WO2010120880A1 - Traitement de maladies hépatiques avec un inhibiteur de caspase - Google Patents

Traitement de maladies hépatiques avec un inhibiteur de caspase Download PDF

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Publication number
WO2010120880A1
WO2010120880A1 PCT/US2010/031031 US2010031031W WO2010120880A1 WO 2010120880 A1 WO2010120880 A1 WO 2010120880A1 US 2010031031 W US2010031031 W US 2010031031W WO 2010120880 A1 WO2010120880 A1 WO 2010120880A1
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steatohepatitis
compound
liver
nash
ash
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PCT/US2010/031031
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English (en)
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John Pollard
John J. Alam
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Vertex Pharmaceuticals Incorporated
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Publication of WO2010120880A1 publication Critical patent/WO2010120880A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4412—Non condensed pyridines; Hydrogenated derivatives thereof having oxo groups directly attached to the heterocyclic ring
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A61K31/355—Tocopherols, e.g. vitamin E
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/74—Synthetic polymeric materials
    • A61K31/765—Polymers containing oxygen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics

Definitions

  • 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].
  • 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).
  • NASH nonalcoholic steatohepatitis
  • NAFLD steatosis
  • One significant difference between NAFLD and NASH is the extent of hepatocyte apoptosis, which is more extensive in NASH.
  • 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].
  • caspase inhibitors to treat disease states associated with an increase in cellular apoptosis has been demonstrated using peptidic caspase inhibitors.
  • peptidic caspase inhibitors 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].
  • 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.
  • NAFLD Non- Alcoholic Fatty Liver Disease
  • NASH nonalcoholic steatohepatitis
  • ASH alcoholic steatohepatitis
  • 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.
  • 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.
  • NAFLD Non- Alcoholic Fatty Liver Disease
  • NASH nonalcoholic steatohepatitis
  • ASH alcoholic steatohepatitis
  • the present disclosure also relates to methods for identifying agents useful for treating these diseases.
  • additional therapeutics that can be used in combination with VX- 166 for the treatment of these
  • 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.
  • 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.
  • FIGURE 2 EFFECTS OF VX-166 ON LIVER HISTOLOGY AND HEPATIC TRIGLYCERIDE CONTENT IN CHOW-FED AND MCD DIET-FED DB/DB MICE.
  • 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.
  • 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.
  • 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.
  • FIGURE 5 EFFECTS OF VX- 166 ON HEPATIC CASPASE ACTIVITY DURING LONG-TERM TREATMENT OF CHOW-FED AND MCD DIET-FED DB/DB MICE.
  • VX caspase 3 cleavage product in mice treated with vehicle or VX- 166 (VX) for 4 weeks (A) or 8 weeks (B).
  • FIGURE 6 EFFECT OF VX- 166 ON HEPATIC ACCUMULATION OF ALPHA- SMOOTH MUSCLE ACTIN (A-SMA).
  • FIGURE 7 EFFECTS OF VX- 166 ON HEPATIC FIBROGENESIS IN CHOW FED- AND MCD DIET-FED DB/DB MICE.
  • 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).
  • 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
  • FIGURE 3X EFFECTS OF VX- 166 ON STEATOHEP ATITIS
  • FIGURE 4X EFFECTS OF VX- 166 TREATMENT ON HEPATIC OXIDATIVE STRESS
  • 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) TNF ⁇ 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.
  • 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.
  • 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.
  • NAFLD Non- Alcoholic Fatty Liver Disease
  • NASH nonalcoholic steatohepatitis
  • ASH alcoholic steatohepatitis
  • diseases involving fibrosis, steatosis, and inflammation of the liver such as Non- Alcoholic Fatty Liver Disease (NAFLD), nonalcoholic steatohe
  • 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.
  • NASH Non- Alcoholic Fatty Liver Disease
  • NASH nonalcoholic steatohepatitis
  • ASH alcoholic steatohepatitis
  • fibrosis steatosis
  • steatosis fibrosis
  • steatosis steatosis
  • inflammation of the liver fibrosis
  • 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 fibro
  • compositions disclosed herein may further comprise another therapeutic agent.
  • 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.
  • agents include, but are not limited to, TPGS (Vitamin E) and PEG.
  • 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.
  • 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.
  • ion exchangers alumina, aluminum stearate, lecithin
  • serum proteins such as human serum albumin
  • buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial gly
  • 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.
  • 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.
  • said therapeutic agent is selected from insulin sensitizers, antioxidants, hepatoprotective agents, or a lipid lowering drug.
  • 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.
  • ACE Angiotensin Converting Enzyme
  • said additional agent is Vitamin E (TPGS). In yet another embodiment, said additional agent is PEG.
  • 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.
  • 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.
  • pharmaceutically acceptable salts those salts are preferably derived from inorganic or organic acids and bases.
  • acid salts include 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
  • 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.
  • 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.
  • 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
  • compositions of this invention 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.
  • a given biological system e.g., blood, lymphatic system, or central nervous system
  • 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.
  • compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
  • parenteral as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection and infusion techniques.
  • the compositions are administered orally or intravenously.
  • 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.
  • a non-toxic parenterally acceptable diluent or solvent for example as a solution in 1,3- butanediol.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • any routine encapsulation is suitable, for example, using the aforementioned carriers in a hard gelatin capsule shell.
  • 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.
  • 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.
  • 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.
  • carriers that are commonly used include lactose and corn starch.
  • Lubricating agents such as magnesium stearate, are also typically added.
  • useful diluents include lactose and dried cornstarch.
  • 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.
  • 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.
  • NAFLD Non- Alcoholic Fatty Liver Disease
  • NASH nonalcoholic steatohepatitis
  • ASH alcoholic steatohepatitis
  • 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.
  • 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).
  • such preparations contain from about 20% to about 80% active compound.
  • 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.
  • 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.
  • 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.
  • 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.
  • One embodiment provides a method for ameliorating, treating, or preventing steatohepatitis in a subject, comprising administering a compound of formula I:
  • the steatohepatitis is non-alcoholic fatty liver disease
  • NAFLD nonalcoholic steatohepatitis
