WO2010120880A1

WO2010120880A1 - Treatment of liver diseases with a caspase inhibitor

Info

Publication number: WO2010120880A1
Application number: PCT/US2010/031031
Authority: WO
Inventors: John Pollard; John J. Alam
Original assignee: Vertex Pharmaceuticals Incorporated
Priority date: 2009-04-14
Filing date: 2010-04-14
Publication date: 2010-10-21

Abstract

The present disclosure provides a method of using VX-166, a pan caspase inhibitor, for treating or reducing the symptoms of liver diseases, such as Non- Alcoholic Fatty Liver Disease (NAFLD), nonalcoholic steatohepatitis (NASH), alcoholic steatohepatitis (ASH), and other diseases involving fibrosis, steatosis, or inflammation of the liver. The present disclosure also relates to methods for identifying agents useful for treating these diseases. The present disclosure also relates to additional therapeutics that can be used in combination with VX-166 for the treatment of these diseases.

Description

TREATMENT OF LIVER DISEASES WITH A CASPASE INHIBITOR

Background of the Invention

[0001] Caspases are a family of cysteine protease enzymes that are key mediators in inflammation and signaling pathways for apoptosis and cell disassembly [WO 99/47545, Thornberry et al, Chem. Biol, 5, 1998 pp. R97-R103].

[0002] Apoptosis contributes to the progression of many liver diseases, such as viral hepatitis, Wilson's disease, cholestatic liver disease and alcohol induced injury [Galle PR, Krammer PH. CD95-induced apoptosis in human liver disease. Semin. Liver Dis. 1998;18: 141-51]. Nonalcoholic Fatty Liver Disease is a potentially progressive liver disease that culminates in cirrhosis. Cirrhosis occurs more often in individuals with nonalcoholic steatohepatitis (NASH) than in those with steatosis (NAFLD). One significant difference between NAFLD and NASH is the extent of hepatocyte apoptosis, which is more extensive in NASH. In all these diseases, hepatocyte apoptosis acts as a contributing mechanism for fibrogenesis and cirrhosis [Yoon JH, Gores GJ "Death receptor-mediated apoptosis and the liver" J Hepatol. 2002;37:400-10]. [0003] The utility of caspase inhibitors to treat disease states associated with an increase in cellular apoptosis has been demonstrated using peptidic caspase inhibitors. However, due to their peptidic nature, such inhibitors are typically characterized by undesirable pharmacological properties, such as poor cellular penetration and cellular activity, poor oral absorption, poor stability and rapid metabolism [JJ. Plattner and D. W. Norbeck, in Drug Discovery Technologies, CR. Clark and W.H. Moos, Eds. (Ellis Horwood, Chichester, England, 1990), pp. 92-126].

[0004] Therefore, there is a need for small molecule, caspase inhibitors that can treat liver diseases by blocking inflammation and/or apoptosis, both of which are known drivers of diseases such as Non- Alcoholic Fatty Liver Disease (NAFLD), nonalcoholic steatohepatitis (NASH), alcoholic steatohepatitis (ASH), and diseases involving fibrosis, steatosis, and inflammation of the liver. Summary of the Invention

[0005] The present disclosure provides a method of using VX- 166, a pan caspase inhibitor, for treating or reducing the symptoms of liver diseases, such as Non- Alcoholic Fatty Liver Disease (NAFLD), nonalcoholic steatohepatitis (NASH), alcoholic steatohepatitis (ASH), and other diseases involving fibrosis, steatosis, or inflammation of the liver. The present disclosure also relates to methods for identifying agents useful for treating these diseases. The present disclosure also relates to additional therapeutics that can be used in combination with VX- 166 for the treatment of these diseases.

Brief Description of the Figures

[0006] FIGURE 1. BODY WEIGHTS IN WILD TYPE C57BL6 MICE (WT) AND DB/DB C57BL6 MICE FED WITH CHOW OR MCD DIET TREATED WITH VEHICLE ± VX-166 (VX). EFFECTS OF VX-166 ON TOTAL LIVER CHOLESTEROL AND NEFA IN CHOW-FED AND MCD DIET-FED DB/DB MICE. (A) Weight difference between age and sex matched WT and db/db mice; (B) Body weight distribution following MCD and VX-166 administration. Mice (n=12/ experimental VX treated group, and n=5/control) were weighed at the beginning of the experiment (Initial Body Weight) and at the time of sacrifice (i.e., after 4 or 8 weeks of diet treatment; Terminal Body Weight). * P < 0.05 versus untreated db/db mice. (E) Total Liver Cholesterol in chow fed and MCD diet fed db/db mice after VX-166 administration (F) Total Liver NEFA in chow fed and MCD diet fed db/db mice after VX-166 administration; for (E) and (F), mean + SEM are graphed. *P < 0.05, **P < 0.005

FIGURE 2. EFFECTS OF VX-166 ON LIVER HISTOLOGY AND HEPATIC TRIGLYCERIDE CONTENT IN CHOW-FED AND MCD DIET-FED DB/DB MICE. [0007] H&E-stained liver sections from representative mice fed MCD diet + vehicle for 4 weeks (A) or 8 weeks (C), or MCD diet + VX-166 for 4 weeks (B) or 8 weeks (D). Liver histology remained constant throughout the study in chow- fed db/db mice, so histology from a representative db/db mouse after 8 weeks of diet treatment is shown (E), with histology from a representative chow- fed wild-type mouse displayed in the insert for comparison. Hepatic triglyceride content was assessed in all mice at the time of sacrifice and mean +/- SEM are graphed (F). *P < 0.05, **P < 0.005 FIGURE 3. EFFECTS OF VX- 166 ON SERUM ALT LEVELS IN CHOW-FED AND MCD DIET-FED DB/DB MICE.

[0008] Serum ALT was assessed in all mice at the time of sacrifice (i.e., after 4- or 8- weeks of treatment with chow or MCD diet +/- VX- 166). ALT values in a group ofage- and gender-matched chow-fed wild type C57BL6 mice are also displayed. Results are shown as Mean +/- SEM. * P < 0.05, ** P< 0.001.

FIGURE 4. EFFECT OF VX- 166 ON CELLULAR ACCUMULATION OF ACTIVE CASPASE 3 IN LIVERS OF CHOW-FED OR MCD DIET-FED DB/DB MICE. [0009] Immunohistochemistry for activated caspase 3 was performed on liver tissues obtained at the time of sacrifice (i.e., after either 4 weeks or 8 weeks of treatment with chow or MCD diet), db/db mice that received chow + vehicle had only very rare caspase 3- stained cells (data not shown). Similar results were noted in db/db mice that were fed chow + VX- 166. Results from representative chow- fed db/db mice that received VX- 166 for 4 weeks (A) or 8 weeks (B) are shown. MCD diet feeding for 4 weeks (C ,D) or 8 weeks (E,F) increased hepatic accumulation of cells that stained for active caspase 3. Sections from representative db/db mice that were treated with MCD diet + vehicle (C,E) had more caspase 3 -stained cells than sections from representative mice that received MCD diet + VX=166 (D,F).

FIGURE 5. EFFECTS OF VX- 166 ON HEPATIC CASPASE ACTIVITY DURING LONG-TERM TREATMENT OF CHOW-FED AND MCD DIET-FED DB/DB MICE. [0010] Representative Western blot and results of net densitometric analysis of caspase 3 cleavage product in mice treated with vehicle or VX- 166 (VX) for 4 weeks (A) or 8 weeks (B). Representative Western blot and results of net densitometric analysis of ILl β cleavage (C, E) and IL 18 cleavage product (D, F) in mice treated with vehicle or VX- 166 for 4 or 8 weeks. Results are shown as Mean +/- SEM. **P < 0.001.

FIGURE 6. EFFECT OF VX- 166 ON HEPATIC ACCUMULATION OF ALPHA- SMOOTH MUSCLE ACTIN (A-SMA).

[0011] Representative Western blot analysis for α-SMA, a marker of myo fibroblastic HSC, in mice treated with vehicle or VX- 166 for 4 weeks (A) or 8 weeks (B). Results in chow-fed or MCD diet-fed db/db mice are compared to α-SMA expression in age- and gender-matched wild type C57BL6 mice. Densitometric analysis of Western blot data from all mice in the various treatment groups at 4 weeks (C) and 8 weeks (D). Results are shown as Mean +/- SEM. * P < 0.05; ** P < 0.001.

FIGURE 7. EFFECTS OF VX- 166 ON HEPATIC FIBROGENESIS IN CHOW FED- AND MCD DIET-FED DB/DB MICE.

[0012] Chow-fed db/db mice had similar findings on Sirius red-stained liver sections as wild type C57BL6 mice (insert), regardless of whether they received vehicle or VX-166, so results from a representative chow- fed db/db mice that received vehicle for 8 weeks are shown (A). Sections from representative db/db mice treated with MCD diets + vehicle for 4 weeks (B) or 8 weeks (C); or MCD diets + VX-166 for 4 weeks (C) or 8 weeks (E). QRT-PCR analysis of collagen lαl mRNA expression (F) and hydroxyproline content (G) in livers of age- and gender-matched wild type C57BL6 mice (NRL) and db/db mice in the various treatment groups at 4 weeks and 8 weeks. Results are shown as Mean +/- SEM. * P < 0.05; **P < 0.001.

