administration,” “administering” and the like, refer to administration to a subject by a medical professional or by self-administration by the subject, as well as to indirect administration, which may be the act of prescribing a composition of the invention. Typically, an effective amount is administered, which amount can be determined by one of skill in the art. Any method of administration may be used. Administration to a subject can be achieved by, for example, oral administration, in liquid or solid form, e.g. in capsule or tablet form; intravascular injection; intramyocardial delivery; or other suitable forms of administration.

[0051] “Effective amount” as used herein refers to an amount of an agent effective in achieving a particular effect, e.g., increasing transcript and / or protein levels of ADIPOR2. In certain embodiments, the increase is about 1.1 to about 10-fold, as compared to the amount prior to or without treatment. In the context of therapeutic treatment of a subject, an effective amount may be, e.g., an amount effective or sufficient to reduce one or more disease symptoms in theAttorney Docket No: RRBS-OIO / OIWO 353016-2018 subject, e.g., a subject with a disease or condition characterized by reduced expression and / or function of ADIPOR2). The amount of a compound or composition of the invention which constitutes an “effective amount” will vary depending on the compound or composition, the disease, injury or condition and its severity, the manner of administration, and the age of the subject to be treated, but can be determined routinely by one of ordinary skill in the art having regard to his own knowledge and to this disclosure.

[0052] As used herein, the term “control subject” refers to a subject who is not known to have the disease or condition for which the subject is administered a composition comprising omavel oxoIone or a pharmaceutically acceptable salt thereof. A “control subject” may be a subject who is not known to have obesity. A “control subject” may be a subject who is not known to have liver disease.

[0053] A “composition” can comprise an active agent and a carrier, inert or active, e.g., a pharmaceutically acceptable carrier, diluent or excipient. A composition may be a pharmaceutical composition. In particular embodiments, the compositions are sterile, substantially free of endotoxins or non-toxic to recipients at the dosage or concentration employed.

[0054] “Pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which 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.

[0055] As used herein, the term “pharmaceutically acceptable salt” refers to any salt of an acidic or a basic group that may be present in a compound of the present invention (e.g., omaveloxolone), which salt is compatible with pharmaceutical administration.

[0056] As is known to those of skill in the art, “salts” of compounds may be derived from inorganic or organic acids and bases. Examples of acids include, but are not limited to, hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic and benzenesulfonic acid. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds described herein and their pharmaceutically acceptable acid addition salts.Attorney Docket No: RRBS-OIO / OIWO 353016-2018

[0057] Examples of bases include, but are not limited to, alkali metal (e.g., sodium and potassium) hydroxides, alkaline earth metal (e.g., magnesium and calcium) hydroxides, ammonia, and compounds of formula NW4+, wherein W is Cl -4 alkyl, and the like.

[0058] Examples of salts include, but are not limited to, acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate, and the like. Other examples of salts include anions of the compounds of the present invention compounded with a suitable cation such as Na+, K+, Ca2+, NH4+, and NW4+ (where W can be a C 1-4 alkyl group), and the like.

[0059] For therapeutic use, salts of the compounds of the present invention (e.g., omaveloxolone) are contemplated as being pharmaceutically acceptable. However, salts of acids and bases that are non-pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound.

[0060] As used herein, “pharmaceutically acceptable excipient” refers to a substance that aids the administration of an active agent to and / or absorption by a subject and can be included in the compositions of the present invention without causing a significant adverse toxicological effect on the subject. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, such as a phosphate buffered saline solution, emulsions (e.g., such as an oil / water or water / oil emulsions), lactated Ringer’s, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer’s solution), alcohols, oils, gelatins, carbohydrates, fatty acid esters, and colors, and the like. Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and / or aromatic substances and the like that do not deleteriously react with the compounds of the invention. For examples of excipients, see Martin, Remington’s Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA (1975).Attorney Docket No: RRBS-OIO / OIWO 353016-2018

[0061] As used herein, “solid dosage form” means a pharmaceutical dose(s) in solid form, e.g., tablets, capsules, granules, powders, sachets, reconstitutable powders, dry powder inhalers and chewables.

[0062] As used herein, the term “mutation” refers to a substitution, a deletion, an insertion, and / or a translocation. The mutation may comprise a missense mutation, a nonsense mutation, a frameshift mutation, a gene fusion, a gain-of-function mutation, a loss-of-function mutation, a duplication mutation, a mutation that introduces a splice site, and / or a mutation that affects an existing splice site. The mutation may be in one or more genes. The mutation may be naturally occurring. Alternatively, the mutation may be induced or engineered.

[0063] “Pharmaceutical compositions” include compositions of one or more compounds disclosed herein and one or more pharmaceutically acceptable carrier, excipient, or diluent.

[0064] “Dose” means a specified quantity of a pharmaceutical agent provided in a single administration, or in a specified time period. In certain embodiments, a dose can be administered in two or more boluses, tablets, or injections. In certain embodiments, a dose can be administered in two or more injections to minimize injection site reaction in an individual. Doses can be stated as the amount of pharmaceutical agent per hour, day, week or month. “Dosage amount” may be used interchangeably with “dose”.

[0065] “Dosage regimen” means a schedule according to which doses of a pharmaceutical agent are provided, e.g., daily, weekly, or other schedule capable of being developed by a person of ordinary skill in the art.

[0066] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology techniques, microbiology, biochemistry and immunology, which are within the scope of those of skill in the art. Such techniques are explained fully in the literature, such as, "Molecular Cloning: A Laboratory Manual", second edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (M. J. Gait, ed., 1984); "Animal Cell Culture" (R. I. Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (D. M. Weir & C. C. Blackwell, eds.); "Gene Transfer Vectors for Mammalian Cells" (J. M. Miller & M. P. Calos, eds., 1987); "Current Protocols in Molecular Biology" (F. M. Ausubel et al., eds., 1987); "PCR: The Polymerase Chain Reaction", (Mullis et al., eds., 1994); and "Current Protocols in Immunology" (J. E. Coligan et al., eds., 1991), each of which is expressly incorporated by reference herein.Attorney Docket No: RRBS-OIO / OIWO 353016-2018

[0067] Several aspects of the invention are described below with reference to example applications for illustration. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the invention. One having ordinary skill in the relevant art, however, will readily recognize that the invention can be practiced without one or more of the specific details or with other methods. The present invention is not limited by the illustrated ordering of acts or events, as some acts may occur in different orders and / or concurrently with other acts or events. Furthermore, not all illustrated acts or events are required to implement a methodology in accordance with the present invention.Adiponectin Receptor 2 (ADIPOR2)

[0068] The disclosure provides compositions and methods of increasing the level of adiponectin receptor 2 (ADIPOR2) transcript and / or protein in a cell, by contacting the cell with an effective amount of a composition comprising omaveloxolone, or a pharmaceutically acceptable salt thereof. The disclosure also provides compositions and methods of treating or preventing a disease or condition characterized by reduced expression and / or function of ADIPOR2 in a subject in need thereof, by contacting the cell with an effective amount of a composition comprising omaveloxolone, or a pharmaceutically acceptable salt thereof.