  • ASH alcoholic steatohepatitis
  • the steatohepatitis is nonalcoholic steatohepatitis (NASH).
  • the steatohepatitis is alcoholic steatohepatitis (ASH).
  • 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.
  • Another embodiment provides a method of reducing inflammation in the liver of a subject with steatohepatitis, comprising administering a compound of formula I:
  • the steatohepatitis is non-alcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), or alcoholic steatohepatitis (ASH).
  • NAFLD non-alcoholic fatty liver disease
  • NASH nonalcoholic steatohepatitis
  • ASH alcoholic steatohepatitis
  • the steatohepatitis is nonalcoholic steatohepatitis (NASH).
  • the steatohepatitis is alcoholic steatohepatitis (ASH).
  • Another embodiment provides a method for reducing apoptosis in the liver of a subject with steatohepatitis, comprising administering a compound of formula I:
  • the steatohepatitis is non-alcoholic fatty liver disease
  • NAFLD nonalcoholic steatohepatitis
  • ASH alcoholic steatohepatitis
  • the steatohepatitis is nonalcoholic steatohepatitis (NASH).
  • the steatohepatitis is alcoholic steatohepatitis (ASH).
  • Another embodiment provides a method for ameliorating, treating, or preventing fibrosis in a subject, comprising administering a compound of formula I:
  • the fibrosis is caused by a fibrotic disease.
  • the fibrosis is liver fibrosis.
  • the fibrosis is caused by non-alcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), or alcoholic steatohepatitis (ASH).
  • NAFLD non-alcoholic fatty liver disease
  • NASH nonalcoholic steatohepatitis
  • ASH alcoholic steatohepatitis
  • the fibrosis is caused by nonalcoholic steatohepatitis (NASH).
  • the fibrosis is caused by alcoholic steatohepatitis (ASH).
  • Another embodiment provides a method for ameliorating, treating, or preventing steatosis in a subject, comprising administering a compound of formula I:
  • the steatosis is caused by obesity, insulin resistance, metabolic syndrome, or viral infection.
  • the steatosis is caused by non-alcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), or alcoholic steatohepatitis (ASH).
  • NAFLD non-alcoholic fatty liver disease
  • NASH nonalcoholic steatohepatitis
  • ASH alcoholic steatohepatitis
  • the steatosis is caused by nonalcoholic steatohepatitis (NASH).
  • the steatosis is caused by alcoholic steatohepatitis (ASH).
  • 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.
  • a pharmaceutically acceptable composition described above.
  • the patient 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.
  • 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.
  • 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.
  • said disease or condition is NASH, ASH, fibrotic disease, or steatosis.
  • said caspase inhibitor is the compound of formula I (VX- 166).
  • 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.
  • said disease or condition is NASH, ASH, fibrotic disease, or steatosis.
  • said caspase inhibitor is the compound of formula I (VX- 166).
  • biomarker is a physical, functional, or biochemical indicator, e.g., the presence of a particular metabolite, of a physiological or disease process.
  • biomarkers related to NASH, ASH, fibrosis, inflammation or steatosis in the liver include, but are not limited to, cytokeratin-18 (CK- 18), ALT, TNF- ⁇ , monocyte macrophage infiltration (MCP-I), hepatocyte proliferating cell nuclear antigen (PCNA), active caspase- 3, active caspase-1, IL-I, IL-18, ⁇ -SMA, TUNEL, and nitrotyrosine.
  • the compound is a compound of formula Ia. In other embodiments, the compound is a compound of formula Ib.
  • the resulting mixture was kept at 0 0 C 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 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.
  • 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.
  • VX- 166 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 ⁇ -SMA, collagenl ⁇ l, hydroxyproline); and reduction of liver injury (determined by biomarker levels such as CK- 18 or ALT). The following studies examine these various factors.
  • NAS NAFLD Activity Score
  • 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.
  • MCD Methionine Choline Deficient mice
  • 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.
  • PEG Poly-ethylene glycol
  • Vitamin E Vitamin E
  • mice were sacrificed at 4 weeks and the remaining mice at 8 weeks. At sacrifice, liver tissue and serum were collected for further analysis.
  • VX- 166 pan-caspase inhibitor was prepared fresh for each daily gavage.
  • 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.
  • 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.
  • ALT Alanine Aminotransferase
  • Target gene levels in treated cells or tissues are shown as a ratio to levels detected in corresponding control tissue, according to the ⁇ Ct method. Following primers were used to amplify target genes: collagenl ⁇ l left, 5'- GAGCGGAGAGTACTGGATCG-3', right, 5'-GCTTCTTTTCCTTGGGGTTC-S'; ribosomal 755 left, 5'-TTGACGGAAGGGCACCACCAG-S', right, 5'- GCACCACCACCCACGGAATCG-3'.
  • liver sample was digested with 500ul of standard RIPA buffer containing Protease Inhibitor Cocktail Tablets from Roche (Indianapolis, IN).
  • Hepatic hydroxyproline content was quantified colorimetrically in flash-frozen liver samples according a method described in Choi SS, Sictician 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.
  • 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.
  • 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).
  • VX-166 retained its pan-caspase inhibitory activity during long-term treatment
  • Caspase-1 is involved in cytokine activation during liver injury and is required for proteolytic cleavage of ILl ⁇ and IL18, two injury related, pro-inflammatory cytokines.
  • Western blot analysis of ILl ⁇ 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- l ⁇ and IL- 18, and that VX- 166 treatment abrogated both processes ( Figures 5C-F).
  • TUNEL staining was performed to investigate the possibility of caspase- independent mechanisms of liver injury.
  • 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.
  • VX-166 Treatment with VX-166 inhibited hepatic fibrogenesis in MCD diet-fed db/db mice
  • 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).
  • HSC hepatic stellate cells
  • ⁇ -SMA staining that is typical of steatohepatitis-related fibrosis in human NASH
  • Fig. 6E inset
  • ⁇ -SMA staining decreased (Fig. 6C), particularly around liver cells and along hepatic sinusoids (Fig. 6F).
  • Morphometry confirmed general reductions in ⁇ -SMA accumulation after VX-166 treatment at both 4 and 8 weeks (Fig. 6D).
  • Fig. 6D To determine if reduced accumulation of ⁇ -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).
  • Example 5 Impact of VX- 166 in early stage steatotic disease and established steatohepatitis
  • NAFLD/NASH Random allocation of animals to different experimental groups within each study-arm was performed at the start of the study.
  • HFD 'Steatosis ' Model 8-week old male C3 ⁇ / ⁇ eN 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.
  • 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.
  • TBARS Thiobarbituric Acid Reactive Substances
  • 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 95 0 C 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 ⁇ M/lOOmg tissue.
  • 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.
  • PCNA Hepatocyte proliferating cell nuclear antigen
  • 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).
  • VX-166 inhibits caspase activity and apoptosis in N ⁇ FLD/N ⁇ SH
  • PCNA staining showed that VX-166 reduced overall apoptotic and non- apoptotic cell death (Figure 2X).
  • 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
  • VX-166 ameliorates established biochemical hepatitis and tissue oxidative stress
  • ALT had stabilised in both the VX-166 and TPGS/PEG treated groups.
  • TPGS/PEG Vehicle appears to contribute significantly to observed anti-inflammatory effects
  • VX-166 shows little anti-fibrotic effect in established disease