FIGURE 8. HISTOLOGICAL ANALYSIS OF STEATOSIS, INFLAMMATION, BALLOONING, AND OVERAL NAS (NAFLD ACTIVITY SCORE) IN MCD- AND VX-166 TREATED DB/DB AND WILD TYPE MICE

FIGURE IX: EFFECTS OF DIET AND TREATMENT ON HEPATIC CK- 18 STAINING

[0013] The %CK-18 positive cells per high powered field were recorded as a measure of caspase-3 activity and apoptosis. Significant increases in CK- 18 cleavage products were seen in both study arms. VX-166 reduced CK-18 in the HFD (2.67±0.32% vs.l.71±0.17%, p=0.05) and MCD arms (4.81±0.51% vs. 1.77±0.38%, pO.0001). VX-166 also significantly lowered CK-18 levels compared to vehicle in the MCD arm (4.09±0.40% vs. 1.77±0.38%, p=0.002) with a similar trend in the HFD arm. No effect of TPGS/PEG vehicle observed. Original magnification x400. * p<0.05. FIGURE 2X: EFFECTS OF DIET AND TREATMENT ON HEPATIC PCNA STAINING

[0014] The %PCNA positive cells per high powered field were recorded as a measure of cell turnover/death. Significant increases in %PCNA positive staining cells over control diet were seen in the HFD-Steatosis (1.34±0.34% vs. 1.87±0.15%, p=0.041) and MCD- Steatohepatitis (1.34±0.18% vs. 3.70±0.39%, p<0.001) arms. Amongst HFD fed animals, VX- 166 significantly reduced PCNA% vs. HFD-only (p=0.004) and vs. TPGS/PEG (p=0.004). A similar trend was observed in the MCD arm. No effect of TPGS/PEG vehicle observed. Original magnification x400. * p<0.05.

FIGURE 3X: EFFECTS OF VX- 166 ON STEATOHEP ATITIS [0015] Representative histological images showing the effects of HFD and differing treatments on NAFLD as assessed by H&E staining. VX- 166 treated HFD-fed mice exhibited significantly less hepatic inflammation than untreated mice (p=0.018). The effect did not alter overall NASH score. Amongst MCD-fed animals, VX- 166 significantly reduced inflammation (p=0.03) and NASH Score (p=0.006). TPGS/PEG vehicle had no significant effect on any one aspect of histological score. Original magnification x400.

FIGURE 4X: EFFECTS OF VX- 166 TREATMENT ON HEPATIC OXIDATIVE STRESS

[0016] Overall oxidative stress was relatively low in the HFD-steatosis arm, changes did not reach statistical significance although VX- 166 appeared to reduce TBARS levels. (B) Significantly higher TBARS levels were detected in MCD fed animals compared to control diet (p<0.0001). VX-166 treated MCD-fed mice exhibited reduced TBARS (p=0.024). A trend in the TPGS/PEG vehicle group was not statistically significant. * p<0.05.

FIGURE 5X: CHANGES IN INFLAMMATORY GENE EXPRESSION [0017] Fold-change in hepatic gene expression relative to Db/m-Control Diet fed (phenotypically normal) mice. MCD feeding produced significant increases in (A) TNFα and (B) MCP-I expression over control diet (* p<0.01). These were significantly reduced by treatment (# p<0.05) however no significant differences between VX-166 and TPGS/PEG vehicle were detected. FIGURE 6X: HEPATIC FIBROSIS IN MCD-STEATOHEPATITIS ARM [0018] Fibrotic changes were minimal across all groups. Histologically, no significant effect of VX- 166 treatment or vehicle was observed although both were associated with a similar reduction in collagen expression. Original magnification x400.

SUPPLEMENTARY FIGURE IX: EFFECTS OF VX- 166 ON STEATOSIS SCORE AND %AREA STEATOSIS IN HFD AND MCD DIET-FED C3H AND DB/DB MICE.

SUPPLEMENTARY FIGURE 2X:

TABLE 1 : STUDY GROUPS & TREATMENT DETAILS

TABLE 2: BIOCHEMICAL CHANGES FROM PRE- AND POST TREATMENT

Detailed Description of the Invention

[0019] The present disclosure provides a method of using VX-166, a pan caspase inhibitor, for treating or reducing the symptoms of liver diseases, such as Non- Alcoholic Fatty Liver Disease (NAFLD), nonalcoholic steatohepatitis (NASH), alcoholic steatohepatitis (ASH), and diseases involving fibrosis, steatosis, and inflammation of the liver. The present disclosure also relates to methods for identifying agents useful for treating these diseases. The present disclosure also relates to additional therapeutics that can be used in combination with VX-166 for the treatment of these diseases.

[0020] One embodiment provides a compound of formula I (VX-166):

I (VX-166); or a pharmaceutical composition comprising said compound to said subject for use in ameliorating, treating and/or preventing certain liver diseases, including, but not limited to, Non- Alcoholic Fatty Liver Disease (NAFLD), nonalcoholic steatohepatitis (NASH), alcoholic steatohepatitis (ASH), and fibrosis, steatosis, and inflammation of the liver. VX- 166 is particularly effective at reducing key drivers of NASH such as hepatitic apoptosis and hepatic inflammation. VX- 166 is effective at improving diet-induced steatosis and inhibiting hepatic fibrogenesis. The compounds disclosed herein, including VX-166, are pan-caspase inhibitors. These compounds can be assayed according to methods known in the art (see Examples herein, WO 2004/106304).

[0021] Another embodiment provides a composition comprising VX-166 or a pharmaceutically acceptable derivative (e.g., salt) thereof, as described above, and a pharmaceutically acceptable carrier for use in treating the diseases mentioned herein. [0022] According to another embodiment, the compositions disclosed herein may further comprise another therapeutic agent. Such agents include, but are not limited to, agents used for treating liver diseases, such as Non-Alcoholic Fatty Liver Disease (NAFLD), nonalcoholic steatohepatitis (NASH), alcoholic steatohepatitis (ASH), and fibrosis, steatosis, and inflammation of the liver. Examples of such agents include, but are not limited to, TPGS (Vitamin E) and PEG.

[0023] The term "pharmaceutically acceptable carrier" refers to a non-toxic carrier that may be administered to a patient, together with a compound of this invention, and which does not destroy the pharmacological activity thereof.

[0024] Pharmaceutically acceptable carriers that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene -polyoxypropylene-block polymers, polyethylene glycol and wool fat.

[0025] In pharmaceutical compositions comprising only a compound of this invention as the active component, methods for administering these compositions may additionally comprise the step of administering to the subject an additional agent. Such agents include, but are not limited to, agents used for treating liver diseases, such as Non-Alcoholic Fatty Liver Disease (NAFLD), nonalcoholic steatohepatitis (NASH), alcoholic steatohepatitis (ASH), and fibrosis, steatosis, and inflammation of the liver. [0026] In some embodiments, said therapeutic agent is selected from insulin sensitizers, antioxidants, hepatoprotective agents, or a lipid lowering drug. In some embodiments, said insulin sensitizer is selected from metformin, pioglitazone, thiazolidninediones, or rosiglitazone; said antioxidant is selected from vitamin E or vitamin C; said hepatoprotective agents is selected from Angiotensin Converting Enzyme (ACE) Inhibitors, betaine, ursodeoxycholic acid, or pentoxyfylline; and said lipid lowering drug is Probucol.

[0027] In yet another embodiments, said additional agent is Vitamin E (TPGS). In yet another embodiment, said additional agent is PEG.

[0028] When a second agent is used, the second agent may be administered either as a separate dosage form or as part of a single dosage form with the compounds or compositions of this invention.

[0029] The amount of compound present in the above-described compositions should be sufficient to cause a detectable decrease in the severity of the disease, or in caspase inhibition, the levels of biomarkers associated with caspase inhibition. [0030] If pharmaceutically acceptable salts are utilized, those salts are preferably derived from inorganic or organic acids and bases. Included among such acid salts are the following: acetate, adipate, alginate, aspartate, benzoate, benzene sulfonate, bisulfate, butyrate, citrate, camphorate, camphor sulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2- hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3 -phenyl-propionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate and undecanoate. Base salts include ammonium salts, alkali metal salts, such as sodium and potassium salts, alkaline earth metal salts, such as calcium and magnesium salts, salts with organic bases, such as dicyclohexylamine salts, N-methyl-D-glucamine, and salts with amino acids such as arginine, lysine, and so forth.

[0031] Also, the basic nitrogen-containing groups can be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates, such as dimethyl, diethyl, dibutyl and diamyl sulfates; long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; aralkyl halides, such as benzyl and phenethyl bromides and others. Water or oil-soluble or dispersible products are thereby obtained.