[0069] Adiponectin receptor 2 (ADIPOR2) is a key regulator of glucose and lipid metabolism, predominantly expressed in the liver. It belongs to the seven-transmembrane domain receptor family, but unlike G protein-coupled receptors, it functions through alternative signaling mechanisms. Upon activation by adiponectin, ADIPOR2 stimulates the PPARa pathway, enhancing fatty acid oxidation and reducing lipid accumulation in hepatocytes, which is critical for maintaining hepatic and metabolic health. Enhanced fatty acid oxidation by ADIPOR2 also influences the phospholipid composition of cell membranes, creating a balanced lipid environment that supports membrane stability and proper receptor function.

[0070] Structural studies have revealed that ADIPOR2 has intrinsic ceramidase activity, which is further enhanced by adiponectin binding. This activity reduces intracellular ceramide levels, increasing membrane fluidity as well as improving insulin sensitivity and overall metabolic function. Dysregulation of ADIPOR2 disrupts these processes, leading to membrane lipid imbalances that contribute to metabolic disorders such as obesity, insulin resistance, and liver disease (e.g., non-alcoholic steatohepatitis (NASH)).Attorney Docket No: RRBS-010 / 01WO 353016-2018

[0071] The human ADIPOR2 gene has 22 transcripts, each of which encode a protein isoform of ADIPOR2. In some embodiments, the increased transcript level of ADIPOR2 comprises transcript NM_024551.3, NM_001375363.1, NM_001375364.1, NM_001375365.1, XM_006719018.3, XM_011521024.2, XM_047429545.1, XM_047429546.1, XM_047429547.1, XM_047429548.1, XM_054332319.1, XM_054332320.1, XM_054332321.1, XM_054332322.1, XM_054332323.1, XM_054332324.1, XM_054373229.1, XM_054373230.1, XM_054373231.1, XM_054373232.1, XM_054373233.1, and / or XM_054373234.1.

[0072] In some embodiments, the increased protein level of ADIPOR2 comprises NP_078827.2, NP_001362292.1, NP_001362293.1, NP_001362294.1, XP_006719081.1, XP_011519326.1, XP_047285501.1, XP_047285502.1, XP_047285503.1, XP_047285504.1, XP_054188294.1, XP_054188295.1, XP_054188296.1, XP_054188297.1, XP_054188298.1, XP_054188299.1, XP_054229204.1, XP_054229205.1, XP_054229206.1, XP_054229207.1, XP_054229208.1, and / or XP_054229209.1.

[0073] In some embodiments, reduced expression of ADIPOR2 comprises reduced transcript levels of ADIPOR2. In some embodiments, reduced expression of ADIPOR2 comprises reduced protein levels of ADIPOR2. In some embodiments, reduced function of ADIPOR2 comprises a mutation that demonstrates reduced effectiveness in one or more functions of ADIPOR2. Non-limiting examples of functions of ADIPOR2 include PPARa signaling, ceramidase activity, fatty acid oxidation, reducing lipid accumulation in adipocytes, increasing membrane fluidity, improving insulin sensitivity, and adiponectin binding.

[0074] In various embodiments, the subject comprises one or more mutations in one or both alleles of the ADIPOR2 gene (NCBI Gene ID: 79602). In some embodiments, the one or more mutations comprise a frameshift mutation. In some embodiments, the one or more mutations comprise a missense mutation. In some embodiments, the one or more mutations comprise a nonsense mutation. In some embodiments, the one or more mutations comprise a mutation that introduces a splice site. In some embodiments, the one or more mutations comprise a mutation that affects an existing splice site. In some embodiments, the one or more mutations comprise a deletion of the ADIPOR2 gene. In some embodiments, the one or more mutations result in a mutant gene encoding a truncated protein.Attorney Docket No: RRBS-OIO / OIWO 353016-2018

[0075] In some embodiments, the one or more mutations in the ADIPOR2 gene correspond to one or more amino acid substitutions or deletions in the ADIPOR2 protein (Uniprot Accession No: Q86V24).

[0076] In some embodiments, the subject is determined to have the one or more mutations by analysis of the subject’s ADIPOR2 genome sequence. In some embodiments, the analysis of the subject’ &ADIPOR2 genome sequence is performed using a method selected from the group consisting of: targeted variant analysis, deletion analysis, mutation scanning of select exons, mutation scanning of the entire ADIPOR2 coding region, sequence analysis of select exons, sequence analysis of the entire ADIPOR2 coding region, or ADIPOR2 RNA analysis.

[0077] In some embodiments, the subject has at least one ADIPOR2 allele encoding a protein with residual function. Residual function of ADIPOR2, as used herein, refers to an ability of a ADIPOR2 protein to perform at least one function of a wild-type ADIPOR2 at a level that is at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%, of that of the wild-type ADIPOR2 protein. In some embodiments, the function of ADIPOR2 is a biological activity. The at least one function of ADIPOR2 may be measured by any suitable method known in the art. In some embodiments, the function is any function described herein.

[0078] In some embodiments, the ADIPOR2 allele encoding a protein with residual function comprises at least an adiponectin binding domain with residual function. In some embodiments, the ADIPOR2 allele encoding a protein with residual function comprises at least a zinc catalytic site with residual function. In some embodiments, the ADIPOR2 allele encoding a protein with residual function comprises at least a ceramide binding domain with residual function. In some embodiments, the ADIPOR2 allele encoding a protein with residual function comprises at least a transmembrane domain with residual function.Methods of Use

[0079] In one aspect, the present disclosure provides methods of increasing the level of ADIPOR2 transcript and / or protein in a cell, wherein the method comprises contacting a cell with an effective amount of a composition comprising omaveloxolone, or a pharmaceutically acceptable salt thereof. In some embodiments, the method comprises increasing the level ofAttorney Docket No: RRBS-OIO / OIWO 353016-2018 ADIPOR2 transcript in the cell. In some embodiments, the method comprises increasing the level of ADIPOR2 protein in the cell.

[0080] Omaveloxolone, also known as N-((4aS,6aR,6bS,8aR,12aS,14aR,14bS)-ll-cyano-2,2,6a,6b,9,9,12a-heptamethyl-10,14-dioxo-l,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,12a,14,14a,14b-octadecahydropicen-4a-yl)-2,2-difluoropropanamide or RTA-408, has the chemical structure of formula (I), as depicted below:

[0081] In some embodiments, the present disclosure provides methods of decreasing ceramide levels, reducing lipogenesis, improving glucose homeostasis, and / or increasing insulin sensitivity in a cell, wherein the method comprises contacting a cell with an effective amount of a composition comprising omaveloxolone, or a pharmaceutically acceptable salt thereof. In some embodiments, the method comprises decreasing ceramide levels in a cell. In some embodiments, the method comprises reducing lipogenesis in a cell. In some embodiments, the method comprises improving glucose homeostasis in a cell. In some embodiments, the method comprises increasing insulin sensitivity in a cell. In some embodiments, the method comprises contacting a cell with an effective amount of a composition comprising omaveloxolone, or a pharmaceutically acceptable salt thereof.

[0082] In some embodiments, the cell has reduced expression and / or activity of ADIPOR2. In some embodiments, the activity of ADIPOR2 is any activity or function described herein. In some embodiments, the cell has reduced ceramidase activity. In some embodiments, the cell has increased ceramide levels compared to a control cell. In some embodiments, the cell has increased membrane rigidity, impaired insulin signaling, increased insulin resistance, and / or increased lipotoxicity compared to a control cell. In some embodiments, the control cell does not comprise reduced expression of ADIPOR2.Attorney Docket No: RRBS-OIO / OIWO 353016-2018

[0083] In some embodiments, the cell comprises one or more mutations in one allele of the ADIPOR2 gene. Alternatively, the cell comprises one or more mutations in both alleles of the ADIPOR2 gene. In some embodiments, the one or more mutations comprise any one or more mutations described herein.