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Abstract

La présente invention concerne un procédé d'utilisation de VX-166, un inhibiteur de caspase Pan, pour traiter ou réduire les symptômes de maladies hépatiques, telles qu'une stéatose hépatique non alcoolique (NAFLD), une stéato-hépatite non alcoolique (NASH), une stéatose hépatique alcoolique (ASH), et d'autres maladies impliquant une fibrose, une stéatose, ou une inflammation du foie. La présente invention concerne en outre des procédés pour identifier des agents utiles pour traiter ces maladies. La présente invention concerne en outre des agents thérapeutiques additionnels qui peuvent être utilisés en combinaison avec VX-166 pour le traitement de ces maladies.
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CN114555076A (zh) * 2019-10-18 2022-05-27 正大天晴药业集团股份有限公司 用于治疗非酒精性脂肪性肝炎的药物

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CN112245419A (zh) * 2014-12-02 2021-01-22 艾菲穆恩有限公司 包括15-hepe的组合物以及使用其治疗或预防纤维化的方法
WO2018065902A1 (fr) * 2016-10-05 2018-04-12 Novartis Ag Compositions de combinaison comprenant des agonistes de fxr pour le traitement ou la prévention d'une maladie ou d'un trouble fibrotique, cirrhotique
CN109789119A (zh) * 2016-10-05 2019-05-21 诺华股份有限公司 用于治疗或预防纤维化、硬化疾病或障碍的含fxr激动剂的复合组合物
CN114555076A (zh) * 2019-10-18 2022-05-27 正大天晴药业集团股份有限公司 用于治疗非酒精性脂肪性肝炎的药物
CN114555076B (zh) * 2019-10-18 2024-02-02 正大天晴药业集团股份有限公司 用于治疗非酒精性脂肪性肝炎的药物

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