[0032] The compounds utilized in the compositions and methods disclosed herein may also be modified by appending appropriate functionalities to enhance selective biological properties. Such modifications are known in the art and include those which increase biological penetration into a given biological system (e.g., blood, lymphatic system, or central nervous system), increase oral availability, increase solubility to allow administration by injection, alter metabolism and/or alter rate of excretion. [0033] According to a preferred embodiment, the compositions of this invention are formulated for pharmaceutical administration to a subject or patient, e.g., a mammal, preferably a human being. Such pharmaceutical compositions are used to ameliorate, treat or prevent liver diseases in a subject and comprise a compound that inhibits caspase and a pharmaceutically acceptable carrier.

[0034] Such pharmaceutical compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection and infusion techniques. Preferably, the compositions are administered orally or intravenously. [0035] Sterile injectable forms of the compositions of this invention may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3- butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or di-glycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil and castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long- chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.

[0036] If a solid carrier is used, the preparation can be in tablet form, placed in a hard gelatin capsule in powder or pellet form, or in the form of a troche or lozenge. The amount of solid carrier will vary, e.g., from about 25 to 800 mg, preferably about 25 mg to 400 mg. When a liquid carrier is used, the preparation can be, e.g., in the form of a syrup, emulsion, soft gelatin capsule, sterile injectable liquid such as an ampule or nonaqueous liquid suspension. Where the composition is in the form of a capsule, any routine encapsulation is suitable, for example, using the aforementioned carriers in a hard gelatin capsule shell. [0037] A syrup formulation can consist of a suspension or solution of the compound in a liquid carrier for example, ethanol, glycerin, or water with a flavoring or coloring agent. An aerosol preparation can consist of a solution or suspension of the compound in a liquid carrier such as water, ethanol or glycerin; whereas in a powder dry aerosol, the preparation can include e.g., a wetting agent.

[0038] Formulations of the present invention comprise an active ingredient together with one or more acceptable carrier(s) thereof and optionally any other therapeutic ingredient(s). The carrier(s) should be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. [0039] The pharmaceutical compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, and aqueous suspensions or solutions. In the case of tablets for oral use, carriers that are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added. [0040] Alternatively, the pharmaceutical compositions of this invention may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols. [0041] It will be recognized by one of skill in the art that the form and character of the pharmaceutically acceptable carrier or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration, and other well- known variables. The above-described compounds and compositions are also useful in therapeutic applications relating to certain diseases. Such certain diseases include liver diseases, such as Non- Alcoholic Fatty Liver Disease (NAFLD), nonalcoholic steatohepatitis (NASH), alcoholic steatohepatitis (ASH), and diseases involving fibrosis, steatosis, and inflammation of the liver.

[0042] According to another embodiment, an active ingredient compound of this invention is administered to a subject at a dose of between about 1 mg to about 10,000 mg per administration. In another embodiment, an active ingredient compound of this invention is administered to a subject at a dose of between about 100 mg to about 2,400 mg per administration.

[0043] Typically, the pharmaceutical compositions of this invention will be administered from about 1 to 5 times per day or alternatively, as a continuous infusion. Such administration can be used as a chronic or acute therapy. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. A typical preparation will contain from about 5% to about 95% active compound (w/w). Preferably, such preparations contain from about 20% to about 80% active compound. [0044] When the compositions disclosed herein comprise a combination of a compound disclosed herein and one or more additional therapeutic agents, both the compound and the additional agent should be present at dosage levels of between about 10% to about 80% of the dosage normally administered in a monotherapy regime. [0045] Upon improvement of a patient's condition, a maintenance dose of a compound, composition or combination of this invention may be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained. When the symptoms have been alleviated to the desired level, treatment may cease. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence or disease symptoms.

[0046] It should also be understood that a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the judgment of the treating physician and the severity of the particular disease being treated. The amount of active ingredients will also depend upon the particular compound and other therapeutic agent, if present, in the composition.

[0047] Accordingly, a method for ameliorating, treating or preventing a disease of this invention in a subject comprises the step of administering to the subject any compound, pharmaceutical composition, or combination described herein.

[0048] One embodiment provides a method for ameliorating, treating, or preventing steatohepatitis in a subject, comprising administering a compound of formula I:

i; or a pharmaceutical composition comprising said compound to said subject.

[0049] In some embodiments, the steatohepatitis is non-alcoholic fatty liver disease

(NAFLD), nonalcoholic steatohepatitis (NASH), or alcoholic steatohepatitis (ASH). In some embodiments, the steatohepatitis is nonalcoholic steatohepatitis (NASH). In other embodiments, the steatohepatitis is alcoholic steatohepatitis (ASH).

[0050] In some embodiments, the compound of formula I ameliorates, treats, or prevents steatohepatitis by reducing inflammation in the liver. In other embodiments, the compound of formula I ameliorates, treats, or prevents steatohepatitis by reducing apoptosis. In yet other embodiments, the compound of formula I ameliorates, treats, or prevents steatohepatitis by reducing liver fibrosis.

[0051] Another embodiment provides a method of reducing inflammation in the liver of a subject with steatohepatitis, comprising administering a compound of formula I:

I; or a pharmaceutical composition comprising said compound to said subject. [0052] In some embodiments, the steatohepatitis is non-alcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), or alcoholic steatohepatitis (ASH). In some embodiments, the steatohepatitis is nonalcoholic steatohepatitis (NASH). In other embodiments, the steatohepatitis is alcoholic steatohepatitis (ASH). [0053] Another embodiment provides a method for reducing apoptosis in the liver of a subject with steatohepatitis, comprising administering a compound of formula I:

i; or a pharmaceutical composition comprising said compound to said subject.

[0054] In some embodiments, the steatohepatitis is non-alcoholic fatty liver disease

[0055] Another embodiment provides a method for ameliorating, treating, or preventing fibrosis in a subject, comprising administering a compound of formula I:

i; or a pharmaceutical composition comprising said compound to said subject. In some embodiments, the fibrosis is caused by a fibrotic disease. In some embodiments, the fibrosis is liver fibrosis. In some embodiments, the fibrosis is caused by non-alcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), or alcoholic steatohepatitis (ASH). In some embodiments, the fibrosis is caused by nonalcoholic steatohepatitis (NASH). In other embodiments, the fibrosis is caused by alcoholic steatohepatitis (ASH).

[0056] Another embodiment provides a method for ameliorating, treating, or preventing steatosis in a subject, comprising administering a compound of formula I:

i; or a pharmaceutical composition comprising said compound to said subject. [0057] In some embodiments, the steatosis is caused by obesity, insulin resistance, metabolic syndrome, or viral infection. In other embodiments, the steatosis is caused by non-alcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), or alcoholic steatohepatitis (ASH). In some embodiments, the steatosis is caused by nonalcoholic steatohepatitis (NASH). In other embodiments, the steatosis is caused by alcoholic steatohepatitis (ASH).

[0058] In a preferred embodiment, the invention provides a method of treating a mammal, having one of the aforementioned diseases, comprising the step of administering to said mammal a pharmaceutically acceptable composition described above. In this embodiment, if the patient is also administered another therapeutic agent, it may be delivered together with the compound of this invention in a single dosage form, or, as a separate dosage form. When administered as a separate dosage form, the other therapeutic agent may be administered prior to, at the same time as, or following administration of a pharmaceutically acceptable composition comprising a compound of this invention. [0059] Another embodiment provides a method for identifying a compound or composition for ameliorating, treating or preventing a disease or condition selected from NASH, ASH, or a disease involving fibrosis, steatosis, or inflammation of the liver in a subject comprises administering to said subject a compound that inhibits caspase or a pharmaceutical composition comprising the compound and comparing the caspase inhibition in the subject before and after treatment with the compound. In some embodiments, said disease or condition is NASH, ASH, fibrotic disease, or steatosis. In some embodiments, said caspase inhibitor is the compound of formula I (VX- 166). [0060] Another embodiment provides a method for identifying a compound or composition for ameliorating, treating or preventing a disease selected from NASH, ASH, or a disease involving fibrosis, steatosis, or inflammation of the liver in a subject comprises administering to said subject a compound that inhibits caspase or a pharmaceutical composition comprising the compound and comparing a biomarker for NASH, ASH, fibrosis, steatosis, or inflammation of the liver in said subject before and after treatment with said compound. In some embodiments, said disease or condition is NASH, ASH, fibrotic disease, or steatosis. In some embodiments, said caspase inhibitor is the compound of formula I (VX- 166).

[0061] The term "biomarker" is a physical, functional, or biochemical indicator, e.g., the presence of a particular metabolite, of a physiological or disease process. Examples of biomarkers related to NASH, ASH, fibrosis, inflammation or steatosis in the liver include, but are not limited to, cytokeratin-18 (CK- 18), ALT, TNF-α, monocyte macrophage infiltration (MCP-I), hepatocyte proliferating cell nuclear antigen (PCNA), active caspase- 3, active caspase-1, IL-I, IL-18, α-SMA, TUNEL, and nitrotyrosine. [0062] In some embodiments, the compound is a compound of formula Ia. In other embodiments, the compound is a compound of formula Ib.

I-b.

[0063] All applications, patents and references disclosed herein are incorporated by reference. In order that this invention be more fully understood, the following preparative and testing examples are set forth. These examples are for the purpose of illustration only and are not to be construed as limiting the scope of the invention in any way. Examples

Synthetic Methods

Example 1

(S, S)-3-[2-(3-Acetylamino-2-oxo-2/f-pyridin-l-yl)-butyrylamino]-4-oxo-5-(2, 3,5,6- tetrafluoro-phenoxy)-pentanoic acid

Method A:

(S)-2-(3-Benzyloxycarbonylamino-2-oxo-2H-pyridin-l-yl)-butyric acid tert-butyl ester [0064] To a cooled (0⁰C) solution of (R)-tert-butyl hydroxybutyrate (1.03 g, 6.43 mmol) in dichloromethane (25 mL), was slowly added 2,6-lutidine (1.38 g, 12.9 mmol) and then trifluoromethanesulfonic anhydride (3.45 g, 12.2 mmol). The resulting mixture was stirred at 0⁰C for 1 hour, then partitioned between tert-butylmethyl ether (150 mL) and an aqueous solution of IM HCl (30 mL). The organic layer was washed with brine (30 mL), dried (sodium sulfate), filtered and concentrated to afford the triflate as a light brown oil.

[0065] To a solution of (2-oxo-l ,2-dihydro-pyridin-3-yl)-carbamic acid benzyl ester (P. Warner et al, J. Med. Chem., 37, 19, 1994, 3090-3099)(1.73 g, 7.07 mmol) in dry THF (60 mL) was added sodium hydride (60% dispersion, 257 mg, 6.43 mmol) and the solution was stirred at room temperature for 45 minutes. The reaction mixture was then slowly transferred with a canula onto a solution of the triflate prepared above in THF (3 mL). The reaction mixture was stirred at room temperature for 90 minutes and quenched with aqueous ammonium chloride (10 mL). Most of the solvent was evaporated and the residue was partitioned between EtOAc and saturated aqueous NH₄Cl. The organic layer was washed with brine (30 mL), dried (MgSO₄), filtered and evaporated. The residue was purified by flash chromatography (10% ethyl acetate/hexane) to afford the title compound as a colourless oil (2.48 g, 100%): ¹H NMR (400 MHz, CDCl₃) δ 0.92 (3H, t), 1.45(9H, s),

1.94(1H, m), 2.25(1H, m), 5.23 (2H, s), 5.47 (IH, dd), 6.32 (IH, t), 7.01 (IH, d), 7.32-7.43

(5H, m), 7.92 (IH, s), 8.06 (IH, br d).

Method B:

(S)-2-(3-Amino-2-oxo-2H-pyridin-l-yl)-butyric acid tert-butyl ester

[0066] To a solution of (S)-2-(3-Benzyloxycarbonylamino-2-oxo-2H-pyridin-l-yl)- butyric acid tert-butyl ester (2.48 g, 6.43 mmol) in a mixture of MeOH (15 mL) and EtOAc (15 mL) was added 10% Pd/C (250 mg). The mixture was degassed and stirred at room temperature for 90 minutes under an atmosphere of hydrogen (balloon pressure). The reaction mixture was filtered through a short pad of silica which was then flushed with MeOH. The combined filtrates were evaporated under reduced pressure to afford the title compound as a white solid (1.62 g, 100%); ¹H NMR (400 MHz, CDCl₃) δ 0.91 (3H, t), 1.44(9H, s), 1.91(1H, m), 2.21(1H, m), 4.24 (2H, br s), 5.50 (IH, dd), 6.11 (IH, t), 6.53 (IH, d), 6.77 (IH, d). Method C: (S)-2-(3-Acetylamino-2-oxo-2H-pyridin-l-yl)-butyric acid tert-butyl ester

[0067] To a cooled (0⁰C) solution of (S)-2-(3-Amino-2-oxo-2H-pyridin- 1 -yl)-butyric acid tert-butyl ester (500 mg, 1.98 mmol) in dichloromethane (5 mL) was added triethylamine (220 mg, 2.18 mmol) followed by acetic anhydride (202 mg, 1.98 mmol). The reaction mixture was stirred at room temperature for 12 hours and then partitioned between EtOAc and aqueous IM HCl. The organic layer was washed with saturated aqueous NaHCO₃, brine (30 mL), dried (MgSO₄), filtered and evaporated. The residue was purified by flash chromatography (40% ethyl acetate/hexane) to afford the title compound as a colourless oil (569 mg, 97%): ¹H NMR (400 MHz, CDCl₃) δ 0.87 (3H, t), 1.40(9H, s), 1.91(1H, m), 2.13 (3H, s), 2.19(1H, m), 5.38 (IH, dd), 6.26 (IH, t), 6.99 (IH, d), 8.33 (IH, d), 8.43 (IH, br s). Method D: (S)-2-(3-Acetylamino-2-oxo-2H-pyridin- 1 -yl)-butyric acid

[0068] A solution of (S)-2-(3-Acetylamino-2-oxo-2H-pyridin-l-yl)-butyric acid tert- butyl ester (569 mg, 1.93 mmol) in dichloromethane (5 mL) was cooled to 0⁰C. Trifluoroacetic acid (5 ml) was added and the resulting mixture allowed to warm to room temperature and stir for 2 hours. The mixture was then concentrated under reduced pressure and the residue redisolved in dichloromethane. This process was repeated several times in order to remove excess trifluoroacetic acid. The resulting solid was slurried in diethyl ether, filtered and washed with more diethyl ether. The solid was then dried to constant weight under vacuum. This gave the title product as a white solid (327 mg, 71%); ¹H NMR (400 MHz, d6-DMSO) δ 0.78 (3H, t), 2.02-2.17 (5H, m), 4.98 (IH, dd), 6.29 (IH, t), 7.35 (IH, d), 8.21 (IH, d), 9.30 (IH, s), 13.07 (IH, vbr s). Method E: (S, S)-3-[2-(3-Acetylamino-2-oxo-2H-pyridin-l-yl)-butyrylamino]-4-hydroxy-5-(2, 3,5,6- tetrafluoro-phenoxy)-pentanoic acid tert-hvXy\ ester

[0069] A stirred mixture of (S)-2-(3-Acetylamino-2-oxo-2H-pyridin-l-yl)-butyric acid (100 mg, 0.42 mmol), 3-amino-5-(2,3,5,6-tetrafluorophenoxy)-4-hydroxy-pentanoic acid tert-hutyl ester (163 mg, 0.462 mmol), ΗOBt (62 mg, 0.462 mmol), DMAP (56 mg, 0.462 mmol)and TΗF (5 mL) was cooled to 0⁰C then EDC (89 mg, 0.462 mmol) was added. The mixture was allowed to warm to room temperature during 16h then concentrated under reduced pressure. The residue was purified by flash chromatography (50-50% ethyl acetate/hexane) to afford the title compound as a white foam (221 mg, 92%); ¹H NMR (400 MHz, CDCl₃) δ 0.88-0.93 (3H, m), 1.37-1.38 (9H, 2s), 1.86-1.96 (IH, m), 2.15-2.25 (4H, m), 2.55-2.71 (2H, m), 3.70-4.64 (5H, m), 5.30-5.39 (IH, m), 6.30-6.35 (IH, m), 6.75-6.86 (IH, m), 7.17-7.31 (2H, m), 8.31-8.47 (2H, m). Method F:

(S, S)-3-[2-(3-Acetylamino-2-oxo-2H-pyridin-l-yl)-butyrylamino]-4-oxo-5-(2, 3,5,6- tetrafluoro-phenoxy)-pentanoic acid tert-butyi ester

[0070] A stirred solution of (S,S)-3-[2-(3-Acetylamino-2-oxo-2H-pyridin-l-yl)- butyrylamino]-4-hydroxy-5-(2,3,5,6-tetrafluoro-phenoxy)-pentanoic acid tert-butyi ester (221 mg, 0.385 mmol) in anhydrous DCM (10 rnL) was treated with l,l,l-triacetoxy-l,l- dihydro-l,2-benziodoxol-3(lΗ)-one (212 mg, 0.5 mmol) at 0⁰C. The resulting mixture was kept at 0⁰C for 2hr, diluted with ethyl acetate, then poured into a 1 : 1 mixture of saturated aqueous sodium hydrogen carbonate and saturated aqueous sodium thiosulfate. The organic layer was removed and the aqueous layer re-extracted with ethyl acetate. The combined organic extracts were dried (Magnesium sulfate) and concentrated. The residue was purified by flash chromatography (50-50% ethyl acetate/hexane) to afford the title compound as a white solid (187 mg, 85%); ¹H NMR (400 MHz, CDCl₃) δ 0.93 (3H, t), 1.36 (3H, s), 1.95 (IH, m), 2.21 (3H, s), 2.25 (IH, m), 2.73 (2H, dd), 2.89 (IH, dd), 4.91 (IH, m), 5.04-5.17 (2H, m), 5.47 (IH, m), 6.34 (IH, t), 6.80 (IH, m), 7.19 (IH, m), 7.68 (IH, d), 8.36-8.41 (2H, m). Method G:

(S,S)-3-[2-(3-Acetylamino-2-oxo-2H-pyridin-l-yl)-butyrylamino]-4-oxo-5-(2,3,5,6- tetrafluoro-phenoxy)-pentanoic acid

[0071] A solution of (S,S)-3-[2-(3-Acetylamino-2-oxo-2H-pyridin-l-yl)- butyrylamino]-4-oxo-5-(2,3,5,6-tetrafluoro-phenoxy)-pentanoic acid tert-butyl ester (187 mg, 0.327 mmol) in dichloromethane (5 niL) was cooled to 0⁰C. Trifluoroacetic acid (5 ml) was added and the resulting mixture allowed to warm to room temperature and stir for 2 hours. The mixture was then concentrated under reduced pressure and the residue redisolved in dichloromethane. This process was repeated several times in order to remove excess trifluoroacetic acid. The resulting solid was slurried in diethyl ether, filtered and washed with more diethyl ether. The solid was then dried to constant weight under vacuum. This gave the title product as a white solid (138 mg, 82%); ¹H NMR (400 MHz, d6-DMSO) δ 0.78 (3H, t), 1.87-2.13 (5H, m), 2.56-2.78 (2H, m), 4.62 (IH, m), 5.18-5.29 (2H, m), 5.40 (IH, m), 6.28 (IH, t), 7.37 (IH, d), 7.53-7.66 (IH, m), 8.17-8.21 (IH, m), 8.92 (IH, d), 9.21 (IH, s), 12.51 (IH, br s); ¹⁹F NMR (376 MHz, d6-DMSO, proton- decoupled) δ -156.9, -141.1; M+H 516.2, M-H 514.2.

Example 2

(S,S)-3-[2-(3-Methoxycarbonylamino-2-oxo-2H-pyridin-l-yl)-butyrylamino]-4-oxo-5- (2,3,5,6-tetrafluoro-phenoxy)-pentanoic acid (VX- 166)

[0072] Prepared from (S)-2-(3-Amino-2-oxo-2H-pyridin-l-yl)-butyric acid tert-butyi ester and methyl chloro formate according to methods C-G; pink solid; IR (solid) 1644, 1661, 1709 cm-1; ¹H NMR (400 MHz, d6-DMSO) δ 0.81 (3H, m), 1.95 (IH, m), 2.09 (IH, m), 2.50-2.98 (2H, m), 3.70 (3H, s), 4.20-5.50 (4H, m), 6.31 (IH, m), 7.40 (IH, m), 7.59 (IH, m), 7.82 (IH, m), 8.20 (IH, s), 8.55-9.00 (IH, d); ¹⁹F NMR (376 MHz, d6-DMSO, proton-decoupled) δ -140.6, -141.0, -141.1, -156.80, -156.9, -157.0, -157.1; M+H 532.3, M-H 530.3.

Biological Methods

Example 3 : Enzyme Assays

[0073] The assays for caspase inhibition are based on the cleavage of a fluorogenic substrate by recombinant, purified human Caspases -1, -3, or -8. The assays can be run in essentially the same way as those reported by Garcia-Calvo et al. (J. Biol. Chem. 273 (1998), 32608-32613), using a substrate specific for each enzyme. The substrate for Caspase-1 is Acetyl-Tyr-Val-Ala-Asp-amino-4-methylcoumarin. The substrate for Caspases -3 and -8 is Acetyl-Asp-Glu-Val-Asp-amino-4-methylcoumarin. Both substrates are known in the art.

[0074] The observed rate of enzyme inactivation at a particular inhibitor concentration, k_obs, is computed by direct fits of the data to the equation derived by Thornberry et al. (Biochemistry 33 (1994), 3943-3939) using a nonlinear least-squares analysis computer program (PRISM 2.0; GraphPad software). To obtain the second order rate constant, k_inact, kobs values are plotted against their respective inhibitor concentrations and k_inact values are subsequently calculated by computerized linear regression.

Animal Studies

[0075] The effectiveness of VX- 166 in the treatment of NASH/ASH and diseases involving fibrosis, steatosis, and inflammation of the liver may be demonstrated by the following examples. Effectiveness of a compound in NASH is determined by a combination of factors, including the following: NAFLD Activity Score (NAS), reduction of liver inflammation, inhibition of liver apoptosis, reduction of liver fibrosis and markers of liver fibrosis (such as α-SMA, collagenlαl, hydroxyproline); and reduction of liver injury (determined by biomarker levels such as CK- 18 or ALT). The following studies examine these various factors. Example 4 is an 8-week long study that demonstrates the effect of VX- 166 dosed at 6 mg/kg in a NASH model where NASH is induced simultaneously with VX- 166 treatment. Example 5 is a 4-week long study that demonstrates the effect of VX- 166 dosed at 2 mg/kg, treatment starting after the disease had been established.

Example 4: Dietary Model of Steatohepatitis

[0076] Male C57BL/6 obese and diabetic db/db (stock no. 699) and WT C57BL/6 (stock no. 664) mice were purchased from Jackson Laboratories (Bar Harbor, ME) and maintained in a temperature- and light-controlled facility. For all experiments, animals were age matched and used at approximately 8-10 weeks of age. To induce NASH and liver fibrosis, 35 db/db mice were fed Methionine Choline Deficient (MCD) diet. Control mice (n=35, db/db) were permitted ad libitum consumption of water and standard rodent food. In each group, 25 mice were gavaged once daily with the pan-caspase inhibitor VX- 166 (6mg/kg/day) in Poly-ethylene glycol (PEG) supplemented with Vitamin E, and the remaining 10 mice received PEG and Vitamin E only. In each group 12 of the mice were sacrificed at 4 weeks and the remaining mice at 8 weeks. At sacrifice, liver tissue and serum were collected for further analysis.

Preparation of Pan-Caspase inhibitor VX- 166

[0077] VX- 166 pan-caspase inhibitor was prepared fresh for each daily gavage. To prepare VX-166, appropriate concentration of the drug was first added to Peg 300 (81162; Sigma Aldrich, St. Louis, MO) and sonicated to allow formation of vehicle colloid. Following, Vitamin E TPGS (57668; Sigma Aldrich, St. Louis, MO) in water was added to form a final formulation consisting of 30% Peg 300, 10% Vitamin E TPGS, and 60% water. Each daily gavage consisted of 200ul Peg vehicle ± 6mg/kg of VX-166.

Histology and Immunohistochemistry

[0078] Formalin-fixed, paraffin-embedded liver sections were stained with hematoxylin and eosin to assess general histology; Sirius red staining was used to evaluate liver fibrosis. Standard IHC with citrate antigen retrieval was performed to localize expression of active Caspase 3 using active caspase 3 antibody at 1 :500 dilution (abl3847; Abeam, Cambridge, MA)

[0079] Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay (BoehringerMannheim, Mannheim, Germany) was performed according to the manufacturer's suggestions. Immunohistochemical quantification for nitrotyrosine, α- smooth muscle actin (α -SMA), and active caspase-3 was assessed using MetaView (Universal Imaging, Downingtown, PA). To obtain statistical significance, at least five x200 magnification random-field images were taken per slide, and at least five animals per group were scored (n = 5).

Serum Analysis

[0080] Following animal sacrifice, blood was collected and placed on ice for approximately 30min to coagulate. Next, samples were centrifuged at 10,000rpm for lOmin to separate serum from blood cloth. Obtained serum was then placed in separate tubes and stored in -80oC until analysis. To determine the extent of liver injury serum Alanine Aminotransferase (ALT) levels were measured using commercially available assay (Biotron Diagnostics; Hemet, CA). Hepatic lipid content was analyzed for total triglycerides (Biotron Diagnostics; Hemet, CA), cholesterol (Amplex Red Cholesterol Assay Kit, Invitrogen), and nonessential fatty acids (NEFAs) (Wako Pure Chemicals Industries, Richmond, VA). All procedures were performed as per manufacturer's instructions.

RNA and Protein Analysis

[0081] RNA was extracted and analyzed by QRT-PCR as previously described.13 Briefly, total RNA was collected using Triazol (Invitrogen). Following DNase I treatment, 5μg of RNA was reverse transcribed to cDNA templates using random hexamers and Superscript II Reverse Transcriptase. Following reverse transcription, cDNA was resuspended in pure water at the ratio of lμg of starting RNA to lOOμl of water. Amplification was performed using a SYBR Green PCR master mix (Applied Biosystems, Foster City, CA). 5μl of diluted cDNA was used per reaction and each sample was analyzed in triplicates. Target gene levels in treated cells or tissues are shown as a ratio to levels detected in corresponding control tissue, according to the ΔΔCt method. Following primers were used to amplify target genes: collagenlαl left, 5'- GAGCGGAGAGTACTGGATCG-3', right, 5'-GCTTCTTTTCCTTGGGGTTC-S'; ribosomal 755 left, 5'-TTGACGGAAGGGCACCACCAG-S', right, 5'- GCACCACCACCCACGGAATCG-3'. For protein analysis, liver sample was digested with 500ul of standard RIPA buffer containing Protease Inhibitor Cocktail Tablets from Roche (Indianapolis, IN). Approximately 50μg of total protein was loaded on Tris-Glycine 4-20% gels. Following standard Western Blot procedure, proteins were analyzed for presence of active caspase 3 (abl3847), ILIb (ab9722), IL18 (sc6179; SantaCruz Biotech., Santa Cruz, CA), α-Smooth Muscle Actin (α-SMA)(M0851; DAKO Cytomation, Carpinteria, CA) and B-Actin(A4700; Sigma-Aldrich, St. Louis, MO). Standard ECL Pico- Sensitivity substrate (Pierce Biotechnology, Rockford, IL) was used for final protein visualization.

Hydroxyproline Analysis

[0082] Hepatic hydroxyproline content was quantified colorimetrically in flash-frozen liver samples according a method described in Choi SS, Sicklick JK, Ma Q, et al. "Sustained activation of Racl in hepatic stellate cells promotes liver injury and fibrosis in mice." Hepatology 2006;44: 1267-77. Concentrations were calculated from a standard curve prepared with high purity hydroxyproline (Sigma- Aldrich, St Louis, MO, USA) and expressed as mg hydroxyproline/g liver.

Statistical Analysis

[0083] Results are expressed as mean ±SD. Significance was established using the student's t-test and analysis of variance when appropriate. Differences were considered significant when p<0.05.

Results

Treatment with VX-166 was well-tolerated and improved diet-induced steatosis in obese db/db mice

[0084] db/db mice were obese (Figure IA), weighing almost twice as much as age- and gender-matched wild type mice at the end of the eight week experiment, regardless of whether they were gavaged daily with vehicle or vehicle + VX- 166 (Figure IB). Although mice that were fed MCD diets lost 20-30% of their body weight during this time period, the degree of weight loss was not influenced by treatment with VX-166. Also, despite significant weight loss, mice in both MCD diet-fed groups remained significantly more obese than wild type control mice (Figure IA and B). Although obese, chow-fed db/db mice had only mild hepatic steatosis, as assessed either by histology (Figure 2A-E) or triglyceride content (Figure 2F). MCD diets dramatically induced hepatic fat accumulation, increasing hepatic triglyceride content about 40 fold by 4 weeks (Figure 2A, F). Hepatic steatosis decreased with further time on the diets. However, it remained about 15 fold greater than that of chowfed db/db controls after 8 weeks of MCD diet treatment (Figure 2C, F). Treatment with VX-166 significantly reduced hepatic triglyceride content at 4 weeks (Figure 2B, F), but not at 8 weeks (Figure 2D, F). It also generally reduced total liver cholesterol in MCD diet-treated animals (Fig. IE), but significantly increased liver NEFAs at 8 weeks (Fig. IF).

Treatment with VX-166 did not result in a reduction of ALT levels in db/db mice [0085] Serum ALT levels in chow-fed db/db mice were about 2 fold higher than in chowfed wild type C57B16 mice at 4 weeks and they increased further at 8 weeks, suggesting that gavage with vehicle alone induced some degree of liver injury in db/db mice (Figure 3). In contrast, feeding db/db mice MCD diets caused significant increases in serum ALT, raising values more than 10 fold above normal. Treatment with VX- 166 did not significantly impact ALT levels in db/db mice, regardless of whether they were fed chow or MCD diet. Thus, feeding MCD diets to db/db mice induced steatohepatitis, and treating such mice with VX- 166 did not improve ALT despite reducing hepatic steatosis. H&E-stained liver sections were carefully reviewed for features of steatohepatitis using criteria described in Brunt et al (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 :1313-21). CD diets increased histologic parameters of liver injury as assessed by NAS (NAFLD Activity Score). VX- 166 treatment did not improve MCD diet- related increases in hepatic inflammation, ballooning, or overall NAS (Figure 8).

VX-166 retained its pan-caspase inhibitory activity during long-term treatment

Caspase-3

[0086] Caspase activity was assessed by performing immunohistochemistry for active caspase 3. Results in all of the various groups of db/db mice were greater than those in wild type C57BL/6 mice, in which fewer than one liver cell/40x field was noted to contain activated caspase 3 (data not shown). Chow- fed db/db mice had only rare liver cells stained, suggesting that those mice also had very low levels of active caspase 3 (Figure 4

A-B, and inset figures). In contrast, the hepatic activity of caspase 3 was substantially increased by feeding db/db mice MCD diets for either 4 or 8 weeks (Figure 4C and E, respectively). Of note, treatment with VX- 166 reproducibly reduced the numbers of liver cells with caspase 3 activity at both time points (Figure 4D and F).

[0087] Western blot analysis of whole liver tissues was also performed. Immunoblot demonstrated that the MCD diet treatment increased hepatic accumulation of the caspase 3 cleavage product at 4 weeks, but not at 8 weeks.

[0088] Treatment with VX- 166 significantly decreased levels of activated caspase 3 at both time points, however (i.e., lowering levels of the caspase 3 cleavage product by nearly

2 fold at 4 weeks and by 50% at 8 weeks; both p<0.05) (Figure 5A and B, respectively). Thus, by immunohistochemistry and immunoblot assessment, the lowest levels of caspase

3 activity were observed in MCD-fed db/db mice that received VX- 166. Caspase-1

[0089] Caspase-1 is involved in cytokine activation during liver injury and is required for proteolytic cleavage of ILlβ and IL18, two injury related, pro-inflammatory cytokines. Western blot analysis of ILl β pre-protein (-3IkDa) and its smaller, biologically-active caspase-1 cleavage product (~17kDa), as well as the caspase 1 cleavage product of IL 18 (-2OkDa), demonstrated that MCD diets increased hepatic accumulation of both truncated IL- lβ and IL- 18, and that VX- 166 treatment abrogated both processes (Figures 5C-F). [0090] TUNEL staining was performed to investigate the possibility of caspase- independent mechanisms of liver injury. In liver, TUNEL staining marks cells that have been killed by both apoptotic and nonapoptotic mechanisms. MCD diets increased cell death by nearly 25-fold at both 4 and 8 weeks (Fig. 5A,B). Treatment with VX- 166 reduced the number of TUNEL-positive cells by 50% (Fig. 5B). Many dead cells were still apparent (Fig. 5A, inset). Effective inhibition of caspase activation by VX-166 was not sufficient to abrogate liver cell death in mice with MCD diet-induced steatohepatitis.

Treatment with VX-166 inhibited hepatic fibrogenesis in MCD diet-fed db/db mice

[0091] Increased apoptosis distinguishes NASH from NAFLD, and this is thought to contribute to the increased risk for liver fibrosis in NASH because phagocytosis of apoptotic bodies promotes myofibroblastic transformation of hepatic stellate cells (HSC). Hence, it was conceivable that the reduced apoptotic activity in VX-166-treated mice might have inhibited activation of HSC, resulting in less liver fibrosis despite no net improvement in MCD diet-related liver injury as measured by ALT. To evaluate this concept, hepatic accumulation of alpha-smooth muscle actin (α-SMA), a marker of myofibroblastic HSC, was examined by Western blot. As shown in Figure 6A-B, treatment with VX-166, reduced α-SMA content in both chow- fed db/db mice, and in MCD diet- fed db/db mice. Effects in MCD diet-fed db/db mice were particularly note worthy. For example, Western blot densitometry (Figure 6C and D) demonstrated 2 fold reductions in α-SMA protein levels at 4 weeks (p<0.05) and nearly 3 fold at 8 weeks (p<0.005). [0092] Further immunohistochemical analysis revealed that α-SMA predominantly accumulated in areas of lobular inflammation following MCD diets (Fig. 6B,E). Additionally, MCD diets induced the chicken wire pattern of pericellular/sinusoidal α- SMA staining that is typical of steatohepatitis-related fibrosis in human NASH (Fig. 6E, inset). Following VX-166 administration, α -SMA staining decreased (Fig. 6C), particularly around liver cells and along hepatic sinusoids (Fig. 6F). Morphometry confirmed general reductions in α -SMA accumulation after VX-166 treatment at both 4 and 8 weeks (Fig. 6D). [0093] To determine if reduced accumulation of α-SMA was accompanied by changes in liver fibrosis, liver sections were stained with picric acid Sirius Red, and deposition of collagen fibrils was analyzed (Figure 7 A-E). Chow-fed db/db mice and chow-fed control mice had comparable collagen deposition peri-portally, and treatment with VX- 166 did not appear to improve this. MCD diets increased collagen deposition in db/db mice, particularly around hepatocytes and along hepatic sinusoids. Treatment with VX- 166 improved periportal, as well as pericellular and sinusoidal fibrosis in MCD diet-fed db/db mice. These changes were most notable after 8 weeks of MCD diet exposure, and they were paralleled by improvements in collagen lαl mRNA expression, as assessed by qRT- PCR analysis (Figure 7F). Indeed, collagen lαl mRNA expression was significantly (p<0.05) reduced at 8 weeks in MCD diet-fed db/db mice that were treated with VX- 166. Biochemical assessment of hepatic collagen by hydroxyproline assay confirmed the effects of treatment on hepatic collagen content in the MCD diet- fed db/db mice (Figure 7G). Treatment of such mice with VX- 166 reduced collagen accumulation back to levels that were observed in chow-fed db/db mice. Therefore, inhibiting hepatic apoptosis by treating obese mice with this pan-caspase inhibitor reduced NASH-related fibrogenesis, despite exerting no apparent net benefit on liver injury as measured by ALT levels.

Example 5: Impact of VX- 166 in early stage steatotic disease and established steatohepatitis

[0094] The study had two arms, each using a different dietary model of

NAFLD/NASH. Random allocation of animals to different experimental groups within each study-arm was performed at the start of the study.

1. HFD 'Steatosis ' Model: 8-week old male C3Η/ΗeN mice (Harlan, UK) were randomised to receive either high fat diet (45%kcal fat derived from lard; D 12451, Research Diets Inc, USA) or standard chow.

2. MCD 'Steatohepatitis ' Model: 8-week old male genetically obese Db/Db mice (BKSCg-m +/+ Leprdb/J (#00642)) and non-obese heterozygote Db/m (BKS-Cg-m +/- Leprdb/J) control mice (Charles River, Belgium) were fed either methionine- choline deficient diet (#A02082002B, Research Diets, USA) or a nutritionally replete control diet (#A02082003B). [0095] Animals started therapy after NAFLD/NASH had become established. Within each study-arm different experimental groups received daily gavage with VX- 166 (2mg/kg/day); gavage with TPGS/PEG vehicle (d-alpha-tocopheryl polyethylene-glycol- 1000 succinate [Vitamin E] and Polyethylene Glycol); or no treatment (Supplementary Figure 2X - Table 1). Animals were inspected daily and weights recorded each week. At the end of the study animals were culled by exsanguination and tissue collected for analysis. Plasma was analysed using an Olympus AU400 (Olympus, UK) automated clinical chemistry analyser to determine ALT, alkaline hosphatase & bilirubin levels.

Thiobarbituric Acid Reactive Substances (TBARS) Assay

[0096] TBARS were measured to quantify lipid peroxidation and tissue oxidative stress using a colorimetric assay. Briefly, lOOmg liver tissue was homogenised in a RIPA buffer, supernatant mixed with 10% trichloroacetic acid and incubated on ice for 5 minutes. Following centrifugation, 0.67% thiobarbituric acid was added and the mixture incubated at 95⁰C for 10 minutes. Duplicate aliquots were pipetted into a 96-well plate and absorbance measured at 532nm. A standard curve was generated using diluted MDA (1,1,3,3- tetramethoxypropane). Results were expressed as μM/lOOmg tissue.

Histological Analysis

[0097] Formalin fixed tissue was processed into paraffin wax and sections were stained with Haematoxylin & Eosin or collagen specific Sirius Red. Immunohistochemical staining for alpha-smooth muscle actin was performed as a marker of stellate cell activation. All sections were examined by light microscopy by a histopathologist who was unaware which study group each sample was from. Steatohepatitis was assessed using a modified semiquantitative Brunt score [15]. This measures degree of steatosis (0-3), fibrosis (0-4), inflammation (0-3) and hepatocyte ballooning degeneration (0-2). Steatosis area and fibrosis area were measured by digital image analysis and averaged over 10 low- power fields per sample.

Assessment of Caspase-Cleaved CK- 18 for Apoptosis/Caspase-3 Activity [0098] Early in apoptosis, the intermediate filament component Cytokeratin-18 (CK- 18) is cleaved by activated caspase-3 to generate a specific neo-epitope. Immunohistochemical detection of this caspase-generated neo-epitope has been shown to closely mirror capase-3 activity and apoptosis (Duan WR, Garner DS, Williams SD, Funckes-Shippy CL, Spath IS, Blomme EA. "Comparison of immunohistochemistry for activated caspase-3 and cleaved cytokeratin 18 with the TUNEL method for quantification of apoptosis in histological sections of PC-3 subcutaneous xenografts." J Pathol 2003 Feb;199(2):221-228). Hepatic caspase-cleaved CK-18 was measured immunohistochemically in sections stained with biotinylated MAb M30 (Roche Diagnostics, UK) according to the manufacturers protocol.

PCNA Staining

[0099] Hepatocyte proliferating cell nuclear antigen (PCNA) was measured by immunohistochemical staining as an indirect marker of overall cell turnover including both apoptotic and necrotic cell death (mouse monoclonal PCNA proliferation marker (Abeam) visualized using dextranpolymer conjugated with secondary antibody and peroxidase (Real EnVision Rabbit/Mouse, DAKO)).

Total RNA Extraction & Quantification of Relative Gene Expression by rt-PCR [00100] Total RNA was extracted from snap frozen liver tissue (RNeasy Mini kit, Qiagen), reverse transcribed (RETROscript, Ambion) and quantitative rtPCR analysis performed in triplicate (TaqMan, Applied Biosystems). Primers specific for TNFα, MCP-I and pro-collagen Ia2 were used. Data were normalised to glyceraldehyde 3-phosphate dehydrogenase (gapdh) and relative fold-change differences in expression calculated (ABI- Biosystems. User Bulletin #2 ABI PRISM 7700 Sequence Detection System.: ABI Biosystems Inc; 1997).

Statistical Analysis

[00101] Analysis performed with SPSSvI 7. Parametric (ANOVA with post-hoc Bonferroni correction or t-test) or non-parametric (Rruskall-Wallis or Mann Whitney U tests) were used as appropriate. Values are presented as mean±SEM for continuous variables. Two-tailed pvalues are shown throughout with p<0.05 taken as significant. RESULTS

General effects of Dietary Manipulation & Treatment with VX-I 66

[00102] The two diets differed on their effects on body weight. Animals in the HFD- Steatosis arm gained weight during the study whilst those in the MCD-Steatohepatitis arm lost weight. One animal from the HFD-steatosis arm was culled on welfare grounds related to a procedural complication during the course of the study. The mean weight of C3H mice increased from 27.07±0.28g to 46.50±0.39g (p<0.0001) on high fat diet, significantly more than those on standard chow whose weight only increased to 37.24±0.95g (p<0.0001). [00103] HFD fed mice who received no additional treatment gained significantly more weight (2.31±0.36g) during the final 5 weeks of the study than those who received TPGS/PEG vehicle or VX- 166 (0.23±0.15 and 0.74±0.26g respectively; ANOVA p<0.0001). Db/Db mice were significantly heavier than Db/m animals at the start of the study and, as expected, consumption of MCD diet was associated with marked weight loss. [00104] The administration of either VX- 166 or TPGS/PEG exacerbated this effect. Oral administration of either VX- 166 or TPGS/PEG was associated with altered bowel habit, suggesting that the vehicle had an osmotic laxative effect. The observed excess weight-loss in animals exposed to these agents may therefore be due to rapid-transit malabsorption. This effect was most apparent in MCD-fed animals that were already consuming a nutritionally deficient diet. As MCD diet is normally associated with substantial weight-loss in the presence of severe steatohepatitis, it is unlikely that the anti- steatohepatitic effects of TPGS/PEG vehicle and VX- 166 are simply due to weight- loss associated reduction in adiposity.

VX-166 inhibits caspase activity and apoptosis in NΛFLD/NΛSH

[00105] Both diets significantly increased caspase activity and tissue apoptosis over chow fed control mice as assessed by caspase-cleaved CK- 18 (Figure IX). Reflecting the relative severity of liver damage and consequent caspase activation in each model, caspase-cleaved CK- 18 levels were greater in the more florid MCD-steatohepatitis arm (1.59±0.28% vs. 4.81±0.51%, p<0.0001) than the HFD-steatosis arm (1.18±0.15% vs. 2.67±0.32%, p=0.007). VX-166 reduced CK-18 in both HFD-Steatosis (1.71±0.17%, p=0.05) and MCD-Steatohepatitis arms (1.77±0.38%, p<0.0001) to levels comparable to control and significantly lower than TPGS/PEG vehicle in the MCD arm (4.09±0.40%, p=0.002). PCNA staining showed that VX-166 reduced overall apoptotic and non- apoptotic cell death (Figure 2X). Amongst HFD fed mice, post-hoc analysis demonstrated that VX-166 significantly reduced PCNA% compaired to HFD-control (1.06±0.08% vs. 1.87±0.85%, p=0.004) and vehicle (1.83±0.20%, p=0.004). In the MCD-Steatohepatitis arm, a similar reduction trend in PCNA% in VX-166 treated animals was observed however this did not reach statistical significance.

VX-166 reduces histological steatohepatitis

[00106] Chow- fed C3H mice showed no histological abnormality. After 15 weeks on HFD, C3H mice exhibited marked steatosis with features of mild steatohepatitis but no fibrosis. The degree of steatosis did not differ between treatment sub-groups (Supplementary Figure IX) however differences in inflammatory infiltrate were detected (Kruskall-Wallis H(2)=10.60, n=9-10 per group, p=0.005). VX-166 treated mice exhibited less hepatic inflammation than untreated HFD mice (Mann- Whitney, U=I 8, r=-0.63, p=0.018) with a more modest effect on overall NASH activity score (NAS), Figure 3X. [00107] MCD-fed Db/Db mice exhibited generalised steatohepatitis including hepatocyte ballooning degeneration, an active inflammatory infiltrate and mild pericellular fibrosis. No differences in hepatic steatosis were detected between groups (Supplementary Figure IX). Significant differences in severity of hepatic inflammation (Kruskall-Wallis H(2)=8.22, p=0.016, n=7-8) and NASH Activity Score (NAS) (H(2)=12.72, p=0.002) were detected between groups. Post-hoc analysis showed that Inflammatory score was significantly reduced by VX-166 (U=6.5, r=- 0.69, p=0.03) over control as was overall NASH activity score (NAS) (U=2, r=-0.81, p=0.006), Figure 3X. VX-166 also reduced overall NASH score (U=2, r=-0.81, p=0.006) significantly more than TPGS/PEG vehicle which showed no significant histological effect.

VX-166 ameliorates established biochemical hepatitis and tissue oxidative stress [00108] Prior to starting treatment after ten weeks on diet, overall HFD fed C3H mice showed a small but significant increase in mean ALT above chow fed littermates (40.10±2.76 vs. 16.76±1.25 IU/ml, p<0.0001). After five weeks therapy, further significant increases in ALT were observed in HFD-only animals (Supplementary Figure 2X - Table 2). In contrast, ALT had stabilised in both the VX-166 and TPGS/PEG treated groups. More strikingly, in the MCD-Steatohepatitis arm, where an overall initial marked ALT rise induced by MCD had been observed (347.99±120.41 vs. 16.02±2.57 IU/ml on control diet, P<0.0001), the VX-166 treated group showed a significant fall in ALT (-140.0±46.1 IU/ml, p=0.039).

[00109] A smaller reduction was observed in mice gavaged with TPGS/PEG vehicle but this did not reach significance. The significance of pre/posttreatment ALT changes were assessed by paired t-test to counter intergroup variability in baseline ALT levels. Lipid peroxidation was assessed as a surrogate for hepatic oxidative stress. Significantly higher levels of TBARS were detected in MCD fed animals compared to control diet (4.40±0.76 vs. 0.66±0.1 lμM/lOOmg tissue, p<0.0001). VX-166 treatment significantly reduced TBARS (2.37±0.16 vs. 4.40±0.76μM/100mg tissue, p=0.024), Figure 4B. A nonsignificant trend was observed in the TPGS/PEG group. Consistent with the very mild inflammatory changes observed in the HFD-Steatosis arm, changes in TBARS levels were minor (Figure 4X) and, although of a similar pattern, did not reach statistical significance.

TPGS/PEG Vehicle appears to contribute significantly to observed anti-inflammatory effects

[00110] To further characterise the changes in inflammation observed in the MCD study arm, hepatic expression of key mediators of inflammation (TNF α) and monocyte/macrophage infiltration (MCP-I) were examined, Figure 5X. MCD feeding alone produced a 10.7-fold increase in hepatic TNFα expression over control which was reduced to 3.1 -fold by VX-166 treatment. However, this was not significantly greater than vehicle (3.6-fold). Similarly, whilst a 29.2-fold increase in MCP-I was observed in MCD fed mice, treatment with VX-166 reduced this to 12.4-fold but TPGS/PEG brought this to 9.7-fold above control.

VX-166 shows little anti-fibrotic effect in established disease

[00111] There was no significant difference in overall morphological fibrosis score or percentage area fibrosis measured by digital image analysis (Figure 6X). Expression of Collagen Ia2 was reduced equally relative to untreated MCD fed mice by both VX-166 and TPGS/PEG. No difference in immunohistochemical alpha-smooth muscle actin positive cells was observed (data not shown).

[00112] While we have described a number of embodiments of this invention, it is apparent that our basic examples may be altered to provide other embodiments, which utilize the compounds and methods of this invention. Therefore, it will be appreciated that the scope of this invention is to be defined by the appended claims rather than by the specific embodiments, which have been represented by way of example.

Claims

We claim:

1. A method for ameliorating, treating, or preventing steatohepatitis in a subject, comprising administering a compound of formula I:

I; or a pharmaceutical composition comprising said compound to said subject.

2. The method of claim 1, wherein the steatohepatitis is non-alcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH) or alcoholic steatohepatitis (ASH).

3. The method of claim 2, wherein the steatohepatitis is nonalcoholic steatohepatitis (NASH).

4. The method of claim 2, wherein the steatohepatitis is alcoholic steatohepatitis (ASH).

5. A method for reducing inflammation in the liver of a subject with steatohepatitis comprising administering a compound of formula I:

i; or a pharmaceutical composition comprising said compound to said subject.

6. The method of claim 5, wherein the steatohepatitis is non-alcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH) or alcoholic steatohepatitis (ASH).

7. The method of claim 6, wherein the steatohepatitis is nonalcoholic steatohepatitis (NASH).

8. The method of claim 6, wherein the steatohepatitis is alcoholic steatohepatitis (ASH).

9. A method for reducing apoptosis in the liver of a subject with steatohepatitis comprising administering a compound of formula I:

I; or a pharmaceutical composition comprising said compound to said subject.

10. The method of claim 9, wherein the steatohepatitis is non-alcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH) or alcoholic steatohepatitis (ASH).

11. The method of claim 10, wherein the steatohepatitis is nonalcoholic steatohepatitis (NASH).

12. The method of claim 10, wherein the steatohepatitis is alcoholic steatohepatitis (ASH).

13. A method for ameliorating, treating, or preventing fibrosis in a subject, comprising administering a compound of formula I:

i; or a pharmaceutical composition comprising said compound to said subject.

14. The method of claim 13, wherein said fibrosis is liver fibrosis.

15. The method of claim 14, wherein the liver fibrosis is cause by non-alcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH) or alcoholic steatohepatitis (ASH).

16. The method of claim 15, wherein the liver fibrosis is cause by nonalcoholic steatohepatitis (NASH).

17. The method of claim 15, wherein the liver fibrosis is cause by alcoholic steatohepatitis (ASH).

18. A method for ameliorating, treating, or preventing steatosis in a subject, comprising administering a compound of formula I:

I; or a pharmaceutical composition comprising said compound to said subject.

19. The method of claim 18, wherein the steatosis is caused by obesity, insulin resistance, metabolic syndrome, or viral infection.

20. The method of claim 18, wherein the steatosis is cause by non-alcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH) or alcoholic steatohepatitis (ASH).

21. The method of claim 20, wherein the steatosis is cause by nonalcoholic steatohepatitis (NASH).

22. The method of claim 20, wherein the steatosis is cause by alcoholic steatohepatitis (ASH).

23. The method of any one of claims 1-22, comprising an additional therapeutic agent.

24. The method of claim 23, wherein said therapeutic agent is an agent for treating NASH, an agent for treating ASH, or an agent for treating a disease involving fibrosis, steatosis, or inflammation of the liver.

25. The method of claim 24, wherein said therapeutic agent is selected from insulin sensitizers, antioxidants, hepatoprotective agents, or lipid lowering drugs.

26. The method of claim 24, wherein said insulin sensitizer is metformin, pioglitazone, thiazolidninediones, or rosiglitazone; said antioxidant is vitamin E or vitamin C; said hepatoprotective agents is Angiotensin Converting Enzyme (ACE) Inhibitors, betaine, ursodeoxycholic acid, or pentoxyfylline; and said lipid lowering drug is Probucol.

27. The method of claim 23, wherein said therapeutic agent is Vitamin E (TPGS) and/or PEG.

28. A method for identifying a compound for ameliorating, treating or preventing a disease or condition selected from NASH, ASH, or a disease involving fibrosis, steatosis, or inflammation of the liver in a subject comprising administering a compound selected from a compound of formula I:

I; or a pharmaceutical composition comprising said compound and comparing caspase inhibition in the subject before and after treatment with said compound.

29. A method for identifying a compound for ameliorating, treating, or preventing a disease or condition selected from NASH, ASH, or a disease involving fibrosis, steatosis, or inflammation of the liver in a subject comprising administering a compound of formula I:

I; or a pharmaceutical composition comprising said compound and comparing a biomarker for NASH, ASH, fibrosis, steatosis, or inflammation of the liver in said subject before and after treatment with said compound.

30. A pharmaceutical composition for ameliorating, treating, or preventing NASH, ASH, or a disease involving fibrosis, steatosis, or inflammation of the liver in a subject, comprising a compound selected from a compound of formula I:

i; and a pharmaceutically acceptable carrier.

31. The method of any one of claims 1 -29, wherein the compound has formula I-a.

I-a.

32. The method of claim 31, wherein the compound has formula I-b:

I-b.