[0084] In some embodiments, the cell has at least one.ADIPOR2 allele encoding a protein with residual function. In some embodiments, the residual function is any ADIPOR2 function described herein.

[0085] In some embodiments, the level of ADIPOR2 transcript and / or protein in the cell is increased by about 1.1 to about 10-fold, compared to control cell. In some embodiments, the level of ADIPOR2 transcript and / or protein in the cell is increased by about 1.1 -fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, about2-fold, about 2.5-fold, about3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold, compared to a control cell.

[0086] In some embodiments, the control cell comprises one or more mutations in one or both alleles of the ADIPOR2 gene. In some embodiments, the control cell does not comprise a mutation in either allele of the ADIPOR2 gene. In some embodiments, the control cell is treated with vehicle / control. In some embodiments, the control cell does not comprise reduced expression of ADIPOR2.

[0087] In some embodiments, the cell is a liver cell. In some embodiments, the contact occurs in vivo or ex vivo.

[0088] In some embodiments, the method of increasing the level of ADIPOR2 transcript and / or protein in a cell results in one or more measurable effects in the cell. In some embodiments, the method results in decreased ceramide levels in the cell. In some embodiments, the method results in reduced lipogenesis in the cell. In some embodiments, the method results in improved glucose homeostasis in the cell. In some embodiments, the method results in increased insulin sensitivity in the cell.

[0089] In some aspects, provided herein is a method of decreasing ceramide levels in a cell. In some aspects, provided herein is a method of reducing lipogenesis in a cell. In some aspects, provided herein is a method of improving glucose homeostasis in a cell. In some aspects, provided herein is a method of increasing insulin sensitivity in a cell. In some embodiments,Attorney Docket No: RRBS-OIO / OIWO 353016-2018 the method comprises contacting a cell with an effective amount of a composition comprising omaveloxolone, or a pharmaceutically acceptable salt thereof.

[0090] In some embodiments, ceramide levels may be measured by any suitable method known in the art. In some embodiments, ceramide levels are reduced by about 5% to about 90%, compared to a control cell. In some embodiments, ceramide levels in the cell are reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, compared to a control cell.

[0091] In some embodiments, lipogenesis may be measured by any suitable method known in the art. In some embodiments, reduced lipogenesis may be measured by quantifying suppression of SREBP-lc and / or decreased expression of one or more lipogenic genes (e.g., FASN, SCD1, and / or ACC1). In some embodiments, transcript and / or protein levels of SREBP-lc, FASN, SCD1, and / or ACC1 in the cell are reduced by about 5% to about 90%, compared to a control cell. In some embodiments, transcript and / or protein levels of SREBP-lc, FASN, SCD1, and / or ACC1 in the cell are reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, compared to a control cell.

[0092] In some embodiments, glucose homeostasis may be measured by any suitable method known in the art. In some embodiments, glucose homeostasis may be measured by increased AMPK activation and / or reduced expression of gluconeogenesis genes (e.g., PEPCK, G6PC expression). In some embodiments, AMPK activation in the cell is increased by about 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold, compared to a control cell. In some embodiments, transcript and / or protein levels of PEPCK and / or G6PC in the cell are reduced by about 5% to about 90%, compared to a control cell. In some embodiments, transcript and / or protein levels of PEPCK and / or G6PC are reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, compared to a control cell.Attorney Docket No: RRBS-OIO / OIWO 353016-2018

[0093] In some embodiments, enhanced insulin sensitivity may be measured by any suitable method known in the art. In some embodiments, insulin sensitivity may be measured by glucose tolerance tests (GTT). In some embodiments, insulin sensitivity may be measured by insulin signaling assays. In some embodiments, insulin sensitivity in the cell is increased by about 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, about 2-fold, about 2.5 -fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold, compared to a control cell.Methods of Treatment

[0094] In one aspect, the present disclosure provides a method of decreasing ceramide levels, reducing lipogenesis, improving glucose homeostasis, and / or improving insulin sensitivity within the liver of a subject in need thereof, comprising administering an effective amount of a composition comprising omaveloxolone, or a pharmaceutically acceptable salt thereof.

[0095] In some embodiments, ceramide levels within the liver of the subject may be measured by any suitable method known in the art. In some embodiments, ceramide levels within the liver of the subject are reduced by about 5% to about 90%, compared to ceramide levels within the liver of the subject prior to administration. In some embodiments, ceramide levels within the liver of the subject are reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, compared to ceramide levels within the liver of the subject prior to administration.

[0096] In some embodiments, lipogenesis may be measured by any suitable method known in the art. In some embodiments, reduced lipogenesis within the liver of the subject may be measured by quantifying suppression of SREBP-lc and / or decreased expression of one or more lipogenic genes (e.g., FASN, SCD1, and / or ACC1). In some embodiments, transcript and / or protein levels of SREBP-lc, FASN, SCD1, and / or ACC1 within the liver of the subject are reduced by about 5% to about 90%, compared to levels within the liver of the subject prior to administration. In some embodiments, transcript and / or protein levels of SREBP-lc, FASN, SCD1, and / or ACC1 within the liver of the subject are reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atAttorney Docket No: RRBS-OIO / OIWO 353016-2018 least 85%, or at least 90%, compared to levels within the liver of the subject prior to administration.

[0097] In some embodiments, glucose homeostasis may be measured by any suitable method known in the art. In some embodiments, glucose homeostasis may be measured by increased AMPK activation and / or reduced expression of gluconeogenesis genes (e.g., PEPCK, G6PC expression). In some embodiments, AMPK activation within the liver of the subject is increased by about 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold, compared to levels within the liver of the subject prior to administration. In some embodiments, transcript and / or protein levels of PEPCK and / or G6PC within the liver of the subject are reduced by about 5% to about 90%, compared to levels within the liver of the subject prior to administration. In some embodiments, transcript and / or protein levels of PEPCK and / or G6PC within the liver of the subject are reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, compared to levels within the liver of the subject prior to administration.

[0098] In some embodiments, enhanced hepatic insulin sensitivity compared to levels within the liver of the subject prior to administration may be measured by any suitable method known in the art. In some embodiments, hepatic insulin sensitivity may be measured by glucose tolerance tests (GTT). In some embodiments, hepatic insulin sensitivity may be measured by insulin signaling assays. In some embodiments, hepatic insulin sensitivity in the subject is increased by about 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold, compared to levels in the subject prior to administration.

[0099] In one aspect, the present disclosure provides a method of reducing adipocyte hypertrophy, comprising administering an effective amount of a composition comprising omaveloxolone, or a pharmaceutically acceptable salt thereof. In some embodiments, adipocyte hypertrophy may be measured by any suitable method known in the art. In some embodiments, adipocyte hypertrophy is reduced by about 5% to about 90%, compared to levelsAttorney Docket No: RRBS-OIO / OIWO 353016-2018 within the subject prior to administration. In some embodiments, adipocyte hypertrophy is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, compared to levels within the subject prior to administration.

[0100] In one aspect, the present disclosure provides a method of reducing plasma levels of triglycerides and / or free fatty acids, comprising administering an effective amount of a composition comprising omaveloxolone, or a pharmaceutically acceptable salt thereof. In some embodiments, plasma levels of triglycerides and / or free fatty acids may be measured by any suitable method known in the art. In some embodiments, plasma levels of triglycerides and / or free fatty acids are reduced by about 5% to about 90%, compared to levels within the subject prior to administration. In some embodiments, plasma levels of triglycerides and / or free fatty acids are reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, compared to levels within the subject prior to administration.

[0101] In one aspect, the present disclosure provides a method of increasing mitochondrial function, comprising administering an effective amount of a composition comprising omaveloxolone, or a pharmaceutically acceptable salt thereof. In some embodiments, mitochondrial function may be measured by any suitable method known in the art. In some embodiments, mitochondrial function is increased by about 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold, compared to levels in the subject prior to administration.

[0102] In one aspect, the present disclosure provides a method of increasing oxygen consumption rates, comprising administering an effective amount of a composition comprising omaveloxolone, or a pharmaceutically acceptable salt thereof. In some embodiments, oxygen consumption rates may be measured by any suitable method known in the art. In some embodiments, oxygen consumption is increased by about 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold,Attorney Docket No: RRBS-OIO / OIWO 353016-2018 about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold, compared to oxygen consumption in the subject prior to administration.

[0103] In one aspect, the present disclosure provides a method of increasing expression of thermogenic markers (e.g., UCP1 and / or PGC-la) in brown adipose tissue, comprising administering an effective amount of a composition comprising omaveloxolone, or a pharmaceutically acceptable salt thereof. In some embodiments, expression of thermogenic markers (e.g., UCP1 and / or PGC-la) in brown adipose tissue may be measured by any suitable method known in the art. In some embodiments, expression of thermogenic markers (e.g., UCP1 and / or PGC-la) in brown adipose tissue is increased by about 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold, compared to expression of thermogenic markers in brown adipose tissue in the subject prior to administration.

[0104] In one aspect, the present disclosure provides a method of reducing liver inflammation in a subject in need thereof, comprising administering an effective amount of a composition comprising omaveloxolone, or a pharmaceutically acceptable salt thereof. In some embodiments, liver inflammation may be measured by any suitable method known in the art. In some embodiments, liver inflammation may be measured by Kupffer cell activation. In some embodiments, liver inflammation may be measured by expression of pro-inflammatory cytokines (e.g., TNF-a, IL-6, and / or MCP-1). In some embodiments, liver inflammation is reduced by about 5% to about 90%, compared to liver inflammation within the subject prior to administration. In some embodiments, liver inflammation is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, compared to liver inflammation within the subject prior to administration.

[0105] In one aspect, the present disclosure provides a method of decreasing fibrosis markers in a subject in need thereof, comprising administering an effective amount of a composition comprising omaveloxolone, or a pharmaceutically acceptable salt thereof. In some embodiments, fibrosis markers may be measured by any suitable method known in the art. In some embodiments, fibrosis markers may be measured by quantifying collagen deposition. In1Attorney Docket No: RRBS-OIO / OIWO 353016-2018 some embodiments, fibrosis markers may be measured by expression of TGF-pi). In some embodiments, fibrosis markers are reduced by about 5% to about 90%, compared to fibrosis markers within the subject prior to administration. In some embodiments, fibrosis markers are reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, compared to fibrosis markers within the subject prior to administration.

[0106] In one aspect, the present disclosure provides a method of improving insulin sensitivity in a subject in need thereof, comprising administering an effective amount of a composition comprising omaveloxolone, or a pharmaceutically acceptable salt thereof. In some embodiments, insulin sensitivity may be measured by any suitable method known in the art. In some embodiments, insulin sensitivity may be measured by quantifying fasting insulin levels. In some embodiments, fasting insulin levels in the subject are increased by about 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold, compared to fasting insulin levels in the subject prior to administration. In some embodiments, insulin sensitivity may be measured HOMA-IR index. In some embodiments, HOMA-IR index for the subject is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, compared to HOMA-IR index for the subject prior to administration. In some embodiments, insulin sensitivity may be measured by AKT phosphorylation in insulin-responsive tissues. In some embodiments, AKT phosphorylation in insulin-responsive tissues of the subject are reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, compared AKT phosphorylation in insulin-responsive tissues within the subject prior to administration.

[0107] In one aspect, the present disclosure provides methods of treating a disease or condition characterized by reduced expression and / or function of ADIPOR2 in a subject in need thereof. In one aspect, the present disclosure provides methods of preventing a disease or condition characterized by reduced expression and / or function of ADIPOR2 in a subject in need thereof.Attorney Docket No: RRBS-OIO / OIWO 353016-2018 In some embodiments, the method comprises administering an effective amount of a composition that increases expression of ADIPOR2 at the transcript and / or protein level. In some embodiments, the composition comprises omaveloxolone or a pharmaceutically acceptable salt thereof.

[0108] In some embodiments, the subject has reduced ceramidase activity compared to a control subject. In some embodiments, the subject has increased ceramide levels compared to a control subject. In some embodiments, the subject has increased membrane rigidity, impaired insulin signaling, insulin resistance, and / or lipotoxicity, compared to a control subject. In some embodiments, the control subject is not known to have the disease or condition for which the subject is administered a composition comprising omaveloxolone or a pharmaceutically acceptable salt thereof. In some embodiments, the control subject is not known to have obesity. In some embodiments, the control subject is not known to have liver disease. In some embodiments, the control subject does not comprise a mutation in either allele of the ADIPOR2 gene.

[0109] In some embodiments, the method results in increased expression and / or activity of ADIPOR2 in the subject. In some embodiments, expression is increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%. In some embodiments, expression is increased within 1 month of beginning administration of omaveloxolone. In some embodiments, expression is increased within 2 months of beginning administration of omaveloxolone.

[0110] In some embodiments, the disease or condition associated with reduced expression and / or function of ADIPOR2 is obesity. In some embodiments, the disease or condition associated with reduced expression and / or function of ADIPOR2 is liver disease.[OHl] In some embodiments, the subject comprises one or more mutations in one allele of the ADIPOR2 gene. Alternatively, the subject comprises one or more mutations in both alleles of the ADIPOR2 gene. In some embodiments, the one or more mutations are any described herein. In some embodiments, the method results in increased expression and / or activity of ADIPOR2 in the subject. The increased expression may be of any amount described herein.

[0112] In some embodiments, the subject in need thereof is administered an effective amount of a composition comprising omaveloxolone, or a pharmaceutically acceptable salt thereof.Attorney Docket No: RRBS-OIO / OIWO 353016-2018

[0113] The dose and dosage regimen of the composition comprising omavel oxoIone, or a pharmaceutically acceptable salt thereof, may depend upon a variety of factors readily determined by a physician, such as the nature of the disease or condition, the characteristics of the subject, and the subject's history.

[0114] In some embodiments, the effective amount of the composition is about 0.5 mg / kg to about 20 mg / kg. In some embodiments, the effective amount of the composition is about 1 mg / kg to about 5 mg / kg. In some embodiments, the effective amount of the composition is about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, or about 10 mg / kg.

[0115] In some embodiments, the effective amount of omaveloxolone is about 1 mg to about 1000 mg per day. In some embodiments, the effective amount of omaveloxolone is about 2 mg to about 500 mg per day. In some embodiments, the effective amount of omaveloxolone is about 4 mg to about 200 mg per day. In some embodiments, the effective amount of omaveloxolone is about 5 mg to about 150 mg per day. In some embodiments, the effective amount of omaveloxolone is about 10 mg, 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, or about 200 mg per day. In some embodiments, the effective amount of omaveloxolone is 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, or about 150 mg per day. In some embodiments, the effective amount of the composition is greater than or equal to about 1 mg, greater than or equal to about 20 mg, greater than or equal to about 40 mg, greater than or equal to about 60 mg, greater than or equal to about 80 mg, or greater than or equal to about 100 mg. In certain embodiments, the effective amount is Img to 500 mg, 5mg to 300 mg, 10 mg to 250 mg, 20 mg to 200 mg, 50 mg to 150 mg, or 60 mg to 120 mg.

[0116] In some embodiments, the composition is administered orally. In some embodiments, the composition is administered parenterally, e.g., intravenously, rectally, or by injection. In some embodiments, the composition is administered locally, e.g., topically or intramuscularly. In some embodiments, the composition is administered to target tissues. The skilled artisan can determine an appropriate site and route of administration based on factors including, but not limited to, the disease or condition being treated.Attorney Docket No: RRBS-OIO / OIWO 353016-2018

[0117] In some embodiments, the composition is administered once or twice per day. The skilled artisan can determine an appropriate dosage regimen based on factors including, but not limited to, the nature of the disease or condition, the characteristics of the subject, and the subject's history.

[0118] In some embodiments, omaveloxolone is administered for at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 2 months, at least about 3 months, at least about 4 months, at least about 6 months, at least about 9 months, at least about 12 months, at least about 15 months, at least about 18 months, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years. The skilled artisan can determine an appropriate duration of treatment based on factors including, but not limited to, the nature of the disease or condition, the characteristics of the subject, and the subject's history.

[0119] The compositions described herein may be administered chronically (“chronic administration”). Chronic administration refers to administration of a compound or pharmaceutical composition thereof over an extended period of time, e.g., for example, over 3 months, 6 months, 1 year, 2 years, 3 years, 5 years, etc., or may be continued indefinitely, for example, for the rest of the subject’s life. In certain embodiments, the chronic administration is intended to provide a constant level of the compound in the blood, e.g., within the therapeutic window over the extended period of time.

[0120] In certain embodiments, the pharmaceutical compositions provided herein are administered to the patient as a solid dosage form. In certain embodiments, the solid dosage form is a capsule.

[0121] In some aspects, the present disclosure provides a composition for use in treating or preventing a disease or disorder disclosed herein. In some aspects, the present disclosure provides use of a composition of the present disclosure in the manufacture of a medicament for treating or preventing a disease or disorder disclosed herein.

[0122] In some embodiments, the method results in reduction or improvement of at least one symptom of the disease or condition. In some embodiments, the method results in prevention of at least one symptom of the disease or condition. In some embodiments, the method results in reduced membrane rigidity, increased insulin signaling, improved insulin sensitivity, and / or reduced lipotoxicity.Attorney Docket No: RRBS-OIO / OIWO 353016-2018

[0123] In some embodiments, the method results in reduced membrane rigidity within one or more cells within the subject, compared to membrane rigidity within cells of the subject prior to administration. In some embodiments, membrane rigidity may be measured by any suitable method known in the art. In some embodiments, membrane rigidity decreases by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, compared to membrane rigidity within cells of the subject prior to administration.

[0124] In some embodiments, the method results in increased insulin signaling within the subject, compared to insulin signaling within the subject prior to administration. In some embodiments, insulin signaling may be measured by any suitable method known in the art. In some embodiments, insulin signaling is increased by about 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold, compared to insulin signaling in the subject prior to administration.

[0125] In some embodiments, the method results in enhanced hepatic insulin sensitivity in the subject. In some embodiments, hepatic insulin sensitivity may be measured by glucose tolerance tests (GTT). In some embodiments, hepatic insulin sensitivity may be measured by insulin signaling assays. In some embodiments, hepatic insulin sensitivity in the subject is increased by about 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold, compared to hepatic insulin sensitivity in the subject prior to administration.

[0126] In some embodiments, the method results in reduced lipotoxicity within one or more cells within the subject, compared to lipotoxicity within cells of the subject prior to administration. In some embodiments, lipotoxicity may be measured by any suitable method known in the art. In some embodiments, membrane rigidity decreases by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at leastAttorney Docket No: RRBS-OIO / OIWO 353016-2018 80%, at least 85%, or at least 90%, compared to lipotoxicity within the cells of the subject prior to administration.

[0127] In one aspect, the present disclosure provides a method of decreasing ceramide levels, reducing lipogenesis, and / or improved glucose homeostasis within the liver of a subject in need thereof, comprising administering an effective amount of a composition comprising omaveloxolone, or a pharmaceutically acceptable salt thereof.

[0128] In some embodiments, ceramide levels within the liver of the subject may be measured by any suitable method known in the art. In some embodiments, ceramide levels within the liver of the subject are reduced by about 5% to about 90%, compared to ceramide levels within the liver of the subject prior to administration. In some embodiments, ceramide levels within the liver of the subject are reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, compared to ceramide levels within the liver of the subject prior to administration.

[0129] In some embodiments, lipogenesis may be measured by any suitable method known in the art. In some embodiments, reduced lipogenesis within the liver of the subject may be measured by quantifying suppression of SREBP-lc and / or decreased expression of one or more lipogenic genes (e.g., FASN, SCD1, and / or ACC1). In some embodiments, transcript and / or protein levels of SREBP-lc, FASN, SCD1, and / or ACC1 within the liver of the subject are reduced by about 5% to about 90%, compared to levels within the liver of the subject prior to administration. In some embodiments, transcript and / or protein levels of SREBP-lc, FASN, SCD1, and / or ACC1 within the liver of the subject are reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, compared to levels within the liver of the subject prior to administration.

[0130] In some embodiments, glucose homeostasis may be measured by any suitable method known in the art. In some embodiments, glucose homeostasis may be measured by increased AMPK activation and / or reduced expression of gluconeogenesis genes (e.g., PEPCK, G6PC expression). In some embodiments, AMPK activation within the liver of the subject is increased by about 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, about 2-fold, about 2.5-fold, about 3-Attorney Docket No: RRBS-OIO / OIWO 353016-2018 fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold, compared to levels within the liver of the subject prior to administration. In some embodiments, transcript and / or protein levels of PEPCK and / or G6PC within the liver of the subject are reduced by about 5% to about 90%, compared to levels within the liver of the subject prior to administration. In some embodiments, transcript and / or protein levels of PEPCK and / or G6PC within the liver of the subject are reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, compared to levels within the liver of the subject prior to administration.

[0131] In some embodiments, the method results in reduced adipocyte hypertrophy in the subject. In some embodiments, adipocyte hypertrophy may be measured by any suitable method known in the art. In some embodiments, adipocyte hypertrophy is reduced by about 5% to about 90%, compared to levels within the subject prior to administration. In some embodiments, adipocyte hypertrophy is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, compared to levels within the subject prior to administration.

[0132] In some embodiments, the method results in reduced plasma levels of triglycerides and / or free fatty acids in the subject. In some embodiments, plasma levels of triglycerides and / or free fatty acids may be measured by any suitable method known in the art. In some embodiments, reducing plasma levels of triglycerides and / or free fatty acids are reduced by about 5% to about 90%, compared to levels within the subject prior to administration. In some embodiments, plasma levels of triglycerides and / or free fatty acids are reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, compared to levels within the subject prior to administration.

[0133] In some embodiments, the method results in increased mitochondrial function in the subject. In some embodiments, mitochondrial function may be measured by any suitable method known in the art. In some embodiments, mitochondrial function is increased by about 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, aboutAttorney Docket No: RRBS-OIO / OIWO 353016-2018 1.7-fold, about 1.8-fold, about 1.9-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold, compared to levels in the subject prior to administration.

[0134] In some embodiments, the method results in increased oxygen consumption rates in the subject. In some embodiments, oxygen consumption rates may be measured by any suitable method known in the art. In some embodiments, oxygen consumption is increased by about 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold, compared to oxygen consumption in the subject prior to administration.

[0135] I In some embodiments, the method results in increased expression of thermogenic markers (e.g., UCP1 and / or PGC-la) in brown adipose tissue of the subject. In some embodiments, expression of thermogenic markers (e.g., UCP1 and / or PGC-la) in brown adipose tissue may be measured by any suitable method known in the art. In some embodiments, expression of thermogenic markers (e.g., UCP1 and / or PGC-la) in brown adipose tissue is increased by about 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold, compared to expression of thermogenic markers in brown adipose tissue in the subject prior to administration.

[0136] In some embodiments, the method results in reduced liver inflammation in the subject. In some embodiments, liver inflammation may be measured by any suitable method known in the art. In some embodiments, liver inflammation may be measured by Kupffer cell activation. In some embodiments, liver inflammation may be measured by expression of pro-inflammatory cytokines (e.g., TNF-a, IL-6, and / or MCP-1). In some embodiments, liver inflammation is reduced by about 5% to about 90%, compared to liver inflammation within the subject prior to administration. In some embodiments, liver inflammation is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, compared to liver inflammation within the subject prior to administration.Attorney Docket No: RRBS-OIO / OIWO 353016-2018

[0137] In some embodiments, the method results in decreased fibrosis markers in the subject. In some embodiments, fibrosis markers may be measured by any suitable method known in the art. In some embodiments, fibrosis markers may be measured by quantifying collagen deposition. In some embodiments, fibrosis markers may be measured by expression of TGF-pi. In some embodiments, fibrosis markers are reduced by about 5% to about 90%, compared to fibrosis markers within the subject prior to administration. In some embodiments, fibrosis markers are reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, compared to fibrosis markers within the subject prior to administration.

[0138] In some embodiments, the method results in improved insulin sensitivity in the subject. In some embodiments, insulin sensitivity may be measured by any suitable method known in the art. In some embodiments, insulin sensitivity may be measured by quantifying fasting insulin levels. In some embodiments, fasting insulin levels in the subject are increased by about 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold, compared to fasting insulin levels in the subject prior to administration. In some embodiments, insulin sensitivity may be measured HOMA-IR index. In some embodiments, HOMA-IR index for the subject is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, compared to HOMA-IR index for the subject prior to administration. In some embodiments, insulin sensitivity may be measured by AKT phosphorylation in insulin-responsive tissues. In some embodiments, AKT phosphorylation in insulin-responsive tissues of the subject are reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, compared AKT phosphorylation in insulin-responsive tissues within the subject prior to administration.

[0139] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the human is an adult human.Attorney Docket No: RRBS-OIO / OIWO 353016-2018

[0140] In some embodiments, the composition additionally comprises a pharmaceutically acceptable carrier. Non-limiting examples of pharmaceutically acceptable carriers include a pharmaceutically acceptable excipient, binder, and / or diluent.

[0141] In some aspects, the present disclosure provides a composition comprising omaveloxolone for use in a method of increasing the level of adiponectin receptor 2 (ADIPOR2 transcript and / or protein in a cell.

[0142] In some aspects, the present disclosure provides a composition comprising omaveloxolone for use in decreasing ceramide levels, reducing lipogenesis, improving glucose homeostasis, and / or increasing insulin sensitivity in a cell.

[0143] In some aspects, the present disclosure provides a composition comprising omaveloxolone for use in treating or preventing a disease or disorder disclosed herein. In some embodiments, the disease or condition is obesity. In some embodiments, the disease or condition is liver disease.

[0144] In some aspects, the present disclosure provides use of a composition comprising omaveloxolone in the manufacture of a medicament for increasing the level of adiponectin receptor 2 (ADIPO R2> transcript and / or protein in a cell.

[0145] In some aspects, the present disclosure provides use of a composition comprising omaveloxolone in the manufacture of a medicament for decreasing ceramide levels, reducing lipogenesis, improving glucose homeostasis, and / or increasing insulin sensitivity in a cell.

[0146] In some aspects, the present disclosure provides use of a composition comprising omaveloxolone in the manufacture of a medicament for treating or preventing a disease or disorder disclosed herein. In some embodiments, the disease or condition is obesity. In some embodiments, the disease or condition is liver disease.Methods of Identifying a Subject in Need of Treatment

[0147] In one aspect, the present disclosure provides a method of identifying a subject in need of treatment with an effective amount of a composition comprising omaveloxolone. In some embodiments, the method comprises analyzing a genome sequence of the subject; and determining that the subject comprises a mutation in the ADIPOR2 gene. The genome sequence may be determined using any method disclosed herein. The genome sequence of the subject may comprise a sequence of the ADIPOR2 gene, or a portion thereof.Attorney Docket No: RRBS-OIO / OIWO 353016-2018

[0148] In some embodiments, the subject comprises one or more mutations including the ADIPOR2 gene. The one or more mutations including the ADIPOR2 gene may comprise any mutations described herein. For example, the subject may comprise one or more mutations in one or both alleles of the ADIPOR2 gene.

[0149] In some embodiments, the method further comprises administering to the subject an effective amount of a composition comprising omaveloxolone, or a pharmaceutically acceptable salt thereof. The effective amount may be any effective amount described herein.Pharmaceutical Compositions

[0150] In various embodiments, a compound of the present invention (e.g., omaveloxolone) or a pharmaceutically acceptable salt thereof is administered to the subject in a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients, carriers, diluents, or reagents. In some embodiments, the compound is combined with one or more pharmaceutically acceptable carriers, diluents, excipients, and reagents useful in preparing a formulation that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for mammalian, e.g., human or primate, use.

[0151] Examples of carriers, diluents and excipients include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Solutions or suspensions used for the formulations can include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates; detergents such as Tween 20 to prevent aggregation; and compounds for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. In particular embodiments, the pharmaceutical compositions are sterile.

[0152] Pharmaceutical compositions may further include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS). In some embodiments, the composition is sterile and may be fluid such that it can be drawn into a syringe or delivered to a subject from a syringe. The carrier can be, e.g., a solvent or dispersion medium containing,Attorney Docket No: RRBS-OIO / OIWO 353016-2018 for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In certain embodiments, the pharmaceutical composition is stable under the conditions of manufacture and storage and is preserved against the contaminating action of microorganisms such as bacteria and fungi. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the internal compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0153] In some embodiments, the pharmaceutical composition further comprises an additional therapeutic agent disclosed herein.

[0154] The pharmaceutical compositions described herein can be administered by a variety of routes including, but not limited to, oral (enteral) administration, parenteral (by injection) administration, rectal administration, transdermal administration, intradermal administration, intrathecal administration, subcutaneous (SC) administration, intravenous (IV) administration, intramuscular (IM) administration, and intranasal administration. In certain embodiments, the pharmaceutical compositions described herein are administered orally.

[0155] The pharmaceutical compositions described herein may be presented in unit dosage forms to facilitate accurate dosing. The term “unit dosage forms” refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient. Typical unit dosage forms include prefilled, premeasured ampules or syringes of the liquid compositions or pills, tablets, capsules or the like in the case of solid compositions.

[0156] In certain embodiments, the pharmaceutical compositions provided herein are administered to the subject as a liquid dosage form. In some embodiments, the liquid dosage form is a syrup, suspension, solution, emulsion, elixir, or drops. In certain embodiments, the pharmaceutical compositions provided herein are administered to the subject as a solid dosageAttorney Docket No: RRBS-OIO / OIWO 353016-2018 form. In certain embodiments, the solid dosage form is a capsule, a tablet, a lozenge, a powder, or granules.

[0157] In some aspects, the present disclosure provides a composition for use in treating or preventing a disease or disorder disclosed herein. In some aspects, the present disclosure provides use of a composition of the present disclosure in the manufacture of a medicament for treating or preventing a disease or disorder disclosed herein.

[0158] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and / or perform such modification with ordinary experimentation. General considerations in the formulation and / or manufacture of pharmaceutical compositions can be found, for example, in Remington: The Science and Practice of Pharmacy 21sted., Lippincott Williams & Wilkins, 2005.

[0159] Examples of carriers, diluents and excipients include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Solutions or suspensions used for the formulations can include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates; detergents such as Tween 20 to prevent aggregation; and compounds for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. In particular embodiments, the pharmaceutical compositions are sterile.

[0160] Pharmaceutical compositions may further include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS). In some embodiments, the composition is sterile and may be fluid such that it can be drawn into a syringe or delivered toAttorney Docket No: RRBS-OIO / OIWO 353016-2018 a subject from a syringe. The carrier can be, e.g., a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In certain embodiments, the pharmaceutical composition is stable under the conditions of manufacture and storage and is preserved against the contaminating action of microorganisms such as bacteria and fungi. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the internal compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.EXAMPLESExample 1. High-Throughput Screening of Small Molecule Modulators of ADIPOR2

[0161] Small molecules were screened using human neurons derived from the induced pluripotent stem cells (iPSCs) of a presumably healthy donor. iPSCs were directly differentiated into motor neurons as described in Wang et al. Stem Cell Reports. 2017 Oct 10; 9(4), 1221-1233. Briefly, iPl IN healthy control iPSCs were directly differentiated into motor neurons by overexpression of NGN2 via doxycycline treatment on Days 1-3 (neuron induction). On day 4, the cells were replated into 384-well plates and cultured in neuron maturation media until day 17.

[0162] A library of 389 FDA-approved and 45 investigational small molecules was screened in the iPSC-derived human motor neurons. Compounds were added at a 10-point half-logarithmic dilution series (lOpM - 0.0005pM) in quadruplicate on day 17 of neuronal differentiation, and treatment was carried out for 24 hours.

[0163] Gene expression was measured by 3’RNA-seq in cells from each treatment condition. Following data analysis, the candidate compound omaveloxolone was identified for further validation were identified. Omaveloxolone increased the expression level of the adiponectin receptor 2 (ADIPOR2) gene relative to vehicle-treated cells in a dose-dependent manner. ThisAttorney Docket No: RRBS-OIO / OIWO 353016-2018 was determined using a non-linear least squares fit to a 4-parameter Hill Equation for the binding of a ligand to a macromolecule (FIG. 1).Example 2. Validation of ADIPOR2 Expression by Immunocytochemistry

[0164] Omaveloxolone was administered to commercially available human HEPG2 liver cells at 125 nM, 250 nM, and 500 nM daily for three days in 2-3 replicate wells per concentration. On day 4, cells were fixed in 4% paraformaldehyde, permeabilized, and stained using an ADIPOR2 polyclonal antibody with a species-specific AlexaFluor-conjugated immunoglobulin secondary antibody. ADIPOR2 protein levels were measured using fluorescence microscopy and quantified as relative ADIPOR2 positive area per live cells (FIG.2A) and relative ADIPOR2 intensity per live cells (FIG. 2B). Omaveloxolone treatment significantly increased ADIPOR2 expression in protein fluorescent intensity in HepG2 liver cells at all tested concentrations: 125 nM (average FC=1.47), 250 nM (average FC=1.39) and 500 nM (average FC=1.82). All fold changes were relative to vehicle control (DMSO). Oneway ANOVA with Dunnett’s multiple comparisons test was performed, n=3. Statistically significance is represented as: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.Example 3. Validation of ADIPOR2 Expression by Western Blot

[0165] Omaveloxolone was administered to commercially available human HEPG2 liver cells at 125 nM, 250 nM, and 500 nM daily for three days in 2-3 replicate wells per concentration. On day 4, cells were lysed and protein extracted. ADIPOR2 protein levels were quantified by western blot using a ADIPOR2 monoclonal antibody with a species-specific horseradish peroxidase-conjugated immunoglobulin secondary antibody. Proteins were visualized by chemiluminescence (FIG. 3A) and quantified by densitometry analysis using the iBright FL1500 Imaging system, normalized to GAPDH loading control (FIG. 3B). Omaveloxolone treatment significantly increased ADIPOR2 expression in human HEPG2 liver cells at 500 nM, compared to vehicle control (DMSO). One-way ANOVA with Dunnetf s multiple comparisons test was performed, n=3. Statistically significance is represented as *p < 0.05.

Claims

Attorney Docket No: RRBS-OIO / OIWO 353016-2018 CLAIMS1. A method of increasing the level of adiponectin receptor 2 (ADIP0R2) transcript and / or protein in a cell, wherein the method comprises contacting a cell with an effective amount of a composition comprising omaveloxolone, or a pharmaceutically acceptable salt thereof.

2. A method of decreasing ceramide levels, reducing lipogenesis, improving glucose homeostasis, and / or increasing insulin sensitivity in a cell, wherein the method comprises contacting a cell with an effective amount of a composition comprising omaveloxolone, or a pharmaceutically acceptable salt thereof.

3. The method of claim 1 or claim 2, wherein the cell has reduced expression or activity of ADIP0R2.

4. The method of any one of claims 1-3, wherein the cell has reduced ceramidase activity.

5. The method of any one of claims 1-4, wherein the cell has increased ceramide levels compared to a control cell.

6. The method of any one of claims 1-5, wherein the cell has increased membrane rigidity, impaired insulin signaling, insulin resistance, and / or lipotoxicity.

7. The method of any one of claims 1-6, wherein the cell comprises one or more mutations in one allele of the ADIP0R2 gene.

8. The method of claim 7, wherein the one or more mutations comprise a missense mutation, a frameshift mutation, a nonsense mutation, a mutation that introduces a splice site, a mutation that affects an existing splice site, or a deletion.

9. The method of claim 7 or claim 8, wherein the one or more mutations result in a mutant gene encoding a protein with reduced function.

10. The method of any one of claims 1-9, wherein the cell has at least one ADIPOR2 allele encoding a protein with residual function.

11. The method of any one of claims 1-10, wherein the level of ADIPOR2 transcript and / or protein in the cell is increased by about 1.1 to about 10-fold, compared to a control cell.Attorney Docket No: RRBS-OIO / OIWO 353016-2018 12. The method of any one of claims 1-11, wherein the level of ADIPOR2 transcript and / or protein in the cell is increased to a level comparable to that of a control cell that does not comprise reduced expression or activity of ADIPOR2.

13. The method of any one of claims 1-12, wherein the cell is a liver cell.

14. The method of any one of claims 1-13, wherein the contact occurs in vivo or ex vivo.

15. A method of decreasing ceramide levels, reducing lipogenesis, and / or improving glucose homeostasis within the liver of a subject in need thereof, comprising administering an effective amount of a composition comprising omaveloxolone, or a pharmaceutically acceptable salt thereof.

16. A method of reducing adipocyte hypertrophy, reducing plasma levels of triglycerides and free fatty acids, increasing mitochondrial function, and / or increasing expression of thermogenic markers in brown adipose tissue in a subject in need thereof, comprising administering an effective amount of a composition comprising omaveloxolone, or a pharmaceutically acceptable salt thereof.

17. A method of reducing liver inflammation, decreasing fibrosis markers, and / or improving insulin sensitivity in a subject in need thereof, comprising administering an effective amount of a composition comprising omaveloxolone, or a pharmaceutically acceptable salt thereof.

18. A method of treating or preventing a disease or condition characterized by reduced expression and / or function of ADIPOR2 in a subject in need thereof, comprising administering an effective amount of a composition comprising omaveloxolone, or a pharmaceutically acceptable salt thereof.

19. The method of claim 18, wherein the subject has reduced ceramidase activity.

20. The method of claim 18 or claim 19, wherein the subject has increased ceramide levels compared to a control subject.

21. The method of any one of claims 18-20, wherein the subject has increased membrane rigidity, impaired insulin signaling, insulin resistance, and / or lipotoxicityAttorney Docket No: RRBS-OIO / OIWO 353016-2018 22. The method of any one of claims 18-21, wherein the subject comprises one or more mutations in one allele of the ADIPOR2 gene.

23. The method of claim 22, wherein the one or more mutations comprise a missense mutation, a frameshift mutation, a nonsense mutation, a mutation that introduces a splice site, a mutation that affects an existing splice site, or a deletion.

24. The method of claim 22 or claim 23, wherein the one or more mutations result in a mutant gene encoding a protein with reduced function.

25. The method of any one of claims 22-24, wherein the subject is determined to have the one or more mutations by analysis of the subject’ &ADIPOR2 genome sequence.

26. The method of claim 25, wherein the analysis of the subject’s ADIPOR2 genome sequence is performed using a method selected from the group consisting of: targeted variant analysis, deletion analysis, mutations canning of select exons, mutation scanning of the entire ADIPOR2 coding region, sequence analysis of select exons, sequence analysis of the entire ADIPOR2 coding region, or ADIPOR2 RNA analysis.

27. The method of any one of claims 22-26, wherein the subject has at least one ADIPOR2 allele encoding a protein with residual function.

28. The method of any one of claims 18-27, wherein the method results in increased expression and / or activity of ADIPOR2 in the subject.

29. The method of claim 28, wherein expression is increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%, compared to levels in the subject prior to administration.

30. The method of claim 28 or claim 29, wherein expression is increased within 1 month of beginning administration of omavel oxoIone.

31. The method of any one of claims 18-30, wherein the method results in reduced ceramide levels in the subject.Attorney Docket No: RRBS-OIO / OIWO 353016-2018 32. The method of any one of claims 18-31, wherein the method results in reduced membrane rigidity, increased insulin signaling, improved insulin sensitivity, and / or reduced lipotoxicity.

33. The method of any one of claims 18-32, wherein the method results in decreased levels of ceramide, reduced lipogenesis, and / or improved glucose homeostasis within the liver of the subject.

34. The method of any one of claims 18-33, wherein the method results in reduced adipocyte hypertrophy, decreased plasma levels of triglycerides and free fatty acids, increased mitochondrial function, and / or increased expression of thermogenic markers in brown adipose tissue of the subject.

35. The method of any one of claims 18-34, wherein the method results in reduced liver inflammation, decreased levels of fibrosis markers, and / or improved insulin sensitivity in the subject.

36. The method of any one of claims 18-35, wherein the disease or condition is obesity.

37. The method of any one of claims 18-35, wherein the disease or condition is liver disease.

38. The method of any one of claims 18-37, wherein the effective amount of the composition is about 1 mg to about 1000 mg per day.

39. The method of claim 38, wherein the effective amount of the composition is about 5 mg to about 150 mg per day.

40. The method of claim 39, wherein the effective amount of the composition is about 10 mg, about 20 mg, about 30 mg, about 40 mg, 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, or about 150 mg per day.

41. The method of any one of claims 18-40, wherein the composition is administered orally or parenterally.Attorney Docket No: RRBS-OIO / OIWO 353016-2018 42. The method of any one of claims 18-41, wherein the composition is administered once or twice per day.

43. The method of any one of claims 18-42, wherein the method results in reduction or improvement of at least one symptom of the disease or condition.

44. The method of claim 43, wherein the method results in prevention of at least one symptom of the disease or condition.

45. The method of any one of claims 18-44, wherein the subject is a mammal.

46. The method of claim 45, wherein the subject is a human.

47. The method of any one of claims 18-46, wherein the composition comprises omaveloxolone.

48. The method of claim 47, wherein the composition additionally comprises a pharmaceutically acceptable carrier.

49. A method of identifying a subject in need of treatment with an effective amount of a composition comprising omaveloxolone, comprising:a) analyzing a genome sequence of the subject; andb) determining that the subject comprises a mutation of the ADIPOR2 gene.

50. The method of claim 49, wherein the genome sequence comprises a sequence of the ADIPOR2 gene, or a portion thereof.

51. The method of claim 49 or 50, wherein the subject comprises one or more mutations in one or both alleles of the ADIPOR2 gene.

52. The method of any one of claims 49-51, wherein the method further comprises administering to the subject an effective amount of a composition comprising omaveloxolone, or a pharmaceutically acceptable salt thereof.

53. A composition comprising omaveloxolone for use in a method of increasing the level of adiponectin receptor 2 (ADIPOR2) transcript and / or protein in a cell.Attorney Docket No: RRBS-OIO / OIWO 353016-2018 54 A composition comprising omaveloxolone for use in decreasing ceramide levels, reducing lipogenesis, improving glucose homeostasis, and / or increasing insulin sensitivity in a cell.

55. A composition comprising omaveloxolone for use in treating or preventing a disease or disorder.

56. Use of a composition comprising omaveloxolone in the manufacture of a medicament for increasing the level of adiponectin receptor 2 (ADIPOR2) transcript and / or protein in a cell.

57. Use of a composition comprising omaveloxolone in the manufacture of a medicament for decreasing ceramide levels, reducing lipogenesis, improving glucose homeostasis, and / or increasing insulin sensitivity in a cell.

58. Use of a composition comprising omaveloxolone in the manufacture of a medicament for treating or preventing a disease or disorder.

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