WO2010028370A1 - Use of ppar gamma modulators to treat cystic liver diseases - Google Patents

Abstract

Compositions comprising compounds that modulate PPARγ activity and methods for using the compositions in treating liver and bile duct disease are described. Compositions and methods for treating polycystic liver disease are also described.

Description

USE OF PPAR GAMMA MODULATORS TO TREAT CYSTIC LIVER DISEASES

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to United States Provisional Patent

Application No. 61/095,175 filed on September 8, 2008. The subject matter disclosed in this provisional application is hereby expressly incorporated by reference into the present application.

TECHNICAL FIELD

The invention described herein pertains to the treatment of liver and bile duct diseases, such as diseases arising from salt and/or water homeostasis dysfunction. In particular, the invention described herein pertains to the treatment of polycystic liver disease and other diseases involving cyst formation in the biliary system.

BACKGROUND AND SUMMARY OF THE INVENTION

Polycystic liver disease (PLD) is a broad spectrum of diseases for which the major treatment, currently, is surgical intervention (Russell, R.T., et al., World J. Gastroent. 13:S052-59, 2008; Barahona-Garrido et al., World J Gastroenterol, 14(20):3195-3200,

2008). The foregoing publication, and each publication cited herein, is incorporated herein by reference. In general, liver cysts are observed in a number of disease states. It some cases the formation of cysts are a symptom of an underlying disease. In other cases, the formation of cysts is a direct consequence of the disease. Both indirect and direct underlying diseases can result in liver cyst formation include. Thus, PLD can occur as an autosomal dominant disease that presents as predominantly liver involvement or it can be a co-morbidity with other diseases, such as polycystic kidney disease (PKD).

PKD is a genetic disorder characterized by the growth of numerous fluid-filled cysts predominately in the kidney. There are 2 forms of PKD which when combined represent the third leading cause of kidney failure in the United States (Torres V.E., et al., Nat Clin Pract Nephrol 2:40-55,, 2006; Torres V.E., et al., I Intern Med 261:17-31, 2007). Autosomal Dominant Polycystic Kidney Disease (ADPKD), the most common form, occurs at a rate of approximately 1 in 800 in the human population and is usually diagnosed after the fourth decade of life. Autosomal Recessive Polycystic Kidney Disease (ARPKD) is a less common, but a more severe, disease that is typically diagnosed in neonates and children. The primary cause of morbidity and mortality in both forms of PKD is the progression of renal pathology. Even so, one of the more common extra-renal manifestations of PKD is PLD. However, PLD arises from a different biological dysfunction, namely from the cholangiocytes lining the hepatic bile duct. As PKD progresses (adolescence to young adults) liver disease becomes an important and severe clinical finding accompanying the underlying disease.

Although PLD is often later manifested in PKD, treatment of PKD will not necessarily result in decreasing PLD. For example, the use of vasopressin V2 receptor antagonists, such as OPC31260, have been reported for potentially treating the renal pathology of both the autosomal dominant and recessive forms of polycystic kidney disease. In three different animal models of renal cystic disease, treatment with the antagonist decreased cAMP levels, prevented renal enlargement, inhibited cystogenesis and protected renal function by halting progression of renal disease (Gattone V.H., et al., Nat Med 9: 1323- 1326, 2003; Torres V.E., et al., Nat Med 10:363-364, 2004). Though previous studies of the mechanisms involved in renal cyst formation have led to potential therapeutic treatment with vasopressin V2 receptor antagonist, it is believed herein that vasopressin V2 receptor antagonist will likely not be effective in treating hepatic biliary cysts because cholangiocytes do not express the V2 receptor. Similarly, it has also been suggested that compounds like rapamycin and chemotherapeutic drugs may have ameliorative effects on renal cysts, but those drugs have multiple side effects and, therefore, may not be ideal for life-long therapy. Similarly, it has been reported that treatments might be based on modulation of ENaC- mediated Na⁺ transport, based on cell culture studies. However, it has also been discovered herein that unlike cultured cyst epithelial cells, the native cells do not express ENaC- mediated Na⁺ transport. Thus additional treatment modalities are needed to treat hepatic biliary disease. Accordingly, compounds, compositions, and methods are needed for treating cystic liver diseases, including autosomal dominant PLD, PLD accompanying PKD, and the like. It has been discovered herein that peroxisome proliferator- activated receptor

(PPAR) modulators are useful in treating liver diseases characterized by water and/or ion homeostasis dysfunction, hi addition, is has been discovered herein that PPAR modulators are useful in treating liver diseases that are characterized by cyst formation. It has also been discovered herein that cholangiocytes are responsive to PPAR modulators, and in particular that PPAR modulation also effects the actions of the cystic fibrosis transmembrane conductance regulator (CFTR) on cholangiocytes. Without being bound by theory, it is believed herein that the CFTR is one or the primary receptors dysregulated in diseases treatable with the pharmaceutical compositions and methods described herein. In addition, but without being bound by theory, it is believed herein that the PPARs exert their effect on CFTRs by downregulating expression of that receptor, resulting in an overall decrease of ion flux across the cholangiocytes. It has also been discovered herein that hepatic biliary cysts isolated as native tissue from a mouse model of PKD/PLD are capable of Cl- secretion via CFTR in response to an agent that increases intracellular cAMP. Without being bound by theory, it is believed herein that salt and/or water homeostasis dysfunction may arise when chloride channels like the CFTR are activated and/or upregulated, allowing chloride ion secretion from cells. Chloride secretion, in turn, is followed by passive water movement across the cell to equalize the osmotic pressure. That water movement leads to a number of disease states, such as cyst formation. Therefore, though a number of seemingly unrelated diseases result in liver cyst formation, it is believed herein that intervention in one process in common, the CFTR, will provide treatment of the various forms of PLD. In particular, inhibition of the CFTR may alleviate the liver diseases described herein. In one embodiment of the invention, compositions and methods for PLD are described that include PPAR gamma modulators, or mixed PPAR gamma modulators such as PPARα/γ modulators, can inhibit CFTR expression or activity and therefore may be used in the treatment of polycystic liver disease and other liver diseases described herein that include liver cysts. As used herein, the term "PPAR gamma modulator" refers to a modulator of PPAR gamma or a modulator of multiple types of PPAR including PPAR gamma, e.g. a PP AR α/γ modulator.

In another embodiment, compositions and methods are described herein for treating diseases that include liver cysts, such as liver cysts in the bile duct. In another embodiment, methods are described herein for treating liver diseases of the hepatic biliary system responsive to inhibition or down regulation of the CFTR. Without being bound by theory, it is believed that the inhibition of the CFTR herein includes interruption or interference with CFTR-mediated anion transport in any indirect fashion where PPARs are involved, hi another embodiment, methods are described herein for treating liver diseases arising from salt and/or water homeostasis dysfunction. In another embodiment, methods are described herein for treating polycystic liver disease. In another embodiment, methods are described herein for treating chronic hepatic liver cyst formation. It is appreciated that in some cases, chronic hepatic liver cyst formation may be accompanied by liver fibrosis or in general the formation of fibrotic tissue. It is further appreciated that the compounds, compositions, and methods described herein may also be used in those forms of diseases. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Light microscopic images of liver sections showing normal and cystic bile ducts of heterozygous (BALB/c-cpk/+) and homozygous (BALB/c-cpk/cpk) mice. All images were taken with a 4OX objective. The bile ducts/cysts are generally closely associated with the portal triads (bile duct-B, portal vein-V and hepatic artery branch-A). The cystic bile ducts (right) from both the cpk/+ heterozygotes (top) and cpk/cpk homozygotes (bottom) are associated with an increase in periportal fibrosis.

Figure 2. Electrophysiology of biliary epithelia lining the liver cysts found in BALB/c cpk/+ mice. The epithelium in each panel represents a large liver cyst (each from a different mouse). