WO2019161211A2

WO2019161211A2 - Compositions and methods for the reduction or prevention of hepatic steatosis and nash

Info

Publication number: WO2019161211A2
Application number: PCT/US2019/018230
Authority: WO
Inventors: Michael ZEMEL; Antje BRUCKBAUER; Jheelam BANERJEE
Original assignee: Nusirt Sciences, Inc.
Priority date: 2018-02-15
Filing date: 2019-02-15
Publication date: 2019-08-22
Also published as: WO2019161211A3

Abstract

Methods useful for reducing or preventing hepatic steatosis and NASH are provided herein. Such methods may comprise administering to a subject in need thereof an FXR agonist in combination with an amount of a branched amino acid in free amino acid form, such as leucine, or a metabolite thereof. Also provided herein are compositions and kits for practicing any of the methods described herein.

Description

COMPOSITIONS AND METHODS FOR THE REDUCTION OR PREVENTION OF

HEPATIC STEATOSIS AND NASH

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 62/631,164, filed on February 15, 2018, which is incorporated herein by reference.

INCORPORATION BY REFERENCE

[0002] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. In case of any inconsistency between the incorporated by reference publications and the instant specification, the instant specification will control.

BACKGROUND OF THE INVENTION

[0003] Hepatic steatosis, also sometimes referred to as fatty liver disease, is a condition generally characterized by an abnormal retention of lipids in cells of the liver. Hepatic steatosis affects millions of people worldwide. For example, the prevalence of fatty liver disease has been estimated to range from 10-24% in various countries around the globe. Fatty liver disease can have various causes. For example, non-alcoholic fatty liver disease (NAFLD) generally refers to a spectrum of hepatic lipid disorders characterized by hepatic steatosis with no known secondary cause. NAFLD can be subcategorized into (a) nonalcoholic fatty liver (NAFL), defined as the presence of steatosis in the absence of histological evidence of hepatocellular injury, and (b) nonalcoholic steatohepatitis (NASH), hepatic steatosis accompanied by hepatocyte injury and inflammation; NASH may occur with or without fibrosis, but may progress to fibrosis and cirrhosis. NAFLD is generally associated with energy metabolism pathologies, including obesity, dyslipidemia, diabetes and metabolic syndrome. The prevalence of NAFLD is high. Prevalence in the general population is estimated at 20%, with prevalence of NASH estimated to be 3-5%. There is an estimated -70% prevalence of NAFLD among patients with obesity or diabetes, and an estimated prevalence of -50% prevalence of NAFLD among patients with dyslipidemias. However, there are presently no approved pharmaceuticals for the treatment of NAFLD/NASH.

[0004] Sirtuins are highly conserved protein deacetylases and/or ADP-ribosyltransferases that have been shown to extend lifespan in lower model organisms, such as yeast, C. elegans , and drosophila. In mammals, sirtuins have been shown to act as metabolic sensors, responding to environmental signals to coordinate the activity of genes that regulate multiple energy homeostasis pathways. For example, studies have shown that sirtuin activation mimics the effects of caloric restriction, an intervention demonstrated to significantly extend lifespan, and activates genes that improve glucose homeostasis and the conversion of fat to energy by fatty acid oxidation.

[0005] The sirtuin pathway may be defined to include any pathway incorporating or converging upon pathways mediated by phosphodiesterases (PDEs). PDEs are enzymes that interact with cyclic adenosine monophosphates (cAMPs) and cyclic guanosine monophosphates (cGMPs). The PDE family of enzymes comprises multiple subclasses, including PDE 1-11 in humans. Inhibitors of these phosphodiesterases can prevent the inactivation of cAMPs and cGMPs, and can have a variety of different physiological effects. The PDE inhibitors can be selective, by preferentially inhibiting one PDE subclass as compared to another subclass, or non- selective, which have a substantially lower degree of selectivity for individual PDE subclasses. Sildenafil is an example of a selective PDE inhibitor that has shown selective inhibition of PDE 5. Sildenafil is a pharmaceutically active agent that has been used to treat pulmonary

hypertension, erectile dysfunction, and altitude sickness.

[0006] The famesoid receptor FXR is a nuclear receptor highly expressed in liver and intestine regulating bile acids synthesis (suppressing CYP7A1) as well as essential bile acid transporters. In addition, FXR is involved in glucose (downregulation of hepatic PEPCK and G6PC) and lipid metabolism (downregulation of SREB-lc). Natural ligands of FXR are bile acids with chenodeoxycholic acid (CDCA) as the most potent one. Obeticholic acid (OCA), a 6a-ethyl derivative of CDCA, is a selective FXR agonist with about lOO-fold greater potency than CDCA. There are several synthetic agonists (GW4064, WAY-362450 and PX-102), which have been used in animal experiments. Studies have suggested that FXR may play an important role in the pathogenesis of alcoholic liver disease and that FXR activation could be of therapeutic benefit.

[0007] Currently, there is no effective medical therapy for NAFLD/NASH. Accordingly, there is a current need for new therapeutics for treating NAFLD/NASH and associated diseases that provide long term efficacy, and a decreased risk for side effects. The present invention addresses this deficiency with compositions, methods, and kits for treating NAFLD/NASH and associated diseases. SUMMARY OF THE INVENTION

[0008] The present invention generally relates to regulation of fat accumulation,

inflammation and fibrosis in cells and/or tissue. In some embodiments, the present invention provides for compositions, methods, and kits for reducing, treating, or preventing hepatic steatosis or NASH in a subject in need thereof. The present invention generally relates to regulation of fat accumulation, inflammation and fibrosis in cells and/or tissue. In some embodiments, the present invention provides for compositions, methods, and kits for reducing, treating, or preventing hepatic steatosis or NASH in a subject in need thereof. The components of the compositions can have a synergistic effect on reducing, treating, or preventing hepatic steatosis or NASH in a subject in need thereof by directly or indirectly targeting the sirtuin and FXR pathways.

[0009] In some aspects, the present invention provides a composition, comprising: (i) leucine, or a metabolite or pharmaceutically acceptable salt thereof; and (ii) at least one farnesoid X receptor (FXR) agonist, or a pharmaceutically acceptable salt thereof. In some embodiments, the FXR agonist is obeticholic acid (OCA), chenodeoxy cholic acid (CDCA), cholic acid (CA), PX- 104, PX-102, GW4064, WAY-362450 or a pharmaceutically acceptable salt thereof. In an exemplary embodiment, the FXR agonist is obeticholic acid (OCA), or a pharmaceutically acceptable salt thereof.

[0010] In some aspects, the present invention provides a composition, comprising: (i) leucine, or a metabolite or pharmaceutically acceptable salt thereof; and (ii) a compound represented by Formula I:

, or a pharmaceutically acceptable salt thereof, wherein:

R¹, R², R³, and R⁴ are independently selected from hydrogen, halogen, -OR¹⁰, -SR¹⁰, - N(R¹⁰)₂, -S(0)R¹⁰, -S(0)₂R¹⁰, -C(0)R¹⁰, -C(0)OR¹⁰, -OC(0)R¹⁰, -N0₂, =0, =S, =N(R¹⁰), -CN, optionally substituted C _MO alkyl, optionally substituted C_2.l0 alkenyl, and optionally substituted C_2-io alkynyl;

R⁵ is selected from -OR¹⁰, -

N(R¹⁰)₂, -C(O)N(R¹⁰)₂, -C(O)N(R¹⁰)₂C(O)O(R¹⁰), -C(O)N(R¹⁰)₂S(O)₃(R¹⁰), -

C(0)R¹⁰, -C(0)OR¹⁰, -OC(0)R¹⁰, -OS(0)₃(R¹⁰), -SR¹⁰, -S(0)R¹⁰, and -S(0)₂R¹⁰; CMO alkyl, C_2- io alkenyl, C₂-io alkynyl, each of which is independently optionally substituted at each occurrence with one or more substituents selected from halogen, -OR¹⁰, -SR¹⁰, -C(O)N(R¹⁰)₂, - N(R¹⁰)C(O)R¹⁰, -N(R¹⁰)C(O)N(R¹⁰)₂, -N(R¹⁰)₂, -C(0)R¹⁰, -C(0)OR¹⁰, -OC(0)R¹⁰, -N0₂, =0,

=S, =N(R¹⁰), -CN, C_3-l2 carbocycle, and 3- to l2-membered heterocycle; and C_3.l2 carbocycle, and 3- to l2-membered heterocycle, wherein each C_3-i2 carbocycle, and 3- to l2-membered heterocycle in R⁴ is independently optionally substituted with one or more substituents selected from halogen, -OR¹⁰, -SR¹⁰, -C(O)N(R¹⁰)₂, -N(R¹⁰)C(O)R¹⁰, -N(R¹⁰)C(O)N(R¹⁰)₂, -N(R¹⁰)₂, - C(0)R¹⁰, -C(0)OR¹⁰, -OC(0)R¹⁰, -N0₂, =0, =S, =N(R¹⁰), -CN, C^ alkyl, C_2-6 alkenyl, and C_2-6 alkynyl; and

R¹⁰ is independently selected at each occurrence from the group consisting of hydrogen, optionally substituted Cn₀ alkyl, optionally substituted C_2-l0 alkenyl, optionally substituted C₂- io alkynyl, optionally substituted C_3-l2 carbocycle, and optionally substituted 3- to l2-membered heterocycle.

[0011] In some embodiments, R¹ is an optionally substituted C_l-l0 alkyl. R¹ can be an optionally substituted Ci_-6 alkyl. R¹ can be an optionally substituted C_2-3 alkyl.

[0012] In some embodiments, R³ is selected from the group consisting of hydrogen, -OR¹⁰, and -N(R¹⁰)₂. R³ can be -OR¹⁰. For example, R³ can be -OH.

[0013] In some embodiments, R² and R⁴ are independently selected from hydrogen, -OR¹⁰, and optionally substituted C_l-l0 alkyl. In certain embodiments, R² is hydrogen. In certain embodiments, R⁴ is hydrogen.

[0014] In some embodiments, R⁵ is selected from the group consisting of -OR¹⁰, - N(R¹⁰)₂, -C(O)N(R¹⁰)₂, -C(O)N(R¹⁰)₂C(O)O(R¹⁰), -C(O)N(R¹⁰)₂S(O)₃(R¹⁰), - C(0)R¹⁰, -C(0)OR¹⁰, -OC(0)R¹⁰, -OS(0)₃(R¹⁰), -SR¹⁰, -S(0)R¹⁰, and -S(0)₂R¹⁰. In certain embodiments, R⁵ is -C(0)OR¹⁰. For example, R⁵ can be -C(0)OH.

[0015] In some embodiments, the compound is:

, or a pharmaceutically acceptable salt thereof.

[0016] For any of the compositions disclosed herein, in some embodiments, the metabolite is b -hydroxy b -methylbutyrate (HMB) or keto-isocaproic acid (KIC). In certain embodiments, the metabolite is HMB. [0017] For any of the compositions disclosed herein, in some embodiments, the composition comprises at least 500 mg of leucine. The composition can comprise at least 1000 mg of leucine. In certain embodiments, the composition comprises about 50-1000 mg of leucine. The composition can comprise about 500-700 mg of leucine. In some embodiments, the composition comprises at least 200 mg of the metabolite.

[0018] For any of the compositions disclosed herein, in some embodiments, the composition comprises a subtherapeutic amount of component (i). In some embodiments, the composition comprises about 0.1-1500 mg of component (ii). The composition can comprise about 0.1-150 mg of component (ii). The composition can comprise about 0.1-25 mg of component (ii). In some embodiments, the composition comprises a subtherapeutic amount of component (ii).

[0019] For any of the compositions disclosed herein, in some embodiments, the amount of leucine is about 90-99.5 wt % of the total weight of components (i) and (ii). In some

embodiments, the amount of the metabolite comprises about 85-99.5 wt % of the total weight of components (i) and (ii). In some embodiments, the amount of component (ii) is about 0.5-15 wt % of the total weight of components (i) and (ii).

[0020] For any of the compositions disclosed herein, in some embodiments, the composition is formulated as a tablet, capsule, pill or solution. The composition can be formulated as a unit dose.

[0021] For any of the compositions disclosed herein, in some embodiments, the composition is formulated as a unit dose comprising about 250-1250 mg of component (i) and about 0.1-100 mg of component (ii). The composition can be formulated as a unit dose comprising about 500- 1250 mg of component (i) and about 0.1-25 mg of component (ii). In certain embodiments, component (i) and component (ii) are formulated in the same tablet, capsule, pill or solution. In other embodiments, component (i) and component (ii) are formulated in separate tablets, capsules, pills or solutions.

[0022] For any of the compositions disclosed herein, in some embodiments, the composition is substantially free of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine in free amino acid form. The composition can be substantially free of alanine, glutamic acid, glycine, isoleucine, valine, and proline in free amino acid form. In certain embodiments, the composition is substantially free of isoleucine and valine in free amino acid form. [0023] In some aspects, the present invention provides a pharmaceutical composition, comprising any composition described herein and at least one pharmaceutically acceptable excipient.

[0024] In some aspects, the present invention provides a method of treating a metabolic inflammation-mediated disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of any composition described herein or any pharmaceutical composition described herein. In some embodiments, the metabolic inflammation-mediated disease or disorder is diabetes, metabolic syndrome, dyslipidemia, obesity, hypertension, insulin resistance, non-alcoholic fatty liver disease (NAFLD), or any combination thereof. NAFLD can be non-alcoholic steatohepatitis (NASH). Diabetes can be diabetes mellitus type 2.

[0025] In some aspects, the present invention provides a method of treating a hepatic disease, disorder, or injury, comprising administering to a subject in need thereof a therapeutically effective amount of any composition described herein or any pharmaceutical composition described herein.

[0026] In any of the methods described herein, the composition can be administered orally. In some embodiments, the composition is administered at least once daily. The composition can be administered at least twice daily. In some embodiments, component (i) and component (ii) are administered simultaneously, approximately simultaneously, or sequentially in any order. In certain embodiments, component (i) and component (ii) are administered simultaneously or approximately simultaneously. In other embodiments, component (i) and component (ii) are administered sequentially. In some embodiments, component (i) is administered before component (ii). In other embodiments, component (i) is administered after component (ii).

[0027] In any of the methods described herein, in some embodiments, the subject in need is human. The subject in need can exhibit at least one or more symptoms associated with a metabolic inflammation-mediated disease or disorder.

[0028] In some aspects, the present invention provides a kit comprising any composition described herein and instructions. In some embodiments, the composition is administered to a subject in need thereof at least once a day. The composition can be administered to a subject in need thereof at least twice a day.

[0029] In some aspects, the present invention provides any pharmaceutical composition disclosed herein and instructions. In some embodiments, the composition is administered to a subject in need thereof at least once a day. The composition can be administered to a subject in need thereof at least twice a day. [0030] In any of the kits described herein, component (i) and component (ii) can be provided in a single composition. Component (i) and component (ii) can be provided in separate compositions. In some embodiments, the kits further comprise a pharmaceutical active agent.

INCORPORATION BY REFERENCE

[0031] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative

embodiments, in which the principles of the invention are utilized, and the accompanying drawing(s) of which:

[0033] FIG. 1 depicts a sirtuin pathway.

[0034] FIG. 2 depicts the interaction between SIRT1 and FXR, and the downstream effects of the interactions.

[0035] FIG. 3 depicts the interaction between SIRT1 and FXR.

[0036] FIG. 4 depicts the synergistic effects of Obeticholic Acid (OCA) and leucine on

Farnesoid X Receptor (FXR) expression in HepG2 cells.

[0037] FIG. 5 depicts the synergistic effects of Obeticholic Acid (OCA) and leucine on Fatty Acid Synthase (FAS) expression in HepG2 cells.

[0038] FIG. 6 depicts the synergistic effects of Obeticholic Acid (OCA) and leucine on Sterol Regulatory Binding Protein 1 (SREBP1) expression in HepG2 cells.

DETAILED DESCRIPTION OF THE INVENTION

[0039] 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. Unless stated otherwise, 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. The concentrations of various components in the disclosed compositions are exemplary and not meant to be limited to the recited concentration per se.

[0040] As used herein, the singular forms“a”,“an” and“the” include plural references unless the context clearly dictates otherwise. For example, the term“a cell” includes a plurality of cells, including mixtures thereof.

[0041] The terms“determining”,“measuring”,“evaluating”,“assessing,”“assaying,” “interrogating,” and“analyzing” are used interchangeably herein to refer to any form of measurement, and include determining if an element is present or not. These terms include both quantitative and/or qualitative determinations. Assessing may be relative or absolute.

[0042] As used herein, the term“subject” or“individual” includes mammals. Non-limiting examples of mammals include humans and mice, including transgenic and non-transgenic mice. The methods described herein can be useful in both human therapeutics, pre-clinical, and veterinary applications. In some embodiments, the subject is a mammal, and in some

embodiments, the subject is human. Other mammals include, and are not limited to, apes, chimpanzees, orangutans, monkeys; domesticated animals (pets) such as dogs, cats, guinea pigs, hamsters, mice, rats, rabbits, and ferrets; domesticated farm animals such as cows, buffalo, bison, horses, donkey, swine, sheep, and goats; or exotic animals typically found in zoos, such as bear, lions, tigers, panthers, elephants, hippopotamus, rhinoceros, giraffes, antelopes, sloth, gazelles, zebras, wildebeests, prairie dogs, koala bears, kangaroo, pandas, giant pandas, hyena, seals, sea lions, and elephant seals.

[0043] As used herein,“agent” or“biologically active agent” refers to a biological, pharmaceutical, or chemical compound or other moiety. Non-limiting examples include simple or complex organic or inorganic molecule, a peptide, a protein, a peptide nucleic acid (PNA), an oligonucleotide (including e.g., aptomer and polynucleotides), an antibody, an antibody derivative, antibody fragment, a vitamin derivative, a carbohydrate, a toxin, a branched chain amino acid in free amino acid form or metabolite thereof, or a chemotherapeutic compound. Various compounds can be synthesized, for example, small molecules and oligomers (e.g., oligopeptides and oligonucleotides), and synthetic organic compounds based on various core structures. In addition, various natural sources can provide compounds for screening, such as plant or animal extracts, and the like. A skilled artisan can readily recognize that there is no limit as to the structural nature of the agents of the present disclosure.

[0044] The terms“administer”,“administered”,“administers” and“administering” are defined as the providing a composition to a subject via a route known in the art, including but not limited to intravenous, intraarterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, or intraperitoneal routes of

administration. In certain embodiments of the subject application, oral routes of administering a composition may be preferred.

[0045] As used herein, the terms“co-administration”,“administered in combination with” and their grammatical equivalents are meant to encompass administration of the disclosure composition and additional therapeutic agent to a single subject. Co-administration can encompass administration of two or more agents to an animal so that both agents and/or their metabolites are present in the subject at the same time. Co-administration can encompass treatment regimens in which the composition and additional therapeutic agent are administered by the same or different route of administration or at the same or different times. Co- administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which both agents are present. Co-administration can include simultaneous administration of the agents in separate compositions, administration at different times in separate compositions, and/or administration in a single composition comprising each of the agents to be co-administered.

[0046] The term“effective amount” or“therapeutically effective amount” refers to that amount of a compound described herein that is sufficient to effect the intended application including but not limited to disease treatment, as defined below. The therapeutically effective amount may vary depending upon the intended application (in vitro or in vivo), or the subject and disease condition being treated, e.g., the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The term also applies to a dose that will induce a particular response in target cells, e.g., reduction of proliferation or down regulation of activity of a target protein. The specific dose will vary depending on the particular compounds chosen, the dosing regimen to be followed, whether it is administered in combination with other compounds, timing of administration, the tissue to which it is administered, and the physical delivery system in which it is carried.

[0047] The term“energy metabolism,” as used herein, refers to the transformation of energy that accompanies biochemical reactions in the body, including cellular metabolism and mitochondrial biogenesis. Energy metabolism can be quantified using the various measurements described herein, for example and without limitations, weight-loss, fat-loss, insulin sensitivity, fatty acid oxidation, glucose utilization, triglyceride content, Sirt 1 expression level, AMPK expression level, oxidative stress, and mitochondrial biomass. [0048] The term“isolated”, as applied to the subject components, for example a PDE 5 inhibitor, including but not limited to sildenafil and icariin, leucine and leucine metabolites (such as HMB), and resveratrol, refers to a preparation of the substance devoid of at least some of the other components that may also be present where the substance or a similar substance naturally occurs or is initially obtained from. Thus, for example, an isolated substance may be prepared by using a purification technique to enrich it from a source mixture. Enrichment can be measured on an absolute basis, such as weight per volume of solution, or it can be measured in relation to a second, potentially interfering substance present in the source mixture. Increasing enrichment of the embodiments of this disclosure are increasingly more preferred. Thus, for example, a 2-fold enrichment is preferred, lO-fold enrichment is more preferred, lOO-fold enrichment is more preferred, 1000-fold enrichment is even more preferred. A substance can also be provided in an isolated state by a process of artificial assembly, such as by chemical synthesis.

[0049] A“modulator” of a pathway refers to a substance or agent which modulates the activity of one or more cellular proteins mapped to the same specific signal transduction pathway. A modulator may augment or suppress the activity and/or expression level or pattern of a signaling molecule. A modulator can activate a component in a pathway by directly binding to the component. A modulator can also indirectly activate a component in a pathway by interacting with one or more associated components. The output of the pathway can be measured in terms of the expression or activity level of proteins. The expression level of a protein in a pathway can be reflected by levels of corresponding mRNA or related transcription factors as well as the level of the protein in a subcellular location. For instance, certain proteins are activated by translocating in or out of a specific subcellular component, including but not limited to nucleus, mitochondria, endosome, lysosome or other membraneous structure of a cell. The output of the pathway can also be measured in terms of physiological effects, such as mitochondrial biogenesis, fatty acid oxidation, or glucose uptake.

[0050] An“activator” refers to a modulator that influences a pathway in a manner that increases the pathway output. Activation of a particular target may be direct (e.g. by interaction with the target) or indirect (e.g. by interaction with a protein upstream of the target in a signaling pathway including the target).

[0051] The term“selective inhibition” or“selectively inhibit” as referred to a biologically active agent refers to the agent’s ability to preferentially reduce the target signaling activity as compared to off-target signaling activity, via direct or interact interaction with the target.

[0052] The term“substantially free”, as used herein, refers to compositions that have less than about 10%, less than about 5%, less than about 1%, less than about 0.5%, less than 0.1% or even less of a specified component. For example a composition that is substantially free of non- branched chain amino acids may have less than about 1% of the non-branched chain amino acid lysine. For example, substantially free of a non-branched chain amino acid can be evidenced by less than 1% of the non-branched chain amino acid when compared to the rest of the amino acids in a given composition.

[0053] A“sub-therapeutic amount” or“subtherapeutic amount” of an agent, an activator or a therapy is an amount less than the effective amount for that agent, activator or therapy, but when combined with an effective or sub-therapeutic amount of another agent or therapy can produce a desired result, due to, for example, synergy in the resulting efficacious effects, and/or reduced side effects. A sub-therapeutic amount of the agent or component can be such that it is an amount below which would be considered therapeutic. For example, FDA guidelines can suggest a specified level of dosing to treat a particular condition, and a sub -therapeutic amount would be any level that is below the FDA suggested dosing level. The sub-therapeutic amount can be about 1, 5, 10, 15, 20, 25, 30, 35, 50, 75, 90, or 95% less than the amount that is considered to be a therapeutic amount. The therapeutic amount can be assessed for individual subjects, or for groups of subjects. The group of subjects can be all potential subjects, or subjects having a particular characteristic such as age, weight, race, gender, or physical activity level.

[0054] A“synergistic” or“synergizing” effect can be such that the one or more effects of the combination compositions are greater than the one or more effects of each component alone at a comparable dosing level, or they can be greater than the predicted sum of the effects of all of the components at a comparable dosing level, assuming that each component acts independently.

The synergistic effect can be about, or greater than about 10, 20, 30, 50, 75, 100, 110, 120, 150, 200, 250, 350, or 500% or even more than the effect on a subject with one of the components alone, or the additive effects as measured when each of the components when administered individually. The effect can be any of the measurable effects described herein.

[0055] The terms“free amino acid form” or“individual amino acid form”, as used herein, can refer to amino acids that are not bound to other amino acids, for example, by peptide bonds. For example,“free” or“individual” leucine refers to leucine not bound to other amino acids by peptide bonds.

OVERVIEW

[0056] The invention provides methods, compositions, and kits for reducing or preventing hepatic steatosis or NASH in a subject in need thereof. For example, the invention provides a method of reducing or preventing hepatic steatosis in a subject in need thereof, comprising administering to the subject a composition comprising leucine or a metabolite thereof, and at least one farsenoid X receptor (FXR) agonist, such as obeticholic acid (OCA). The compositions of the present invention can synergistically reduce or prevent hepatic steatosis, inflammation, and/or fibrosis. In an exemplary embodiment, subtherapeutic amounts of leucine or a metabolite thereof, and subtherapeutic amounts of at least one FXR agonist, such as OCA, can be administered to a subject in need thereof to synergistically reduce hepatic steatosis and to treat NASH.

Assessment of hepatic steatosis and NASH

[0057] Any of the methods described herein can include an assessment of hepatic steatosis, inflammation, ballooning injury or fibrosis in a subject. For example, assessment of hepatic steatosis in a subject can be used to determine if a subject is in need of reduction of hepatic steatosis. Assessment of hepatic steatosis in a subject can be used to determine if and/or to what extent hepatic steatosis is prevented or reduced in a subject. Hepatic steatosis can be assessed by any means known to those of skill in the art or otherwise described herein. Hepatic steatosis in a subject can be evidenced, e.g., by an accumulation of fat in the liver of the subject (e.g., by an accumulation of fat in hepatic cells of the subject). Accumulation of fat in the liver can be indicated by several means, for example, by ultrasonography, computed tomography (CT), magnetic resonance imaging, measurement of serum alanine transaminase and aspartate transaminase, measurement of liver size or weight, or biopsy.

[0058] Ultrasonography methods for assessing liver can be as known to those of skill in the art or otherwise described herein. Ultrasonography assessment of hepatic steatosis (e.g., fat accumulation in liver) can comprise use of conventional B-mode ultrasonography. Assessment of various hepatic ultrasonography parameters can be used for the assessment of hepatic steatosis. Exemplary ultrasonography parameters for the assessment of hepatic steatosis include, but are not limited to (1) parenchymal brightness, (2) liver-to-kidney contrast, (3) deep beam attenuation, (4) bright vessel walls, and (5) gallbladder wall definition. Assessment of such ultrasonography parameters can be used to calculate an ultrasonographic steatosis score (USS). USS can be calculated, e.g., as follows: absent (score 0) steatosis was defined as normal liver echotexture; mild (score 1) steatosis as slight and diffuse increase in fine parenchymal echoes with normal visualization of diaphragm and portal vein borders; moderate (score 2) steatosis as moderate and diffuse increase in fine echoes with slightly impaired visualization of portal vein borders and diaphragm; and severe (score 3) steatosis as fine echoes with poor or no

visualization of portal vein borders, diaphragm, and posterior portion of the right lobe. [0059] CT methods for assessing liver can be any known to those of skill in the art or otherwise described herein. CT images can be assessed by, e.g., a radiologist. CT images of the liver of a subject can be assessed by, e.g., measuring density of regions of interest in the images. Regions of interest within images can be selected so as not to contain blood vessels or other artifacts (e.g., motion artifacts). Density of regions of interest in a CT image can be measured in Hounsfield units (HU). Normal liver tissue can have a HU measurement of 40-60 HU. By contrast, fat typically has a lower density. For example, fat can have an HU measurement of, e.g., about -100 to about -500. Hepatic steatosis can be evidenced by an HU measurement less than 40 HU. Hepatic steatosis can be evidenced by an HU measurement that is between -500 and 40 HU, for example, an HU measurement that is -500-1 HU, -100-10 HU, 0-20 HU, 5-30 HU, or 20-39.9 HU. A subject can be diagnosed with hepatic steatosis is the subject exhibits an HU measurement of 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23,22, 21, 20,

19, 18, 17, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0, or less than 0 HU. Hepatic steatosis can be evidenced by a difference in HU measurement between spleen and liver (e.g., HU_spieen - HUii_ver) For example, hepatic steatosis can be evidenced if HU_spieen - HUii_veris greater than 0, for example, if HU_spieen - HUii_veris between 1-10, 10-20, or more than 20. In some embodiments, a difference in HU measurement between spleen and liver of 18.5 is used to diagnose hepatic steatosis.

[0060] MRI methods for assessing liver can be any known to those of skill in the art or otherwise described herein. Exemplary methods of using MRI to determine steatosis, e.g., hepatic steatosis, are described in US Patent Application Pub. No. 20050215882, which is hereby incorporated by reference.

[0061] Hepatic steatosis can be evidenced by measurement of serum alanine transaminase (ALT) and/or aspartate transaminase levels. Methods of measuring serum alanine transaminase and/or aspartate transaminase levels can be any known to those of skill in the art or otherwise described herein. Hepatic steatosis can be indicated by an increase in serum ALT levels as compared to a control subject without hepatic steatosis. In some cases, hepatic steatosis can be indicated by an increase in serum ALT levels and serum aspartate transaminase levels as compared to a control subject without hepatic steatosis. In some cases, hepatic steatosis can be indicated by a aspartate transaminase to alanine transaminase ratio that is greater than one.

[0062] Hepatic steatosis can be evidenced by measurement of liver weight and/or size.

Methods of measuring liver weight and/or size can be any known to those of skill in the art or otherwise described herein. Hepatic steatosis can be indicated by an increase in liver weight and/or size as compared to a control subject without hepatic steatosis. In some embodiments, an increase in liver weight/size by 10% or more, 15% or more, 20%, or more, 25% or more, 30%, or more, 35% or more, 40% or more, 45% or more, 50%, or more, 55% or more, 60%, or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90%, or more, 95% or more, 100%, or more than 100% as compared to a control subject without hepatic steatosis can indicate hepatic steatosis in a subject.

[0063] Hepatic steatosis can be evidenced by tissue biopsy. A liver biopsy sample can be obtained by any means known to those of skill in the art, for example, by needle biopsy. The sample can be processed by any means known to those of skill in the art or otherwise described herein. The sample can be fixed (e.g., with formalin) or may be unfixed. The sample may be snap-frozen. For example, the sample may be sectioned into thin sections. The sections may be stained, e.g., with hematoxylin and eosin. Accumulation of fat in the liver can be evidenced by appearance of vacuoles which are filled with lipids such as, by way of example only,

triglycerides. Such vacuoles can appear to be optically“empty”, since fats can dissolve during histological tissue processing. Accordingly, levels of hepatic steatosis can be determined by measuring the number, size, or density of hepatic lipid vacuoles.

[0064] Levels of hepatic steatosis can be determined using any of the assessment methods described herein. Levels of hepatic steatosis can be quantified, by way of non-limiting example only, as a percentage of fat accumulation (e.g., fat content) in the liver. In some embodiments, hepatic steatosis is scored according to a 0-3 score, with 0= <5% fat accumulation in the liver, l=5%-33% fat accumulation in the liver, 2=33%-66% fat accumulation in the liver, and 3=

>66% fat accumulation in the liver. Liver fat content can be assessed by any means known to those of skill in the art, including, e.g., by proton magnetic resonance spectroscopy, by biopsy, or by any other methods described herein.

[0065] Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic steatosis in the absence of a history of significant alcohol use or other known liver disease. Nonalcoholic steatohepatitis (NASH) is the progressive form of NAFLD. The spectrum of NAFLD can be evaluated by a system of histological evaluation, as described in Kleiner. See , Kleiner DE, Brunt EM, Van Natta M, et al, Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology 2005; 41 : 1313-1321. The feature scoring system can use the recognized lesions of NAFLD and NASH and can identify a core group of histological features for evaluation, including: steatosis (0-3), lobular inflammation (0-2), hepatocellular ballooning (0-2), and fibrosis (0-4). The semi-quantitative scoring system can be useful for assessing the range of histological features of NAFLD. The system requires only routine histochemical stains (H&E and Masson trichrome stains), so that the system can be used by practicing pathologists. Other special stains for additional analyses may be used at the discretion of each pathologist. Weighted kappa statistics can be used for scoring and a diagnostic categorization (“NASH,” “borderline,” or“not NASH”). Inter-rater agreement on adult cases can be: 0.84 for fibrosis, 0.79 for steatosis, 0.56 for injury, and 0.45 for lobular inflammation. Agreement on diagnostic category can be 0.61. Using multiple logistic regression, five features can be independently associated with the diagnosis of NASH in adult biopsies: steatosis ( P = .009), hepatocellular ballooning ( P = .0001), lobular inflammation ( P = .0001), fibrosis ( P = .0001), and the absence of lipogranulomas ( P = .001). NAFLD Activity Score (NAS), which specifically includes features of active injury that are potentially reversible in the short term, can be used. The score is defined as the unweighted sum of the scores for steatosis (0-3), lobular inflammation (0-3), and ballooning (0-2); thus ranging from 0 to 8. Fibrosis, which is both less reversible and generally thought to be a result of disease activity, may not be included as a component of the activity score. The separation of fibrosis from other features of activity is an accepted paradigm for staging and grading for both NASH and chronic hepatitis. Cases with NAS of 0 to 2 can be largely considered not diagnostic of steatohepatitis; on the other hand, most cases with scores of >5 can be diagnosed as steatohepatitis. Cases with activity scores of 3 and 4 can be divided almost evenly between the 3 diagnostic categories. NAS of >5 correlated with a diagnosis of NASH, and biopsies with scores of less than 3 were diagnosed as“not NASH.”

[0066] MRE and TE methods for assessing fibrosis and NASH can be known to those skilled in the art or otherwise described herein. Magnetic resonance elastography (MRE) can be a highly accurate, non-invasive technique for staging liver fibrosis in patients with NAFLD. Transient elastography (FibroScan^®, TE) with controlled attenuation parameter (CAP) can be accurate in quantifying the levels of liver steatosis and fibrosis in patients with NAFLD, the factors associated with the diagnosis and NAFLD progression.

Reduction or treatment of hepatic steatosis and NASH in a subject in need thereof

[0067] Administration and/or co-administration of any of the compounds or compositions described herein can reduce hepatic steatosis, resolve NASH and/or regress hepatic fibrosis in a subject in need thereof. Exemplary subjects in need of hepatic steatosis reduction can include subjects which are diagnosed with hepatic steatosis. Any of the methods described herein for assessment of NASH, or otherwise known in the art, may be used for the diagnosis of NASH in a subject. For example, a subject may be diagnosed with hepatic steatosis if the subject exhibits a hepatic fat content of 5% or higher, a hepatic fat content of 10% or higher, a hepatic fat content of 20% or higher, a hepatic fat content of 30% or higher, a hepatic fat content of 40% or higher, a hepatic fat content of 50% or higher, a hepatic fat content of 60% or higher, or a hepatic fat content of 70% or higher. A subject can be diagnosed with stage 1 hepatic steatosis if the subject exhibits 5%-33% fat accumulation in liver. A subject can be diagnosed with stage 2 hepatic steatosis if the subject exhibits 33%-66% fat accumulation in liver. A subject can be diagnosed with stage 3 hepatic steatosis if the subject exhibits over 66% fat accumulation in liver. As described above, a subject can be diagnosed with hepatic steatosis or NASH by a system of histological evaluation, as described in Kleiner.

[0068] The subject may exhibit a non-alcoholic fatty liver disease (NAFLD). Diagnosis of NAFLD can comprise a diagnosis of hepatic steatosis. Diagnosis of NAFLD can further comprise determination of an average daily amount of alcohol consumption by the subject. In some cases, NAFLD is diagnosed in a subject if the subject is diagnosed with hepatic steatosis and is determined to consume an average of less than 20 grams of alcohol per day (e.g., an average of less than 25 ml alcohol/day). NAFLD in a subject may progress to non-alcoholic steatohepatitis (NASH).

[0069] The subject may exhibit NASH. Diagnosis of NASH can comprise a diagnosis of NAFLD. Diagnosis of NASH can further comprise a determination of inflammation in the liver of a subject concurrent with hepatic steatosis. NASH can be diagnosed in a subject, for example, upon detection of hepatic fat accumulation (steatosis) and one or more of the following liver conditions: inflammation, ballooning degeneration of hepatocytes (sometimes with identifiable Mallory bodies), glycogenated hepatocyte nuclei, and pericellular fibrosis. Pericellular fibrosis can be identfied by trichome strain. Pericellular fibrosis can exhibit upon trichrome staining, for example, a characteristic“chicken wire” pattern. NASH in a subject may progress to cirrhosis.

[0070] A subject in need of hepatic steatosis reduction can be suffering from a symptom of hepatic steatosis. Exemplary symptoms include, but are not limited to fatigue, malaise, unexplained weight loss, weakness, lack of appetite, nausea, appearance of small, red spider veins under the skin, easy bruising, jaundice, internal bleeding (e.g., bleeding from engorged veins in the esophagus or intestines), loss of sex drive, ascites, itching, edema, mental confusion, and pain or ache (e.g., pain or ache of the upper right abdomen). In some embodiments, the subject is asymptomatic.

[0071] Reduction of hepatic steatosis can be determined by comparison to a control subject and/or control population. Hepatic steatosis in a subject can be considered reduced if any one or more of hepatic fat content, as measured by any of the methods described herein, liver size or weight, liver vacuole number, size of a liver vacuole, liver vacuole density, serum alanine transaminase (ALT) and/or aspartate transaminase levels in the subject are reduced as compared to a control subject and/or control population. Hepatic steatosis in subject can be considered reduced if NASH resolution is reduced, such as reduced by at least one stage as described above by Kleiner. Hepatic steatosis in subject can be considered reduced if regression of fibrosis is increased, such as increased by at least one stage as described by Kleiner. A control subject can be an individual that has not been administered one or more compounds described herein.

Likewise, a control population can encompass a plurality of individuals that have not been administered one or more compounds described herein. The control subject can be a subject having hepatic steatosis, that is not administered one or more compounds described herein. The control subject can be a different subject.

[0072] It is not necessary for the control subject to be a different individual from said subject. For example, the control subject can be the same subject at an earlier time point, for example, prior to receiving a first dose of any of the compounds described herein. In some embodiments, a level of hepatic steatosis in the subject following administration of one or more compounds described herein is compared to a level of hepatic steatosis in the subject prior to first administration of the one or more compounds.

[0073] An exemplary method of assessing hepatic steatosis and/or NASH reduction in a subject in need thereof can comprise measuring a level of hepatic steatosis in the subject or in a biological sample derived from the subject at a first time point. The first time point may be a time point prior to administration of one or more compounds described herein. The method may further comprise measuring a level of hepatic steatosis in the subject or in a biological sample derived from the subject at a second time point. The second time point may follow

administration of the one or more compounds described herein. The level measured at the second time point may be compared to the level measured at the first time point to determine whether reduction has occurred. Reduction of hepatic steatosis can indicate clinical efficacy of the administration of the one or more compounds described herein. The method may further comprise administering additional doses of the one or more compounds described herein if the level of hepatic steatosis in the subject is reduced.

[0074] Practice of any one of the methods of the invention can reduce hepatic steatosis by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more than 99%. For example, practice of any one of the methods of the invention can reduce hepatic steatosis by 5-20%, 10-40%, 30-60%, 40-80%, 60-95%, or 75-99%. In some embodiments, administration of any combination of agents described herein reduces hepatic steatosis in about six weeks or less. Administration of any combination of agents described herein can, for example, reduce liver weight or liver fat by 25% in about six weeks. The compositions of the present disclosure can reduce liver fat by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% in a subject in need thereof in about six weeks. Administration of any combination of agents described herein can, by way of example only, reduce liver vacuole number, size, and/or density by over 25%, over 50%, over 75%, or over 90% in about six weeks. Administration of any of the compositions or combination of agents described herein can reduce or resolve NASH. In some embodiments, the compositions of the present disclosure can reduce NASH by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% in a subject in need thereof in about six months. Administration of any

compositions or combination of agents described herein can, by way of example only, reduce hepatocellular ballooning and lobular inflammation. Administration of any of the compositions or combination of agents described herein can increase fibrosis regression. In some

embodiments, the compositions of the present disclosure can reduce fibrosis by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% in a subject in need thereof in about six months.

Prevention of hepatic steatosis and NASH

[0075] Administration and/or co-administration of any of the compounds or compositions described herein can reduce hepatic steatosis and regress NASH and/or fibrosis in a subject in need thereof. Exemplary subjects in need of hepatic steatosis reduction can include subjects exhibiting a propensity for, or having a high risk of developing, hepatic steatosis or NASH.

[0076] A number of environmental and genetic risk factors have been found to increase propensity of a subject to develop hepatic steatosis and NASH. A subject can exhibit a propensity for developing hepatic steatosis if the subject exhibits any combination of risk factors described herein. A greater number of risk factors exhibited by a subject can indicate a higher propensity for developing hepatic steatosis, as compared to a subject that exhibits a lower number of risk factors. It is understood that a subject can be considered to exhibit a propensity for developing hepatic steatosis and/or NASH if the subject exhibits even one, two, three, or more of the risk factors described herein.

[0077] An exemplary risk factor for developing hepatic steatosis and NASH is obesity.

Accordingly, a subject can be considered to exhibit an increased propensity to develop hepatic steatosis if the subject is obese. Obesity in a subject can be assessed using a body mass index

(BMI) measurement. A subject’s BMI can be calculated by dividing the subject’s body weight

(in kg) by the square of the subject’s height (m2). A subject can be considered obese if the subject exhibits a BMI that is over 25 kg/m2, over 26kg/m2, over 27 kg/m2, over 28 kg/m2, over

29 kg/m2, over 30 kg/m2, over 31 kg/m2, over 32 kg/m2, over 33 kg/m2, over 34 kg/m2, over

35 kg/m2, over 36 kg/m2, over 37 kg/m2, over 38 kg/m2, over 39 kg/m2, or over 40 kg/m2. A subject can also be considered to exhibit a propensity to develop hepatic steatosis if the subject exhibits abdominal obesity. Abdominal obesity can be assessed by measuring the circumference of the subject’s waist. For example, if the subject is an adult male, the subject can be considered to exhibit abdominal obesity if the subject exhibits a waist circumference of 102 cm or greater. For other example, if the subject is an adult female, the subject can be considered to exhibit abdominal obesity if the subject exhibits a waist circumference of 88 cm or greater.

[0078] Another exemplary risk factor for developing hepatic steatosis and NASH is high cholesterol. Accordingly, a subject can be considered to exhibit an increased propensity to develop hepatic steatosis if the subject exhibits high cholesterol. Cholesterol in a subject can be assessed by measuring total blood cholesterol levels in the subject. A subject can be considered to exhibit high cholesterol if the subject exhibits, e.g., over 200 mg/dl total blood cholesterol, over 210 mg/dl total blood cholesterol, over 220 mg/dl total blood cholesterol, over 230 mg/dl total blood cholesterol, or over 240 mg/dl total blood cholesterol. A subject can be considered to exhibit high cholesterol if the subject exhibits, 200-220 mg/dl total blood cholesterol, 220-240 mg/dl total blood cholesterol, or 240 mg/dl total blood cholesterol or higher. Cholesterol in a subject can also be assessed by measuring blood low density lipoprotein (LDL) levels in the subject. A subject can be considered to exhibit high cholesterol if the subject exhibits, e.g., 130 mg/dl LDL or higher, 135 mg/dl LDL or higher, 140 mg/dl LDL or higher, 145 mg/dl LDL or higher, 150 mg/dl LDL or higher, 155 mg/dl LDL or higher, 160 mg/dl LDL or higher, 165 mg/dl LDL or higher, 170 mg/dl LDL or higher, 175 mg/dl LDL or higher, 180 mg/dl LDL or higher, 185 mg/dl LDL or higher, or 190 mg/dl LDL or higher. Cholesterol in a subject can also be assessed by measuring blood high density lipoprotein (HDL) levels in the subject. A subject can be considered to exhibit high cholesterol if the subject exhibits, e.g. ,60 mg/dl HDL or lower, 55 mg/dl HDL or lower, 50 mg/dl HDL or lower, 45 mg/dl HDL or lower, or 40 mg/dl HDL or lower.

[0079] Another exemplary risk factor for developing hepatic steatosis and NASH is insulin resistance. Accordingly, a subject can be considered to exhibit an increased propensity to develop hepatic steatosis if the subject exhibits insulin resistance. Insulin resistance in a subject can be assessed by any means known in the art or otherwise described herein. By way of example only, insulin resistance may be assessed by measuring Homeostatic Assessment of Insulin Resistance (HOMA_IR) in the subject. HOMA_IR can be determined by any means known in the art, for example, by the following equation: HOMA_IR = Insulin (uU/mL) X glucose (mM)]/22.5. The HOMA_IR value increases with increasing insulin resistance, and values above (2.6) are generally considered to be insulin resistant. A subject can be considered to exhibit insulin resistance if the subject exhibits a HOMA_IR above the the 75^th percentile, or a HOMA_IR value of over 2.6. Insulin resistance may also be assessed by measuring fasting serum insulin levels in the subject. A subject may be considered to exhibit insulin resistance if the subject exhibits a fasting serum insulin level of 60 pmol/L or higher. Insulin resistance may also be assessed by measuring a quantitative insulin sensitivity check index (QUICKI) in the subject. QUICKI can be determined by the following equation: QUICKI = l/(log(fasting insulin pU/mL) + log(fasting glucose mg/dL)). A subject may be considered to exhibit insulin resistance if the subject exhibits a QUICKI of 0.30 or less. For other example, insulin resistance can be determined by measuring blood glucose levels in a subject, in some cases over a period of time, following administration of a bolus of insulin. Subjects with insulin resistance typically exhibit an attenuated drop in blood glucose levels following insulin administration, as compared to subject without insulin resistance.

[0080] Another exemplary risk factor for developing hepatic steatosis and NASH is diabetes. Accordingly, a subject can be considered to exhibit an increased propensity to develop hepatic steatosis if the subject has or is diagnosed with diabetes. The diabetes can be Type I or Type II diabetes. Diabetes may be diagnosed by any means known to those of skill in the art, or otherwise described herein. For example, a subject may be diagnosed with diabetes if the subject exhibits high fasting plasma glucose levels (e.g., 126 mg/dl or higher). A subject may be diagnosed with diabetes if the subject exhibits high plasma glucose levels following

administration of a bolus of glucose (e.g., in a glucose tolerance test). For example, a subject may be diagnosed with diabetes if the subject exhibits 200 mg/dl plasma glucose or higher two hours after administration of a 75 g bolus of glucose.

[0081] Genetics may be a risk factor for developing hepatic steatosis and NASH. For example, males of Indian, Asian, and/or Mexican descent may have a higher risk of developing hepatic steatosis. Accordingly, a subject can be considered to exhibit an increased propensity to develop hepatic steatosis if the subject is a male of African, Asian, and/or Mexican descent. Genetic polymorphisms may also be associated with an increased propensity to develop hepatic steatosis. For example, a subject may be considered to exhibit an increased propensity to develop NASH if the subject exhibits an I148M polymorphism in the PNPLA3 gene. Other genetic polymorphisms that may contribute to NASH include GCKR P446L.

[0082] Certain medications increase risk for developing hepatic steatosis and NASH. Such medications include, but are not limited to oral corticosteroids (e.g., prednisone, hydrocortisone, among others), synthetic estrogens (e.g., Premarin, Ortho-Est, tamoxifen, among others), amiodarone (Cordarone, Pacerone), diltiazem, anti-retroviral drugs such as, e.g., indinavir, and methotrexate. Accordingly, a subject may be considered to exhibit an increased propensity to develop hepatic steatosis if the subject has taken any of the medications described herein as increasing risk for developing hepatic steatosis.

[0083] In some embodiments, the subject exhibiting a propensity for developing hepatic steatosis and NASH has not yet developed hepatic steatosis. For example, the subject may not have exhibited a symptom of hepatic steatosis. For other example, the subject may not exhibit increased fat content of the liver. In some embodiments, the subject has not been diagnosed with hepatic steatosis. In some embodiments, the subject does not exhibit an increase in serum ALT levels as compared to a control subject without hepatic steatosis.

[0084] Practice of any one of the methods of the invention can prevent or reduce occurrence of hepatic steatosis in at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,

60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more than 99% of treated subjects. For example, practice of any one of the methods of the invention can prevent hepatic steatosis or reduce occurrence of hepatic steatosis by 5-20%, 10-40%, 30-60%, 40-80%, 60-95%, or 75-99%. In some embodiments, administration of any combination of agents described herein for about, less than about, or greater than about 1, 2, 3, 4, 5, 6, 7, 8 weeks or 2, 4, 6, 12 or 24 months and such administration prevents or reduces occurrence of hepatic steatosis in administered subjects. Prevention of hepatic steatosis can be determined by comparison to a reference subject and/or reference population. Hepatic steatosis in a subject can be considered to be prevented or to have reduced occurrence if any one or more of hepatic fat content, as measured by any of the methods described herein, liver size or weight, liver vacuole number, size of a liver vacuole, liver vacuole density, serum alanine transaminase (ALT) and/or aspartate transaminase levels in the subject does not increase or increases to a lesser extent as compared to a reference subject and/or reference population. The reference subject and/or reference population can be another subject or population of subjects exhibiting a comparable propensity for developing hepatic steatosis who has not developed hepatic steatosis, and who is not treated with one or more compounds or compositions described herein.

[0085] Practice of any one of the methods of the invention can prevent or reduce occurrence of NASH in at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more than 99% of treated subjects. For example, practice of any one of the methods of the invention can prevent NASH or reduce occurrence of NASH by 5-20%, 10-40%, 30-60%, 40-80%, 60-95%, or 75-99%. In some embodiments, administration of any combination of agents described herein for about, less than about, or greater than about 1, 2, 3, 4, 5, 6, 7, 8 weeks or 2, 4, 6, 12 or 24 months and such administration prevents or reduces occurrence of NASH in administered subjects. Prevention of NASH can be determined by comparison to a reference subject and/or reference population. NASH in a subject can be considered to be prevented or to have reduced occurrence if any one or more of hepatic fat content, as measured by any of the methods described herein, liver size or weight, liver vacuole number, size of a liver vacuole, liver vacuole density, serum alanine transaminase (ALT) and/or aspartate transaminase levels in the subject does not increase or increases to a lesser extent as compared to a reference subject and/or reference population. The reference subject and/or reference population can be another subject or population of subjects exhibiting a comparable propensity for developing NASH who has not developed NASH, and who is not treated with one or more compounds or compositions described herein.

Reduction or treatment of a metabolic inflammation-mediated disease or disorder in a subject

[0086] Administration and/or co-administration of any of the compounds or compositions described herein can reduce or treat a metabolic inflammation-mediated disease or disorder in subject thereof in a subject in need thereof. In some embodiments, the metabolic inflammation- mediated disease or disorder is diabetes, metabolic syndrome, dyslipidemia, obesity,

hypertension, insulin resistance, non-alcoholic fatty liver disease (NAFLD), or any combination thereof. For example, the metabolic inflammation-mediated disease or disorder is non-alcoholic steatohepatitis (NASH). The metabolic inflammation-mediated disease or disorder can be diabetes mellitus type 2.

Exemplary Compositions

[0087] The invention provides compositions comprising a branched amino acid, such as leucine, or metabolites thereof, and a FXR agonist. The compositions of the present disclosure can be administered to a subject in thereof for the reduction and/or prevention of hepatic steatosis or fibrosis and/or resolution or prevention of NASH.

Branched chain amino acids

[0088] Branched chain amino acids may have aliphatic side chains with a branch carbon atom that is bound to two or more other atoms. The other atoms may be carbon atoms. Examples of branched chain amino acids include leucine, isoleucine, and valine. Branched chain amino acids may also include other compounds, such as 4-hydroxyisoleucine. Such branched chain amino acids may be administered to a subject in free amino acid form. In some embodiments, the branched chain amino acid in free amino acid form is leucine in free amino acid form. In some embodiments, a composition comprising a branched chain amino acid in free amino acid form is substantially free of one or more, or all of non-branched chain amino acids. In some embodiments, the compositions are substantially free of one or more, or all of non-branched chain amino acids in free amino acid form. For example, the composition can be substantially free of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, and/or tyrosine. The composition can be substantially free of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, and/or tyrosine in free amino acid form. The composition can be substantially free of alanine, glutamic acid, glycine, isoleucine, valine, and proline. In some embodiments, the composition is substantially free of alanine, glutamic acid, glycine, isoleucine, valine, and proline. In some embodiments, the composition is substantially free of isoleucine and/or valine. In some embodiments, the composition is substantially free of isoleucine and/or valine in free form.

[0089] In some embodiments, a method described herein can include administration of a salt, derivative, metabolite, catabolite, anabolite, precursor, and/or analog of any branched chain amino acids. The metabolite can be a metabolite of leucine, such as HMB. Metabolites of branched chain amino acids can include hydroxymethylbutyrate (HMB), a-hydroxyisocaproic acid, and keto-isocaproic acid (KIC), keto isovalerate, and keto isocaproate. Metabolites of branched chain amino acids can include hydroxymethylbutyrate (HMB), and keto-isocaproic acid (KIC). Non-limiting exemplary anabolites of branched chain amino acids can include glutamate, glutamine, threonine, a-ketobytyrate, a-aceto-a-hydroxy butyrate, a, -dihydroxy- - methylvalerate, a-keto- -methyl valerate, a,b-dihydroxy isovalerate, and a-keto isovalerate.

[0090] In certain embodiments of the invention, any of the compositions to be administered to a subject can be formulated such that they do not contain (or exclude) one or more amino acids selected from the group consisting of lysine, glutamate, proline, arginine, valine, isoleucine, aspartic acid, asparagine, glycine, threonine, serine, phenylalanine, tyrosine, histidine, alanine, tryptophan, methionine, glutamine, taurine, carnitine, cystine and cysteine. Any of the compositions disclosed herein can be formulated such that they do not contain (or exclude) one or more free amino acids selected from the group consisting of lysine, glutamate, proline, arginine, valine, isoleucine, aspartic acid, asparagine, glycine, threonine, serine, phenylalanine, tyrosine, histidine, alanine, tryptophan, methionine, glutamine, taurine, carnitine, cystine and cysteine. In some cases, a composition does not contain any non-branched chain amino acids.

In some cases, a composition does not contain any non-branched chain amino acids in free amino acid form. The mass or molar amount of a non-branched chain amino acid in a composition can be less than 0.01, 0.1, 0.5, 1, 2, or 5 % of the total composition. The mass or molar amount of a non-branched chain amino acid in free amino acid form can be less than 0.01, 0.1, 0.5, 1, 2, or 5 % of the total composition. The mass or molar amount of any branched-chain amino acid or metabolite thereof, aside from leucine or its metabolites can be less than 0.01, 0.1, 0.5, 1, 2, or 5 % of the total composition. The mass or molar amount of any branched-chain amino acid in free amino acid form or metabolite thereof, aside from leucine or its metabolites can be less than 0.01, 0.1, 0.5, 1, 2, or 5 % of the total composition.

FXR Agonists

[0091] Any composition of the present disclosure can be administered to a subject comprises a famesoid X receptor (FXR) agonist. Any agents that regulate FXR activity or expression can used in the compositions, methods and kits of the present disclosure. FXR agonists include, but are not limited to, obeticholic acid (OCA), chenodeoxycholic acid (CDCA), cholic acid (CA), PX-104, PX-102, GW4064, WAY-362450, derivatives thereof, and pharmaceutically acceptable salts thereof. In some embodiments, the FXR agonist is OCA or a derivative thereof. In an exemplary embodiment, the FXR agonist is OCA.

[0092] The FXR agonist may comprise a steroid core, in which any substitutable carbon on the steroid core is optionally substituted. In some embodiments, the steroid core is optionally substituted by a substituent independently selected from halogen, -OR¹⁰, -SR¹⁰, -N(R¹⁰)₂, - S(0)R¹⁰, -S(0)₂R¹⁰, -C(0)R¹⁰, -C(0)OR¹⁰, -OC(0)R¹⁰, -N0₂, =0, =S, =N(R¹⁰), -CN, optionally substituted C_MO alkyl, optionally substituted C_2.l0 alkenyl, and optionally substituted C_2.l0 alkynyl.

[0093] In some aspects, the present disclosure provides a FXR agonist represented by a compound represented by Formula I:

pharmaceutically acceptable salt thereof, wherein:

R¹, R², R³, and R⁴ are independently selected from hydrogen, halogen, -OR¹⁰, -SR¹⁰, - N(R¹⁰)₂, -S(0)R¹⁰, -S(0)₂R¹⁰, -C(0)R¹⁰, -C(0)OR¹⁰, -OC(0)R¹⁰, -N0₂, =0, =S, =N(R¹⁰), -CN, optionally substituted C _O alkyl, optionally substituted C_2-l0 alkenyl, and optionally substituted

C_2-io alkynyl; R⁵ is selected from -OR¹⁰, -

N(R¹⁰)₂, -C(O)N(R¹⁰)₂, -C(O)N(R¹⁰)₂C(O)O(R¹⁰), -C(O)N(R¹⁰)₂S(O)₃(R¹⁰), - C(0)R¹⁰, -C(0)OR¹⁰, OC(0)R¹⁰, -OS(0)₃(R¹⁰), -SR¹⁰, -S(0)R¹⁰, and -S(0)₂R¹⁰; C _O alkyl, C_2.10 alkenyl, C_2-l0 alkynyl, each of which is independently optionally substituted at each occurrence with one or more substituents selected from halogen, -OR¹⁰, -SR¹⁰, -C(O)N(R¹⁰)₂, - N(R¹⁰)C(O)R¹⁰, -N(R¹⁰)C(O)N(R¹⁰)₂, -N(R¹⁰)₂, -C(0)R¹⁰, -C(0)OR¹⁰, -OC(0)R¹⁰, -N0₂, =0,

=S, =N(R¹⁰), -CN, C_3-l2 carbocycle, and 3- to l2-membered heterocycle; and C_3.l2 carbocycle, and 3- to l2-membered heterocycle, wherein each C_3-l2 carbocycle, and 3- to l2-membered heterocycle in R⁴ is independently optionally substituted with one or more substituents selected from halogen, -OR¹⁰, -SR¹⁰, -C(O)N(R¹⁰)₂, -N(R¹⁰)C(O)R¹⁰, -N(R¹⁰)C(O)N(R¹⁰)₂, -N(R¹⁰)₂, - C(0)R¹⁰, -C(0)OR¹⁰, -OC(0)R¹⁰, -N0₂, =0, =S, =N(R¹⁰), -CN, C_M alkyl, C_2-6 alkenyl, and C_2-6 alkynyl; and

R¹⁰ is independently selected at each occurrence from the group consisting of hydrogen, optionally substituted Ci-io alkyl, optionally substituted C_2-l0 alkenyl, optionally substituted C₂- io alkynyl, optionally substituted C_3-l2 carbocycle, and optionally substituted 3- to l2-membered heterocycle.

[0094] In some embodiments, R¹ is selected from hydrogen, -OR¹⁰, -N(R¹⁰)₂ and optionally substituted C _O alkyl. In some embodiments, R¹ is hydrogen. In some embodiments, R¹ is an optionally substituted C _O alkyl. R^xcan be an optionally substituted C_M alkyl. R¹ can be an optionally substituted C_2-3 alkyl. In an exemplary embodiment, R¹ is ethyl.

[0095] In some embodiments, R³ is selected from hydrogen, -OR¹⁰, -N(R¹⁰)₂ and optionally substituted C _O alkyl. R³ can be selected from the group consisting of hydrogen, -OR¹⁰, and - N(R¹⁰)₂. R³ can be -OR¹⁰. In certain embodiments, R³ is -OH.

[0096] In some embodiments, R² and R⁴ are independently selected from hydrogen, -OR¹⁰, - N(R¹⁰)₂ and optionally substituted C_MO alkyl. R² and R⁴ can be independently selected from hydrogen, -OR¹⁰, and optionally substituted C_MO alkyl. In certain embodiments, R² is hydrogen. In certain embodiments, R⁴ is hydrogen. In other embodiments, R⁴ is -OR¹⁰, such as R⁴ is -OH.

[0097] In some embodiments, R⁵ is selected from the group consisting of -OR¹⁰, - N(R¹⁰)₂, -C(O)N(R¹⁰)₂, -C(O)N(R¹⁰)₂C(O)O(R¹⁰), -C(O)N(R¹⁰)₂S(O)₃(R¹⁰), - C(0)R¹⁰, -C(0)OR¹⁰, -OC(0)R¹⁰, -OS(0)₃(R¹⁰), -SR¹⁰, -S(0)R¹⁰, and -S(0)₂R¹⁰. In certain embodiments, R⁵ is selected

from -C(O)N(R¹⁰)₂, -C(O)N(R¹⁰)₂C(O)O(R¹⁰), -C(O)N(R¹⁰)₂S(O)₃(R¹⁰), -C(0)R¹⁰, -C(0)OR¹⁰, - OC(0)R¹⁰, and -OS(0)₃(R¹⁰). In certain embodiments, R⁵ is -C(0)OR¹⁰. R⁵ can be -C(0)OH. [0098] In some embodiments, the compound is selected from:

, and a pharmaceutically acceptable salt thereof. In an exemplary

embodiment, the compound

[0099] In certain embodiments, for a compound or salt of Formula (I):

R¹ is selected from hydrogen, -OR¹⁰, and optionally substituted C_l-l0 alkyl;

R² and R⁴ are independently selected from hydrogen, -OR¹⁰, and optionally substituted C_l-l0 alkyl;

R³ is selected from the group consisting of hydrogen, -OR¹⁰, and -N(R¹⁰)₂; and

R⁵ is selected from -C(O)N(R¹⁰)₂, -C(O)N(R¹⁰)₂C(O)O(R¹⁰), -C(O)N(R¹⁰)₂S(O)₃(R¹⁰), -

C(0)R¹⁰, -C(0)OR¹⁰, -OC(0)R¹⁰, and -OS(0)₃(R¹⁰).

[00100] In certain embodiments, for a compound or salt of Formula (I):

R¹ is an optionally substituted C_MO alkyl;

R² and R⁴ are independently selected from hydrogen, and -OR¹⁰;

R³ is selected from the group consisting of hydrogen, and -OR¹⁰; and

R⁵ is -C(0)OR¹⁰.

[00101] In certain embodiments, for a compound or salt of Formula (I):

R¹ is an optionally substituted Ci_-6 alkyl; R² is hydrogen;

R³ is selected from the group consisting of hydrogen, and -OR¹⁰;

R⁴ is hydrogen; and

R⁵ is -C(0)OR¹⁰.

[00102] In certain embodiments, for a compound or salt of Formula (I):

R¹ is an optionally substituted C2-3 alkyl;

R² is hydrogen;

R³ is -OR¹⁰;

R⁴ is hydrogen; and

R⁵ is -C(0)OR¹⁰.

[00103] In certain embodiments, for a compound or salt of Formula (I):

R¹ is hydrogen;

R² is hydrogen;

R³ is selected from the group consisting of hydrogen, and -OR¹⁰;

R⁴ is selected from hydrogen and -OR¹⁰; and

R⁵ is -C(0)OR¹⁰.

Exemplary sirtuin pathway activators

[00104] Sirtuin pathway activators can include any agents which activate one or more components of a sirtuin pathway. The sirtuin pathway includes, without limitation, signaling molecules such as, Sirtl, Sirt3, and AMPK. The output of the pathway can be determined by the expression level and/or the activity of the pathway and/or a physiological effect. In some embodiments, activation of the Sirtl pathway includes stimulation of PGCl-a and/or subsequent stimulation of mitochondrial biogenesis and fatty acid oxidation. An increase or activation of a sirtuin pathway can be observed by an increase in the activity of a pathway component protein.

For example, the protein can be Sirtl, PGCl-a, AMPK, Epacl, Adenylyl cyclase, Sirt3, or any other proteins and their respective associated proteins along the signaling pathway depicted in

FIG. 1 (Park et. ak,“Resveratrol Ameliorates Aging-Related Metabolic Phenotypes by Inhibiting cAMP Phosphodiesterases,” Cell 148, 421-433 February 3, 2012. Non-limiting examples of physiological effects that can serve as measures of sirtuin pathway output include mitochondrial biogenesis, fatty acid oxidation, glucose uptake, palmitate uptake, oxygen consumption, carbon dioxide production, weight loss, heat production, visceral adipose tissue loss, respiratory exchange ratio, insulin sensitivity, inflammation marker level, vasodilation, browning of fat cells, and irisin production. Examples of indicia of browning of fat cells include, without limitation, increased fatty acid oxidation, and expression of one or more brown-fat-selective genes (e.g. Ucpl, Cidea, Prdml6, and Ndufsl). In some embodiments, changes in one or more physiological effects that can serve as measures of sirtuin pathway output are induced by increasing irisin production.

[00105] An increase in mitochondrial biogenesis can be evidenced by an increase in the formation of new mitochondria and/or by an increase in mitochondrial functions, such as increased fatty acid oxidation, increased heat generation, increased insulin sensitivity, increased in glucose uptake, increased in vasodilation, decreased in weight, decreased in adipose volume, and decreased inflammatory response or markers in a subject.

[00106] In some embodiments the sirtuin pathway activator is a sirtuin activator. The sirtuin activator can be a Sirtl activator, a Sirt2 activator, and/or Sirt3 activator. Sirtl activity can be determined by measuring deacetylation of a substrate, which can be detected using a

fluorophore. An increase in sirtl, sirt2, or sirt3 is observed by applying a corresponding substrate in a deacylation assay conducted in vitro. The substrate for measuring SIRT1 activity can be any substrate known in the art (for example a peptide containing amino acids 379-382 of human p53 (Arg-His-Lys-Lys[Ac]). The substrate for measuring SIRT3 activity can be any substrate known in the art (for example a peptide containing amino acids 317-320 of human p53 (Gin-Pro— Lys- Lys[Ac])).

[00107] Exemplary sirtuin activators can include those described in Howitz et al. (2003)

Nature 425: 191 and include, for example, resveratrol (3,5,4'-Trihydroxy-trans-stilbene), butein (3,4,2',4'-Tetrahydroxychalcone), piceatannol (3,5,3 ',4'-Tetrahydroxy-trans-stilbene), isoliquiritigenin (4,2 ',4 '-Trihydroxy chalcone), fisetin (3,7,3 ',4'-Tetrahyddroxyflavone), quercetin (3,5,7,3',4'-Pentahydroxyflavone), Deoxyrhapontin (3,5-Dihydroxy-4'-methoxystilbene 3-O-b- D-glucoside); trans-Stilbene; Rhapontin (3,3',5-Trihydroxy-4'-methoxystilbene 3-O-b-ϋ- glucoside); cis-Stilbene; Butein (3,4,2',4'-Tetrahydroxychalcone); 3,4,2'4'6'- Pentahydroxychalcone; Chalcone; 7,8,3 ',4'-Tetrahydroxyflavone; 3,6,2',3'-Tetrahydroxyflavone; 4'-Hydroxyflavone; 5,4'-Dihydroxyflavone 5,7-Dihydroxyflavone; Morin (3, 5, 7,2', 4'- Pentahydroxyflavone); Flavone; 5-Hydroxyflavone; (-)-Epicatechin (Hydroxy Sites: 3, 5, 7, 3 ',4'); (-)-Catechin (Hydroxy Sites: 3, 5, 7, 3', 4'); (-)-Gallocatechin (Hydroxy Sites: 3, 5, 7, 3', 4', 5') (+)- Catechin (Hydroxy Sites: 3, 5, 7, 3', 4'); 5,7,3',4',5'-pentahydroxyflavone; Luteolin (5,7,3 ',4'- Tetrahydroxyflavone); 3,6,3 ',4'-Tetrahydroxyflavone; 7,3 ',4',5'-Tetrahydroxyflavone;

Kaempferol (3,5,7,4'-Tetrahydroxyflavone); 6-Hydroxyapigenin (5,6,7,4'-Tetrahydoxyflavone); Scutellarein); Apigenin (5,7,4'-Trihydroxyflavone); 3,6,2',4'-Tetrahydroxyflavone; 7,4'- Dihydroxyflavone; Daidzein (7,4 '-Dihydroxyi soil avone); Genistein (5,7,4'- Trihydroxyflavanone); Naringenin (5,7,4'-Trihydroxyflavanone); 3, 5, 7, 3 ',4'- Pentahydroxyflavanone; Flavanone; Pelargonidin chloride (3,5,7,4'-Tetrahydroxyflavylium chloride); Hinokitiol (b-Thujaplicin; 2-hydroxy-4-isopropyl-2,4,6-cycloheptatrien-l-one); L-(+)- Ergothioneine ((S)-a-Carboxy-2,3-dihydro-N,N,N-trimethyl-2-thioxo-lH-imidazole-4- ethanaminium inner salt); Caffeic Acid Phenyl Ester; MCI-186 (3-Methyl-l-phenyl-2-pyrazolin- 5-one); HBED (N,N'-Di-(2-hydroxybenzyl) ethylenediamine-N,N'-diacetic acid-H20);

Ambroxol (trans-4-(2-Amino-3,5-dibromobenzylamino) cyclohexane-HCl; and Ei-83836E ((-)- 2-((4-(2,6-di-l-Pyrrolidinyl-4-pyrimidinyl)-l-piperzainyl)methyl)-3,4-dihydro-2, 5,7,8- tetramethyl-2H-l-benzopyran-6-ol.2HCl). Analogs and derivatives thereof can also be used.

Synergistic effect of compositions

[00108] The compositions of the present disclosure comprise components, such leucine or a metabolite thereof, and an FXR agonist, that can have a synergistic effect on reducing, treating, or preventing hepatic steatosis in a subject in need thereof.

[00109] Without being limited to theory, a composition comprising leucine or a metabolite thereof, and an FXR agonist, such as obeticholic acid (OCA), can stimulate Sirtl and FXR. As depicted in FIG. 2, Leucine activates Sirtl, which in turn activates FXR and stimulates PGC1- a, which in turn, activates FXR. OCA directly activates FXR. The activation of Sirtl corresponds to an effect on mitochondrial biogenesis and stress resistance. The activation of FXR

corresponds to an effect on BA metabolism. The activation of both Sirt 1 and FXR correspond to an effect on lipid metabolism, such as fatty acid oxidation and triglyceride synthesis,

inflammation, cholesterol metabolism, and glucose homeostasis.

[00110] In particular, as depicted in FIG. 3, Sirtl de-acetylates and thereby activates FXR.

Sirtl activates PGC1 alpha, which in turn also activates FXR. FXR induces an indirect feed- forward activation of Sirtl, as follows: miR-34A, which is induced by energy overload/obesity, transcriptionally represses Sirtl; FXR, via SHP, suppresses miR-34A and thereby de-represses (i.e. activates) Sirtl; and Sirtl and FXR (via induction of SHP) both inhibit Cyp7Al, the rate limiting step in bile acid synthesis. Thus, Sirtl activates FXR (the OCA target), but FXR (and therefore OCA) indirectly stimulates Sirtl via the feed-forward mechanism described herein.

[00111] In some embodiments, the particular choice of compounds or compositions depends upon the diagnosis of the attending physicians and their judgment of the condition of the individual and the appropriate treatment protocol. The compounds are optionally administered concurrently (e.g., simultaneously, essentially simultaneously or within the same treatment protocol) or sequentially, depending upon the nature of the disease, disorder, or condition, the condition of the individual, and the actual choice of compounds used. In certain instances, the determination of the order of administration, and the number of repetitions of administration of each therapeutic agent during a treatment protocol, is based on an evaluation of the disease being treated and the condition of the individual.

[00112] In some embodiments, therapeutically-effective dosages vary when the drugs are used in treatment combinations. Methods for experimentally determining therapeutically-effective dosages of drugs and other agents for use in combination treatment regimens are described in the literature.

[00113] In some embodiments of the combination therapies described herein, dosages of the co-administered compounds or compositions vary depending on the type of co-drug employed, on the specific drug employed, on the disease or condition being treated and so forth. In addition, when co-administered with one or more biologically active agents, the compound or composition provided herein is optionally administered either simultaneously with the biologically active agent(s), or sequentially. In certain instances, if administered sequentially, the attending physician will decide on the appropriate sequence of therapeutic compound or composition described herein in combination with the additional therapeutic agent.

[00114] The multiple therapeutic agents are optionally administered in any order or even simultaneously. If simultaneously, the therapeutic agents are optionally provided in a single, unified form, or in multiple forms (by way of example only, either as a single pill or as two separate pills). In certain instances, one of the therapeutic agents is optionally given in multiple doses. In other instances, both are optionally given as multiple doses. If not simultaneous, the timing between the multiple doses is any suitable timing, e.g., from more than zero weeks to less than four weeks. In addition, the combination methods, compositions and formulations are not to be limited to the use of only two agents; the use of multiple therapeutic combinations is also envisioned (including two or more compounds described herein).

[00115] In certain embodiments, a dosage regimen to treat, prevent, or ameliorate the condition(s) for which relief is sought, is modified in accordance with a variety of factors. These factors include the disorder from which the subject suffers, as well as the age, weight, sex, diet, and medical condition of the subject. Thus, in various embodiments, the dosage regimen actually employed varies and deviates from the dosage regimens set forth herein.

[00116] In some embodiments, the pharmaceutical agents that make up the combination therapy described herein are provided in a combined dosage form or in separate dosage forms intended for substantially simultaneous administration. In certain embodiments, the

pharmaceutical agents that make up the combination therapy are administered sequentially, with either therapeutic compound being administered by a regimen calling for two-step

administration. In some embodiments, two-step administration regimen calls for sequential administration of the active agents or spaced-apart administration of the separate active agents.

In certain embodiments, the time period between the multiple administration steps varies, by way of non-limiting example, from a few minutes to several hours, depending upon the properties of each pharmaceutical agent, such as potency, solubility, bioavailability, plasma half-life and kinetic profile of the pharmaceutical agent.

[00117] In various other embodiments, compositions for use in practicing one or more methods of the invention are formulated such that they do not contain (or exclude) one or more of the following ingredients: caffeine, green tea extract or extracts from guarana seed or guarana plants. Compositions can also be formulated such that they are substantially free of high glycemic index carbohydrate, such as, e.g., simple carbohydrates, including sugars such as but not limited to sucrose, glucose, dextrose, maltose, fructose, and galactose, among others.

Dosing amounts

[00118] The amount of pharmaceutical agent, or any other component used in a combination composition described herein, can be an amount that is therapeutically effective. The amount of pharmaceutical agent, or any other component used in a combination composition described herein, can be an amount that is sub-therapeutic. In some embodiments, using sub-therapeutic amounts of an agent or component can reduce the side-effects of the agent. Use of sub- therapeutic amounts can still be effective, particularly when used in synergy with other agents or components.

[00119] A sub-therapeutic amount of the agent or component can be such that it is an amount below which would be considered therapeutic. For example, FDA guidelines can suggest a specified level of dosing to treat a particular condition, and a sub -therapeutic amount would be any level that is below the FDA suggested dosing level. The sub-therapeutic amount can be about 1, 5, 10, 15, 20, 25, 30, 35, 50, 75, 90, or 95% less than the amount that is considered to be a therapeutic amount. The therapeutic amount can be assessed for individual subjects, or for groups of subjects. The group of subjects can be all potential subjects, or subjects having a particular characteristic such as age, weight, race, gender, or physical activity level.

[00120] For example, a physician suggested starting dose for a given therapeutic can be 1000 mg daily, with subject specific dosing having a range of 500 mg to a maximum of 2550 mg daily. The particular dosing for a subject can be determined by a clinician by titrating the dose and measuring the therapeutic response. The therapeutic dosing level can be determined by measuring fasting plasma glucose levels and measuring glycosylated hemoglobin. A sub- therapeutic amount can be any level that would be below the recommended dosing of the given therapeutic. For example, if a subject’s therapeutic dosing level is determined to be 700 mg daily, a dose of 600 mg would be a sub-therapeutic amount. Alternatively, a sub -therapeutic amount can be determined relative to a group of subjects rather than an individual subject. For example, if the average therapeutic amount of a given therapeutic for subjects with weights over 300 lbs is 2000 mg, then a sub-therapeutic amount can be any amount below 2000 mg. In some embodiments, the dosing can be recommended by a healthcare provider including, but not limited to a patient’s physician, nurse, nutritionist, pharmacist, or other health care

professional. A health care professional may include a person or entity that is associated with the health care system. Examples of health care professionals may include surgeons, dentists, audiologists, speech pathologists, physicians (including general practitioners and specialists), physician assistants, nurses, midwives, pharmaconomists/pharmacists, dietitians, therapists, psychologists, physical therapists, phlebotomists, occupational therapists, optometrists, chiropractors, clinical officers, emergency medical technicians, paramedics, medical laboratory technicians, radiographers, medical prosthetic technicians social workers, and a wide variety of other human resources trained to provide some type of health care service.

[00121] The invention provides for compositions that can comprise any combination of agents, such as leucine, metabolites of leucine, such as HMB or KIC, and FXR agonists (e.g. obeti cholic acid), that have been isolated from one or more sources. The agents can be isolated from natural sources or created from synthetic sources and then enriched to increase the purity of the components. Obeticholic acid can be created from a synthetic source and then enriched by one or more purification methods. Additionally, leucine (e.g., free leucine), can be isolated from a natural source and then enriched by one or more separations. The isolated and enriched components, such as, e.g., obeticholic acid, metformin, resveratrol, icariin, sildenafil, free leucine, HMB, and KIC can then be formulated for administration to a subject in any

combination.

Dosage of branched chain amino acid

[00122] Any of the compositions described herein can comprise an amount or dose of a branched chain amino acid, such as leucine, in free amino acid form, and/or a metabolite thereof.

Any methods described herein can comprise administering a dose of a branched chain amino acid in free amino acid form, and/or a dose of a metabolite thereof. The dose of the branched chain amino acid in free amino acid form, or metabolite thereof, can be a therapeutic dose. The dose of the branched chain amino acid in free amino acid form or metabolite thereof can be a sub-therapeutic dose. A sub-therapeutic dose of leucine in free amino acid form can be about, less than about, or more than about 0.25 - 3.0 g (e.g. 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, or more g). A sub-therapeutic dose of leucine in free amino acid form can be about, less than about, or more than about 0.25 - 3.0 g/day (e.g. 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, or more g/day). In some embodiments, the method comprises administering less than 3.0 g leucine in free amino acid form per day. In some embodiments, the composition comprises at least 500 mg of leucine. The composition may comprise at least 1000 mg of leucine. In some

embodiments, the composition comprises about 50-1000 mg of leucine. For example, the composition may comprise about 500-700 mg of leucine. In some embodiments, the composition comprises at least 50 mg of the metabolite. In certain embodiments, the composition comprises at least 200 mg of the metabolite. A sub -therapeutic dose of HMB can be about, less than about, or more than about 0.05 - 3.0 g (e.g. 0.05, 0.1, 0.2, 0.4, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, or more g). A sub-therapeutic dose of HMB can be about, less than about, or more than about 0.05 - 3.0 g/day (e.g. 0.05, 0.1, 0.2, 0.4, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, or more g/day). A sub-therapeutic dose of KIC can be about, less than about, or more than about 0.1 - 3.0 g (e.g. 0.1, 0.2, 0.4, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, or more g). A sub-therapeutic dose of KIC can be about, less than about, or more than about 0.1 - 3.0 g/day (e.g. 0.1, 0.2, 0.4, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, or more g/day).

Dosage of FXR agonist

[00123] Any compositions described herein can comprise an amount or dose of a FXR agonist, such as obeti cholic acid (OCA), chenodeoxycholic acid (CDCA), cholic acid (CA), PX-104, PX- 102, GW4064, WAY-362450, a compound of Formula (I), a pharmaceutically acceptable salt thereof, or a derivative thereof. Any methods described herein can comprise administering a dose of a FXR agonist disclosed herein. In some embodiments, the composition comprises about 0.1- 1500 mg of the FXR agonist. In certain embodiments, the composition comprises about 0.1-150 mg of the FXR agonist. In other embodiments, the composition comprises about 500-1000 mg.

In certain embodiments, the composition comprises about 0.1-25 mg. In some embodiments, the composition may comprise about 0.1-1500 mg/day of the FXR agonist. In certain embodiments, the composition comprises about 0.1-150 mg/day of the FXR agonist. In other embodiments, the composition comprises about 500-1000 mg/day. In certain embodiments, the composition comprises about 0.1-25 mg/day. The composition may comprise about 0.1-5 mg or 5-25 mg of the FXR agonist. In some embodiments, the composition may comprise less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 mg of the FXR agonist. In some embodiments, the composition comprises about 0.1-25 mg/kg of the FXR agonist, such as about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 mg/kg, which can also be the daily dosage. [00124] The dose of the FXR agonist can be a therapeutic dose. The therapeutic dose can be about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, 100 mg, 150 mg, 300 mg, 500 mg, 1000 mg, or 1500 mg. The therapeutic dose can be about 5 mg/day, about 10 mg/day, about 15 mg/day, about 20 mg/day, about 25 mg/day, about 30 mg/day, about 35 mg/day, about 40 mg/day, about 45 mg/day, about 50 mg/day, 100 mg/day, 150 mg/day, 300 mg/day, 500 mg/day, 1000 mg/day, or 1500 mg/day.

[00125] The dose of the FXR agonist can be a sub -therapeutic dose. In some embodiments, the sub-therapeutic dose is about 0.1 mg, about 0.5 mg, about 1 mg, about 1.5 mg, about 2 mg, about 2.5 mg, about 3 mg, about 3.5 mg, about 4 mg, or about 4.5 mg. The sub-therapeutic dose can be about 0.1 mg/day, about 0.5 mg/day, about 1 mg/day, about 1.5 mg/day, about 2 mg/day, about 2.5 mg/day, about 3 mg/day, about 3.5 mg/day, about 4 mg/day, or about 4.5 mg/day.

[00126] The FXR agonist can be administered as a unit dose. The unit dose of the FXR agonist can be about 0.1, 1, 5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 500, 1000, 1250, or 1500 mg.

[00127] In some embodiments, the composition comprises about 1-150 mg of OCA. The composition can comprise about 1-50 mg of OCA. In certain embodiments, the composition comprises about 1-25 mg of OCA, such as 1, 5, 10, 15, 20, or 25 mg. The composition can comprise less than or around about 5, 10, 25, or 50 mg. In some embodiments, the composition comprises about 1-150 mg/day of OCA. The composition can comprise about 1-50 mg/day of OCA. In certain embodiments, the composition comprises about 1-25 mg/day of OCA, such as 1, 5, 10, 15, 20, or 25 mg/day. The composition can comprise less than or around about 5, 10, 25, or 50 mg/day.

[00128] In some embodiments, the composition comprises about 100-1000 mg of CDCA. The composition can comprise about 500-1000 mg of CDCA, such as 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 800, 825, 850, 900, 950, or 1000 mg. In some embodiments, the composition comprises about 100-1000 mg of CDCA. The composition can comprise about 500- 1000 mg/day of CDCA, such as 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 800, 825, 850, 900, 950, or 1000 mg/day.

[00129] In some embodiments, the composition comprises about 500-1500 mg of CA. In certain embodiments, the composition comprises about 750-1250 mg of CA, such as 750, 800, 825, 850, 900, 950, 1000, 1050, 1100, 1150, 1200 or 1250 mg. In some embodiments, the composition comprises about 500-1500 mg/day of CA. In certain embodiments, the composition comprises about 750-1250 mg/day of CA, such as 750, 800, 825, 850, 900, 950, 1000, 1050, 1100, 1150, 1200 or 1250 mg/day. [00130] In some embodiments, the composition comprises about 0.1-10 mg of PX-104. The composition can comprise about 3-7 mg of PX-10, such as 3, 4, 5, 6, or 7 mg. In certain embodiments, the composition comprises about 5 mg of PX-104. In some embodiments, the composition comprises about 0.1-10 mg/day of PX-104. The composition can comprise about 3- 7 mg/day of PX-10, such as 3, 4, 5, 6, or 7 mg/day. In certain embodiments, the composition comprises about 5 mg/day of PX-104.

[00131] In some embodiments, the composition comprises about 1-500 mg of WAY-362450.

In certain embodiments, the composition comprises about 10-450 mg of WAY-362450, such as 10, 50, 100, 150, 200, 250, 300, 350, 400, or 450 mg. In some embodiments, the composition comprises about 1-500 mg/kg of WAY-362450. In certain embodiments, the composition comprises about 10-450 mg/kg of WAY-362450, such as 10, 50, 100, 150, 200, 250, 300, 350, 400, or 450 mg/kg.

[00132] Any of the above agents can be administered in unit doses. Any of the above agents in the amounts described herein can be administered in unit doses. A unit dose can be a fraction of the daily dose, such as the daily dose divided by the number of unit doses to be administered per day. A unit dose can be a fraction of the daily dose that is the daily dose divided by the number of unit doses to be administered per day and further divided by the number of unit doses (e.g. tablets) per administration. The number of unit doses per administration may be about, less than about, or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. The number of doses per day may be about, less than about, or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. The number of unit doses per day may be determined by dividing the daily dose by the unit dose, and may be about, less than about, or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, 20, or more unit doses per day. For example, a unit dose can be about the daily dose or about 1/2, 1/3, 1/4, 1/5, 1/6, 1/7, 1/8, 1/9, 1/10 of the daily dose. A unit dose can be about one-third of the daily amount and administered to the subject three times daily. A unit dose can be about one- half of the daily amount and administered to the subject twice daily. A unit dose can be about one-fourth of the daily amount with two unit doses administered to the subject twice daily. In some embodiments, a unit dose comprises about, less than about, or more than about 1, 2, 3, 4, 5, 10, 15, 25, or 50 mg of OCA. In some embodiments, a unit dose comprises about, less than about, or more than about 500, 750, or 1000 mg of leucine. In some embodiments, a unit dose comprises about, less than about, or more than about 200 mg of one or more leucine metabolites. In some embodiments, a unit dose (e.g. a unit dose comprising leucine) is administered as two unit doses two times per day. In some embodiments, a unit dose (e.g. a unit dose comprising one or more leucine metabolites, such as HMB) is administered as one unit dose two times per day. In some embodiments, a unit dose comprises about 250-1250 mg of leucine or its metabolite, and about 0.1-100 mg of the FXR agonist. In certain embodiments, a unit dose comprises about 500- 1250 mg of leucine or its metabolite, and about 0.1-25 mg of the FXR agonist.

[00133] In some embodiments, the amount of leucine is about 90-99.5 wt% of the total weight of the composition, such as 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 99.5 wt%. In some embodiments, the amount of the leucine metabolite is about 85-99.5 wt% of the total weight of the composition, such as 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 99.5 wt%. In some embodiments, the amount of the FXR agonist (such as OCA) is about 0.5-15 wt% of the total weight of composition.

[00134] The agents described herein (e.g., branched chain amino acid in free amino acid form, metabolites thereof, FXR agonists) can be administered to a subject orally or by any other methods. Methods of oral administration include administering the composition as a liquid, a solid, or a semi-solid that can be taken in the form of a dietary supplement or food stuff.

[00135] The agents described herein can be co-administered. The agents can be administered simultaneously, e.g., in a single composition, or can be administered sequentially. The agents can be administered sequentially within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 30, 60, 90, or 120 minutes from each other.

[00136] The agents described herein can be administered periodically. For example, the agents can be administered one, two, three, four times a day, or even more frequent. The subject can be administered every 1, 2, 3, 4, 5, 6 or 7 days. In some embodiments, the agents are administered three times daily. The administration can be concurrent with meal time of a subject. The period of treatment or diet supplementation can be for about 1, 2, 3, 4, 5, 6, 7, 8, or 9 days, 2 weeks, 1- 11 months, or 1 year, 2 years, 5 years or even longer. In some embodiments, the subject is administered the agents for six weeks or more. In some embodiments of the invention, the dosages that are administered to a subject can change or remain constant over the period of treatment. For example, the daily dosing amounts can increase or decrease over the period of administration.

[00137] The length of the period of administration and/or the dosing amounts can be determined by a physician, a nutritionist, or any other type of clinician. The physician, nutritionist, or clinician can observe the subject’s response to the administered compositions and adjust the dosing based on the subject’s performance. For example, dosing for subjects that show reduced effects in energy regulation can be increased to achieve desired results. Formulations and Pharmaceutical Compositions

[00138] Any of the agents described herein can be administered to the subject in one or more compositions. In some aspects, the present disclosure provides a pharmaceutical composition comprising a composition disclosed herein and at least one pharmaceutically acceptable excipient. A composition for use in practicing any of the methods of the invention can comprise any combination of the agents described herein. For example, an invention composition can comprise one, two, three, four, or more than four of the agents described herein.

[00139] Compositions described herein can be compounded into a variety of different dosage forms. For example, compositions can be formulated for oral administration, e.g., as a tablet, chewable tablet, caplets, capsule, soft gelatin capsules, lozenges or solution. Compositions can be formulated as a nasal spray or for injection when in its solution form. In some embodiments, the composition is a liquid composition suitable for oral consumption. In some embodiments, the components of the composition are formulated in the same tablet, capsule, pill or solution.

Alternatively, the components of the composition are formulated in separate tablets, capsules, pills or solutions.

[00140] In some embodiments, the agents are formulated into a composition suitable for oral administration. Compositions of the invention suitable for oral administration can be presented as discrete dosage forms, such as capsules, cachets, or tablets, or liquids or aerosol sprays each containing a predetermined amount of an active ingredient as a powder or in granules, a solution, or a suspension in an aqueous or non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil liquid emulsion, including liquid dosage forms (e.g., a suspension or slurry), and oral solid dosage forms (e.g., a tablet or bulk powder). Oral dosage forms may be formulated as tablets, pills, dragees, capsules, emulsions, lipophilic and hydrophilic suspensions, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by an individual or a patient to be treated. Such dosage forms can be prepared by any of the methods of formulation. For example, the active ingredients can be brought into association with a carrier, which constitutes one or more necessary ingredients. Capsules suitable for oral administration include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. Optionally, the inventive composition for oral use can be obtained by mixing a composition a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).In general, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation. For example, a tablet can be prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as powder or granules, optionally mixed with an excipient such as, but not limited to, a binder, a lubricant, an inert diluent, and/or a surface active or dispersing agent. Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

[00141] The compositions may be in liquid form. Exemplary liquid forms, which may be formulated for oral administration or for administration by injection, include aqueous solution, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil as well as elixirs and similar pharmaceutical vehicles. Suitable dispersing or suspending agents for aqueous suspensions include synthetic natural gums, such as tragacanth, acacia, alginate, dextran, sodium

carboxymethyl cellulose, methylcellulose, polyvinylpyrrolidone or gelatin. A method of the invention may comprise administering to a subject a combination of an injectable composition and a composition for oral administration.

[00142] Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for reconstitution with water or other suitable vehicles before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters or ethyl alcohol); preservatives (e.g., methyl or propyl p-hydroxybenzoates or sorbic acid); and artificial or natural colors and/or sweeteners.

[00143] The preparation of pharmaceutical compositions of this invention can be conducted in accordance with generally accepted procedures for the preparation of pharmaceutical

preparations. See, for example, Remington's Pharmaceutical Sciences 18th Edition (1990), E. W. Martin ed., Mack Publishing Co., PA. Depending on the intended use and mode of

administration, it may be desirable to process the magnesium-counter ion compound further in the preparation of pharmaceutical compositions. Appropriate processing may include mixing with appropriate non-toxic and non-interfering components, sterilizing, dividing into dose units, and enclosing in a delivery device.

[00144] Embodiments of the invention further encompass anhydrous compositions and dosage forms comprising an active ingredient, since water can facilitate the degradation of some compounds. For example, water may be added (e.g., 5%) in the arts as a means of simulating long-term storage in order to determine characteristics such as shelf-life or the stability of formulations over time. Anhydrous compositions and dosage forms of the invention can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions. Compositions and dosage forms of the invention which contain lactose can be made anhydrous if substantial contact with moisture and/or humidity during manufacturing, packaging, and/or storage is expected. An anhydrous composition may be prepared and stored such that its anhydrous nature is maintained. Accordingly, anhydrous compositions may be packaged using materials known to prevent exposure to water such that they can be included in suitable formulary kits. Examples of suitable packaging include, but are not limited to, hermetically sealed foils, plastic or the like, unit dose containers, blister packs, and strip packs.

[00145] An agent described herein can be combined in an intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier can take a wide variety of forms depending on the form of preparation desired for administration. In preparing the compositions for an oral dosage form, any of the usual pharmaceutical media can be employed as carriers, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, and the like in the case of oral liquid preparations (such as suspensions, solutions, and elixirs) or aerosols; or carriers such as starches, sugars, micro-crystalline cellulose, diluents, granulating agents, lubricants, binders, and disintegrating agents can be used in the case of oral solid preparations, in some embodiments without employing the use of lactose. For example, suitable carriers include powders, capsules, and tablets, with the solid oral preparations. If desired, tablets can be coated by standard aqueous or nonaqueous techniques.

[00146] Some examples of materials which may serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

[00147] Binders suitable for use in dosage forms include, but are not limited to, corn starch, potato starch, or other starches, gelatin, natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose, pre-gelatinized starch, hydroxypropyl methyl cellulose, microcrystalline cellulose, and mixtures thereof.

[00148] Lubricants which can be used to form compositions and dosage forms of the invention include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, com oil, and soybean oil), zinc stearate, ethyl oleate, ethylaureate, agar, or mixtures thereof. Additional lubricants include, for example, a syloid silica gel, a coagulated aerosol of synthetic silica, or mixtures thereof. A lubricant can optionally be added, in an amount of less than about 1 weight percent of the composition.

Lubricants can be also be used in conjunction with tissue barriers which include, but are not limited to, polysaccharides, polyglycans, seprafilm, interceed and hyaluronic acid.

[00149] Disintegrants may be used in the compositions of the invention to provide tablets that disintegrate when exposed to an aqueous environment. Too much of a disintegrant may produce tablets which may disintegrate in the bottle. Too little may be insufficient for disintegration to occur and may thus alter the rate and extent of release of the active ingredient(s) from the dosage form. Thus, a sufficient amount of disintegrant that is neither too little nor too much to detrimentally alter the release of the active ingredient(s) may be used to form the dosage forms of the compounds disclosed herein. The amount of disintegrant used may vary based upon the type of formulation and mode of administration, and may be readily discernible to those of ordinary skill in the art. About 0.5 to about 15 weight percent of disintegrant, or about 1 to about 5 weight percent of disintegrant, may be used in the pharmaceutical composition. Disintegrants that can be used to form compositions and dosage forms of the invention include, but are not limited to, agar-agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrilin potassium, sodium starch glycolate, potato or tapioca starch, other starches, pre-gelatinized starch, other starches, clays, other algins, other celluloses, gums or mixtures thereof.

[00150] Examples of suitable fillers for use in the compositions and dosage forms disclosed herein include, but are not limited to, talc, calcium carbonate (e.g., granules or powder), microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof.

[00151] When aqueous suspensions and/or elixirs are desired for oral administration, the active ingredient therein may be combined with various sweetening or flavoring agents, coloring matter or dyes and, if so desired, emulsifying and/or suspending agents, together with such diluents as water, ethanol, propylene glycol, glycerin and various combinations thereof.

[00152] The tablets can be uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate can be employed. Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin or olive oil.

[00153] Compositions may include a solubilizer to ensure good solubilization and/or dissolution of the compound of the present invention and to minimize precipitation of the compound of the present invention. This can be useful for compositions for non-oral use, e.g., compositions for injection. A solubilizer may also be added to increase the solubility of the hydrophilic drug and/or other components, such as surfactants, or to maintain the composition as a stable or homogeneous solution or dispersion.

[00154] The composition can further include one or more pharmaceutically acceptable additives and excipients. Such additives and excipients include, without limitation, detackifiers, anti-foaming agents, buffering agents, polymers, antioxidants, preservatives, chelating agents, viscomodulators, tonicifiers, flavorants, colorants, odorants, opacifiers, suspending agents, binders, fillers, plasticizers, lubricants, and mixtures thereof. A non-exhaustive list of examples of excipients includes monoglycerides, magnesium stearate, modified food starch, gelatin, microcrystalline cellulose, glycerin, stearic acid, silica, yellow beeswax, lecithin,

hydroxypropylcellulose, croscarmellose sodium, and crospovidone.

[00155] The compositions described herein can also be formulated as extended-release, sustained-release or time-release such that one or more components are released over time.

Delayed release can be achieved by formulating the one or more components in a matrix of a variety of materials or by microencapsulation. The compositions can be formulated to release one or more components over a time period of 4, 6, 8, 12, 16, 20, or 24 hours. The release of the one or more components can be at a constant or changing rate.

[00156] Using the controlled release dosage forms provided herein, the one or more cofactors can be released in its dosage form at a slower rate than observed for an immediate release formulation of the same quantity of components. In some embodiments, the rate of change in the biological sample measured as the change in concentration over a defined time period from administration to maximum concentration for an controlled release formulation is less than about 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the rate of the immediate release formulation. Furthermore, in some embodiments, the rate of change in concentration over time is less than about 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the rate for the immediate release formulation.

[00157] In some embodiments, the rate of change of concentration over time is reduced by increasing the time to maximum concentration in a relatively proportional manner. For example, a two-fold increase in the time to maximum concentration may reduce the rate of change in concentration by approximately a factor of 2. As a result, the one or more cofactors may be provided so that it reaches its maximum concentration at a rate that is significantly reduced over an immediate release dosage form. The compositions of the present invention may be formulated to provide a shift in maximum concentration by 24 hours, 16 hours, 8 hours, 4 hours, 2 hours, or at least 1 hour. The associated reduction in rate of change in concentration may be by a factor of about 0.05, 0.10, 0.25, 0.5 or at least 0.8. In certain embodiments, this is accomplished by releasing less than about 30%, 50%, 75%, 90%, or 95% of the one or more cofactors into the circulation within one hour of such administration.

[00158] Optionally, the controlled release formulations exhibit plasma concentration curves having initial (e.g., from 2 hours after administration to 4 hours after administration) slopes less than 75%, 50%, 40%, 30%, 20% or 10% of those for an immediate release formulation of the same dosage of the same cofactor.

[00159] In some embodiments, the rate of release of the cofactor as measured in dissolution studies is less than about 80%, 70%, 60% 50%, 40%, 30%, 20%, or 10% of the rate for an immediate release formulation of the same cofactor over the first 1, 2, 4, 6, 8, 10, or 12 hours.

[00160] The controlled release formulations provided herein can adopt a variety of formats. In some embodiments, the formulation is in an oral dosage form, including liquid dosage forms (e.g., a suspension or slurry), and oral solid dosage forms (e.g., a tablet or bulk powder), such as, but not limited to those, those described herein. [00161] The controlled release tablet of a formulation disclosed herein can be of a matrix, reservoir or osmotic system. Although any of the three systems is suitable, the latter two systems can have more optimal capacity for encapsulating a relatively large mass, such as for the inclusion of a large amount of a single cofactor, or for inclusion of a plurality of cofactors, depending on the genetic makeup of the individual. In some embodiments, the slow-release tablet is based on a reservoir system, wherein the core containing the one or more cofactors is encapsulated by a porous membrane coating which, upon hydration, permits the one or more cofactors to diffuse through. Because the combined mass of the effective ingredients is generally in gram quantity, an efficient delivery system can provide optimal results.

[00162] Thus, tablets or pills can also be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate. In some embodiments, a formulation comprising a plurality of cofactors may have different cofactors released at different rates or at different times. For example, there can be additional layers of cofactors interspersed with enteric layers.

[00163] Methods of making sustained release tablets are known in the art, e.g., see U.S. Patent Publications 2006/051416 and 2007/0065512, or other references disclosed herein. Methods such as described in U.S. Patent Nos. 4,606,909, 4,769,027, 4,897,268, and 5,395,626 can be used to prepare sustained release formulations of the one or more cofactors determined by the genetic makeup of an individual. In some embodiments, the formulation is prepared using OROS® technology, such as described in U.S. Patent Nos. 6,919,373, 6,923,800, 6,929,803, and 6,939,556. Other methods, such as described in U.S. Patent Nos. 6,797,283, 6,764,697, and 6,635,268, can also be used to prepare the formulations disclosed herein.

[00164] In some embodiments, the compositions can be formulated in a food composition. For example, the compositions can be a beverage or other liquids, solid food, semi-solid food, with or without a food carrier. For example, the compositions can include a black tea supplemented with any of the compositions described herein. The composition can be a dairy product supplemented any of the compositions described herein. In some embodiments, the compositions can be formulated in a food composition. For example, the compositions can comprise a beverage, solid food, semi-solid food, or a food carrier.

[00165] In some embodiments, liquid food carriers, such as in the form of beverages, such as supplemented juices, coffees, teas, sodas, flavored waters, and the like can be used. For example, the beverage can comprise the formulation as well as a liquid component, such as various deodorant or natural carbohydrates present in conventional beverages. Examples of natural carbohydrates include, but are not limited to, monosaccharides such as, glucose and fructose; disaccharides such as maltose and sucrose; conventional sugars, such as dextrin and cyclodextrin; and sugar alcohols, such as xylitol and erythritol. Natural deodorant such as taumatin, stevia extract, levaudioside A, glycyrrhizin, and synthetic deodorant such as saccharin and aspartame may also be used. Agents such as flavoring agents, coloring agents, and others can also be used. For example, pectic acid and the salt thereof, alginic acid and the salt thereof, organic acid, protective colloidal adhesive, pH controlling agent, stabilizer, a preservative, glycerin, alcohol, or carbonizing agents can also be used. Fruit and vegetables can also be used in preparing foods or beverages comprising the formulations discussed herein.

[00166] Alternatively, the compositions can be a snack bar supplemented with any of the compositions described herein. For example, the snack bar can be a chocolate bar, a granola bar, or a trail mix bar. In yet another embodiment, the present dietary supplement or food

compositions are formulated to have suitable and desirable taste, texture, and viscosity for consumption. Any suitable food carrier can be used in the present food compositions. Food carriers of the present invention include practically any food product. Examples of such food carriers include, but are not limited to food bars (granola bars, protein bars, candy bars, etc.), cereal products (oatmeal, breakfast cereals, granola, etc.), bakery products (bread, donuts, crackers, bagels, pastries, cakes, etc.), beverages (milk-based beverage, sports drinks, fruit juices, alcoholic beverages, bottled waters), pastas, grains (rice, corn, oats, rye, wheat, flour, etc.), egg products, snacks (candy, chips, gum, chocolate, etc.), meats, fruits, and vegetables. In an embodiment, food carriers employed herein can mask the undesirable taste (e.g., bitterness). Where desired, the food composition presented herein exhibit more desirable textures and aromas than that of any of the components described herein. For example, liquid food carriers may be used according to the invention to obtain the present food compositions in the form of beverages, such as supplemented juices, coffees, teas, and the like. In other embodiments, solid food carriers may be used according to the invention to obtain the present food compositions in the form of meal replacements, such as supplemented snack bars, pasta, breads, and the like. In yet other embodiments, semi-solid food carriers may be used according to the invention to obtain the present food compositions in the form of gums, chewy candies or snacks, and the like.

[00167] The dosing of the combination compositions can be administered about, less than about, or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times a daily. A subject can receive dosing for a period of about, less than about, or greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days, weeks or months. A unit dose can be a fraction of the daily dose, such as the daily dose divided by the number of unit doses to be administered per day. A unit dose can be a fraction of the daily dose that is the daily dose divided by the number of unit doses to be administered per day and further divided by the number of unit doses (e.g. tablets) per administration. The number of unit doses per administration may be about, less than about, or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. The number of doses per day may be about, less than about, or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. The number of unit doses per day may be determined by dividing the daily dose by the unit dose, and may be about, less than about, or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, 20, or more unit doses per day. For example, a unit dose can be about 1/2, 1/3, 1/4, 1/5, 1/6, 1/7, 1/8, 1/9, 1/10. A unit dose can be about one-third of the daily amount and administered to the subject three times daily. A unit dose can be about one-half of the daily amount and administered to the subject twice daily. A unit dose can be about one-fourth of the daily amount with two unit doses administered to the subject twice daily. In some embodiments, a unit dose comprises about, less than about, or more than about 50 mg resveratrol. In some embodiments, a unit dose comprises about, less than about, or more than about 550 mg leucine. In some embodiments, a unit dose comprises about, less than about, or more than about 200 mg of one or more leucine metabolites. In some embodiments, a unit dose (e.g. a unit dose comprising leucine) is administered as two unit doses two times per day. In some embodiments, a unit dose (e.g. a unit dose comprising one or more leucine metabolites, such as HMB) is administered as one unit dose two timer per day. Compositions disclosed herein can further comprise a flavorant and can be a solid, liquid, gel or emulsion.

Kits

[00168] The invention also provides kits. The kits include one or more compositions described herein, in suitable packaging, and written material that can include instructions for use, discussion of clinical studies, listing of side effects, and the like. Such kits may also include information, such as scientific literature references, package insert materials, clinical trial results, and/or summaries of these and the like, which indicate or establish the activities and/or advantages of the composition, and/or which describe dosing, administration, side effects, drug interactions, or other information useful to the health care provider. Such information may be based on the results of various studies, for example, studies using experimental animals involving in vivo models and studies based on human clinical trials. In some embodiments, the compound or components of composition of the present disclosure are provided as separate compositions in separate containers within the kit. In some embodiments, the compound or components of the composition of the present disclosure are provided as a single composition within a container in the kit. The kit may further contain another agent. In some embodiments, the compound or components of the composition of the present disclosure and the agent are provided as separate compositions in separate containers within the kit. In some embodiments, the compound or components of the composition of the present disclosure and agent are provided as a single composition within a container in the kit. Suitable packaging and additional articles for use (e.g., measuring cup for liquid preparations, foil wrapping to minimize exposure to air, and the like) are known in the art and may be included in the kit. Kits described herein can be provided, marketed and/or promoted to health providers, including physicians, nurses, pharmacists, formulary officials, and the like. Kits may also, in some embodiments, be marketed directly to the consumer.

[00169] In some embodiments, the molar ratio of (i) a branched chain amino acids and/or metabolites thereof to (ii) an FXR agonist (for example, leucine to OCA, or HMB to OCA) is less or greater than about 1-5000 (e.g. about 1, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700,

800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3250, 3300, 3500, 4000, 4500, or 5000). In some embodiments, the molar ratio of (i) to (ii) is less or greater than about 100-3210 such as about 130, 320, 640, 800, 1070, 1600, or 3210.

[00170] In some embodiments, the dosing of leucine, any metabolites of leucine, the FXR agonist (e.g. OCA) can be designed to achieve a specified physiological concentration or circulating level of leucine, metabolites of leucine and/or a FXR agonist. The physiological concentration can be a circulating level as measured in the blood stream of a subject. The subject can be a human or an animal. A selected dosing can be altered based on the characteristics of the subject, such as weight, rate of energy metabolism, genetics, ethnicity, height, or any other characteristic. The amount of leucine in a unit dose can be such that the circulating level of leucine in a subject is about or greater than about 0.25 mM, 0.5 mM, 0.75 mM, or 1 mM. A dosing of about 1,125 mg leucine (e.g., free leucine), can achieve a circulating level of leucine in a subject that is about 0.5 mM. A dosing of about 300 mg leucine (e.g., free leucine), can achieve a circulating level of leucine in a subject that is about 0.25 mM. The amount of OCA in a unit dose can be such that the circulating level of OCA in a subject is about or greater than about 25 nM, 50 nM, 70 nM, 90 nM, 100 nM, 125 nM, 145 nM, 175 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM or 600 nM. A dosing of about 5 mg OCA can achieve a circulating level of OCA in a subject that is about 68.5 nM. A dosing of about 10 mg OCA can achieve a circulating level of OCA in a subject that is about 144.1 nM. A dosing of about 25 mg OCA can achieve a circulating level of OCA in a subject that is about 298.4 nM. In some embodiments, the circulating molar ratio of leucine to OCA is about or greater than 100, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000. In certain embodiments, the circulating molar ratio of leucine to OCA is about or greater than 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000. In an embodiment, the circulating molar ratio of leucine to OCA is about or greater than 7,300. In some embodiments, the molar or mass ratios are circulating molar or mass ratios achieved after administration one or more compositions to a subject. The compositions can be a combination composition described herein. The molar ratio of a combination composition in a dosing form can be adjusted to achieve a desired circulating molar ratio. The molar ratio can be adjusted to account for the bioavailability, the uptake, and the metabolic processing of the one or more components of a combination composition. For example, if the bioavailability of a component is low, then the molar amount of a that component can be increased relative to other components in the combination composition. In some embodiments, the circulating molar or mass ratio is achieved within about 0.1, 0.5, 0.75, 1, 3, 5, or 10, 12, 24, or 48 hours after administration. The circulating molar or mass ratio can be maintained for a time period of about or greater than about 0.1, 1, 2, 5, 10, 12, 18, 24, 36, 48, 72, or 96 hours.

[00171] The compositions can be administered to a subject such that the subject is

administered a selected total daily dose of the composition. The total daily dose can be determined by the sum of doses administered over a 24 hour period. The total daily dose of the composition can include about 0.1, 1, 5, 10, 15, 20, 25, 50, 100, 150, 300, 400, 500, 750, 1000, or 1500 mg of OCA. The total daily dose of the composition can include at least about 250, 500, 750, 1000, 1125, 2000, 2250 mg, 2550 mg or more of a branched chain amino acid or metabolite thereof. The branched chain amino acid can be leucine, HMB, or any other branched chain amino acid described herein. EXAMPLES

[00172] Example 1 : Effects of chronic high fat diet on liver mass and hepatic lipid accumulation.

[00173] Amylin Liver NASH (AMLN) mice are used as an experimental model of NASH. Male 5-week old C57BL/6J mice, housed five animals/cage under standard experimental rodent environmental conditions, are fed ad libitum for 35 weeks either control (standard chow) or AMLN diet (D09100301, Research Diets): 40% fat (18% trans fat); 40% carbohydrate (20% fructose); and 2% cholesterol with ad libitum access to water. Following 35 weeks of NASH induction, blood is drawn for baseline biochemistry and baseline biopsies are obtained; the animals are then randomized to treatment groups, with stratification of the NASH groups to balance for NAFLD Activity Score (NAS) score and fibrosis. As shown in Table 1, the animals are then randomized to the treatment groups, with stratification of the NASH groups to balance for NAS score and fibrosis.

Table 1. Treatment Groups

Animals remain on these treatments for eight weeks, following which blood was drawn for plasma biochemistry and animals were then euthanized and liver collected for histological, biochemical and molecular analysis.

[00174] Data for all examples are analyzed via one-way analysis of variance; when ANOVA showed significant differences, means are separated via Tukey’s multiple comparison test (Graphpad Prism, Version 6.0).

[00175] Treatment with leucine in the absence of obeticholic is expected to exert no significant effect on fibrosis and to regress steatosis, inflammation, and ballooning by at least one stage in 25-50% of the animals. Treatment with low-dose obeticholic acid (10 mg/kg) is expected to exert no significant effect on any element of NASH, while the higher dose (30 mg/kg) is expected to regress steatosis in 80% of the animals, inflammation in 50% of the animals, ballooning in 10% of the animals, and fibrosis in 30%. In contrast, combination of the leucine with low-dose (10 mg/kg) obeti cholic acid is expected to exhibit a synergistic effect, regressing steatosis in 80% of the animals, fibrosis in 50-60% of the animals, and ballooning and inflammation in 50-70% of the animals. Similarly, combining leucine with the higher dose of obeticholic acid is expected to increase the efficacy of obeticholic acid, as demonstrated by regressing fibrosis in 60-80% of the animals and ballooning and inflammation in 70-90% of the animals.

[00176] Example 2: OCA-Leucine Synergy

[00177] Cell Culture: HepG2 cells were seeded with 20,000 cells per well in 96 well plates in low (5.5 mM) glucose DMEM containing 10% fetal bovine serum (FBS) and antibiotics (1% penicillin-streptomycin (PS)) and grown at 37°C in 5% C0₂ in air. When cells were -80% confluent, media was changed to high glucose (25 mM) DMEM (10% FBS, 1% PS) plus 100 nM insulin for 48 hours for lipid accumulation except the low glucose control wells.

[00178] Treatment: Cells were treated with either 100 nM, 1 uM or 10 uM obeticholic acid (OCA) with or without 0.5 mM Leucine for 48 hours in high glucose DMEM, except the low glucose controls.

[00179] Analysis: At the end of the treatment time, media was removed and cell lysis, reverse transcription, and RT-PCR were performed using the TaqMan® Gene Expression Cells-to™ Kit (Life Technologies, Cat # 4399002) according to the manufacturer’s instructions. Gene expression was assessed by RT-PCR using StepOnePlus™ PCR system (Thermo Fisher

Scientific) and TaqMan Gene expression assays for FXR (Life Technologies, Cat #

Hs0l026590_ml), SREBP1 (Life Technologies, Cat # HS01088679), and FAS (Life

Technologies, Cat # Hs00236330_ml).

[00180] Results: The lowest dose of OCA (100 nM) exerted no significant effect on Famesoid X Receptor (FXR) expression. However, addition of leucine resulted in a 76% increase in FXR expression vs. control (FIG. 4), although leucine exerted no independent effect. Similar effects were seen at 1 mM OCA. 10 mM OCA caused a 2.24 fold increase in FXR expression in the absence of leucine, and the addition of leucine markedly enhanced this effect with a 3.8-fold increase, confirming a synergistic effect of leucine and OCA on FXR expression (FIG. 4).

[00181] The lowest dose of OCA (100 nM) exerted no significant effect on the expression of either of the two key lipogenic genes studied, fatty acid synthase (FAS) and Sterol Regulatory Element Binding Protein 1 (SREBP1). However, addition of leucine to the lowest dose of OCA (100 nM) resulted in a 52% reduction in FAS (FIG. 5) and a 78% reduction in SREBP1 expression (FIG. 6). Higher doses of OCA were sufficient to maximally inhibit FAS and SREBP1 expression and thus there was no further augmentation of the effect with the addition of leucine. However, the effects at the lowest concentration of OCA demonstrate synergy between leucine and OCA in suppressing these lipogenic enzymes (FIG. 5 and FIG.6).

[00182] It should be understood from the foregoing that, while particular implementations have been illustrated and described, various modifications can be made thereto and are contemplated herein. It is also not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the preferable embodiments herein are not meant to be construed in a limiting sense. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions,

configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. Various modifications in form and detail of the embodiments of the invention will be apparent to a person skilled in the art. It is therefore contemplated that the invention shall also cover any such modifications, variations and equivalents.

Claims

What is claimed is:

1 A composition, comprising:

i) leucine, or a metabolite or pharmaceutically acceptable salt thereof; and ii) at least one famesoid X receptor (FXR) agonist, or a pharmaceutically acceptable salt thereof.

2. The composition of claim 1, the FXR agonist is obeti cholic acid (OCA), chenodeoxycholic acid (CDCA), cholic acid (CA), PX-104, PX-102, GW4064, WAY-362450 or a pharmaceutically acceptable salt thereof.

3. The composition of claim 1 or 2, wherein the FXR agonist is obeti cholic acid (OCA), or a pharmaceutically acceptable salt thereof.

4. A composition, comprising:

i) leucine, or a metabolite or pharmaceutically acceptable salt thereof; and ii) a compound represented by Formula I:

pharmaceutically acceptable salt thereof, wherein:

R¹, R², R³, and R⁴ are independently selected from hydrogen, halogen, -OR¹⁰, -SR¹⁰, - N(R¹⁰)₂, -S(0)R¹⁰, -S(0)₂R¹⁰, -C(0)R¹⁰, -C(0)OR¹⁰, -OC(0)R¹⁰, -N0₂, =0, =S, =N(R¹⁰), -CN, optionally substituted C_MO alkyl, optionally substituted C_2-lo alkenyl, and optionally substituted

C_2-io alkynyl;

R⁵ is selected from -OR¹⁰, -

N(R¹⁰)₂, -C(O)N(R¹⁰)₂, -C(O)N(R¹⁰)₂C(O)O(R¹⁰), -C(O)N(R¹⁰)₂S(O)₃(R¹⁰), - C(0)R¹⁰, -C(0)OR¹⁰, -OC(0)R¹⁰, -OS(0)₃(R¹⁰), -SR¹⁰, -S(0)R¹⁰, and -S(0)₂R¹⁰; C _O alkyl, C_2- io alkenyl, C_2-i0 alkynyl, each of which is independently optionally substituted at each occurrence with one or more substituents selected from halogen, -OR¹⁰, -SR¹⁰, -C(O)N(R¹⁰)₂, - N(R¹⁰)C(O)R¹⁰, -N(R¹⁰)C(O)N(R¹⁰)₂, -N(R¹⁰)₂, -C(0)R¹⁰, -C(0)OR¹⁰, -OC(0)R¹⁰, -N0₂, =0,

=S, =N(R¹⁰), -CN, C 3-i2 carbocycle, and 3- to 12-ineinbered heterocycle, and C3-12 carbocycle, and 3- to 12-membered heterocycle, wherein each C₃_i2 carbocycle, and 3- to 12-membered heterocycle in R⁴ is independently optionally substituted with one or more substituents selected from halogen, -OR¹⁰, -SR¹⁰, -C(O)N(R¹⁰)₂, -N(R¹⁰)C(O)R¹⁰, -N(R¹⁰)C(O)N(R¹⁰)₂, -N(R¹⁰)₂, - C(0)R¹⁰, -C(0)OR¹⁰, -OC(0)R¹⁰, -N0₂, =0, =S, =N(R¹⁰), -CN, C^ alkyl, C_2-6 alkenyl, and C_2-6 alkynyl; and

R¹⁰ is independently selected at each occurrence from the group consisting of hydrogen, optionally substituted Ci-io alkyl, optionally substituted C_2-l0 alkenyl, optionally substituted C₂- _!o alkynyl, optionally substituted C_3-i2 carbocycle, and optionally substituted 3- to l2-membered heterocycle.

5. The composition of claim 4, wherein R¹ is an optionally substituted C_l-l0 alkyl.

6. The composition of claim 4 or 5, wherein R¹ is an optionally substituted Ci_-6 alkyl.

7. The composition of any one of claims 4-6, wherein R¹ is an optionally substituted C_2-3 alkyl.

8. The composition of any one of claims 4-7, wherein R³ is selected from the group consisting of hydrogen, -OR¹⁰, and -N(R¹⁰)₂.

9. The composition of claim 8, wherein R³ is -OR¹⁰.

10. The composition of claim 9, wherein R³ is -OH.

11. The composition of any one of claims 4-10, wherein R² and R⁴ are independently selected from hydrogen, -OR¹⁰, and optionally substituted C_MO alkyl.

12. The composition of claim 11, wherein R² is hydrogen.

13. The composition of claim 11, wherein R⁴ is hydrogen.

14. The composition of any one of claims 4-13, wherein R⁵ is selected from the group consisting of -OR¹⁰, -N(R¹⁰)₂, -C(O)N(R¹⁰)₂, -C(O)N(R¹⁰)₂C(O)O(R¹⁰), -C(O)N(R¹⁰)₂S(O)₃(R¹⁰), -C(0)R¹⁰, -C(0)OR¹⁰, -OC(0)R¹⁰, -OS(0)₃(R¹⁰), -SR¹⁰, -S(0)R¹⁰, and -S(0)₂R¹⁰.

15. The composition of claim 14, wherein R⁵ is -C(0)OR¹⁰.

16. The composition of claim 14 or 15, wherein R⁵ is -C(0)0H.

17. The composition of any one of claims 4-16, wherein the compound is:

, or a pharmaceutically acceptable salt thereof.

18. The composition of any one of claims 1-17, wherein the metabolite is b -hydroxy b -methylbutyrate (HMB) or keto-isocaproic acid (KIC).

19. The composition of claim 18, wherein the metabolite is HMB.

20. The composition of any one of claims 1-19, wherein the composition comprises at least 500 mg of leucine.

21. The composition of any one of claims 1-19, wherein the composition comprises at least 1000 mg of leucine.

22. The composition of any one of claims 1-19, wherein the composition comprises about 50-1000 mg of leucine.

23. The composition of any one of claims 1-19, wherein the composition comprises about 500-700 mg of leucine.

24. The composition of any one of claims 1-19, wherein the composition comprises at least 200 mg of the metabolite.

25. The composition of any one of claims 1-19, wherein the composition comprises a subtherapeutic amount of component (i).

26. The composition of any one of claims 1-19, wherein the composition comprises about 0.1-1500 mg of component (ii).

27. The composition of any one of claims 1-19, wherein the composition comprises about 0.1-150 mg of component (ii).

28. The composition of any one of claims 1-19, wherein the composition comprises about 0.1-25 mg of component (ii).

29. The composition of any one of claims 1-19, wherein the composition comprises a subtherapeutic amount of component (ii).

30. The composition of any one of claims 1-19, wherein the amount of leucine is about 90-99.5 wt % of the total weight of components (i) and (ii).

31. The composition of any one of claims 1-19, wherein the amount of the metabolite is about 85-99.5 wt % of the total weight of components (i) and (ii).

32. The composition of any one of claims 1-19, wherein the amount of component (ii) is about 0.5-15 wt % of the total weight of components (i) and (ii).

33. The composition of any one of claims 1-32, wherein the composition is formulated as a tablet, capsule, pill or solution.

34. The composition of any one of claims 1-33, wherein the composition is formulated as a unit dose.

35. The composition of claim 34, wherein the composition is formulated as a unit dose comprising about 250-1250 mg of component (i) and about 0.1-100 mg of component (ii).

36. The composition of claim 34, wherein the composition is formulated as a unit dose comprising about 500-1250 mg of component (i) and about 0.1-25 mg of component (ii).

37. The composition of any one of claims 1-36, wherein component (i) and component (ii) are formulated in the same tablet, capsule, pill or solution.

38. The composition of any one of claims 1-36, wherein component (i) and component (ii) are formulated in separate tablets, capsules, pills or solutions.

39. The composition of any one of claims 1-38, wherein the composition is substantially free of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine in free amino acid form.

40. The composition of any one of claims 1-38, wherein the composition is substantially free of alanine, glutamic acid, glycine, isoleucine, valine, and proline in free amino acid form.

41. The composition of any one of claims 1-38, wherein the composition is substantially free of isoleucine and valine in free amino acid form.

42. A pharmaceutical composition, comprising a composition of any one of claims 1- 41 and at least one pharmaceutically acceptable excipient.

43. A method of treating a metabolic inflammation-mediated disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of a composition of any one of claims 1-41 or a pharmaceutical composition of claim 42.

44. The method of claim 43, wherein the metabolic inflammation-mediated disease or disorder is diabetes, metabolic syndrome, dyslipidemia, obesity, hypertension, insulin resistance, non-alcoholic fatty liver disease (NAFLD), or any combination thereof.

45. The method of claim 44, wherein the NAFLD is non-alcoholic steatohepatitis (NASH).

46. The method of claim 44, wherein the diabetes is diabetes mellitus type 2.

47. A method of treating a hepatic disease, disorder, or injury, comprising

administering to a subject in need thereof a therapeutically effective amount of a composition of any one of claims 1-41 or a pharmaceutical composition of claim 42.

48. The method of any one of claims 43-47, wherein the composition is administered orally.

49. The method of any one of claims 43-48, wherein the composition is administered at least once daily.

50. The method of any one of claims 43-48, wherein the composition is administered at least twice daily.

51. The method of any one of claims 43-50, wherein component (i) and component (ii) are administered simultaneously, approximately simultaneously, or sequentially in any order.

52. The method of claim 51, wherein component (i) and component (ii) are administered simultaneously or approximately simultaneously.

53. The method of claim 51, wherein component (i) and component (ii) are administered sequentially.

54. The method of claim 53, wherein component (i) is administered before component (ii).

55. The method of claim 53, wherein component (i) is administered after component (ii).

56. The method of any one of claims 43-55, wherein the subject in need is human.

57. The method of any one of claims 43-56, wherein the subject in need exhibits at least one or more symptoms associated with a metabolic inflammation-mediated disease or disorder.

58. A kit comprising a composition of any one of claims 1-41 and instructions.

59. The kit of claim 58, wherein the composition is administered to a subject in need thereof at least once a day.

60. The kit of claim 58, wherein the composition is administered to a subject in need thereof at least twice a day.

61. A kit comprising a pharmaceutical composition of claim 42 and instructions.

62. The kit of claim 61, wherein the composition is administered to a subject in need thereof at least once a day.

63. The kit of claim 61, wherein the composition is administered to a subject in need thereof at least twice a day.

64. The kit of any one of claims 58-63, wherein component (i) and component (ii) are provided in a single composition.

65. The kit of any one of claims 58-63, wherein component (i) and component (ii) are provided in separate compositions.

66. The kit of any one of claims 58-65, further comprising a pharmaceutical active agent.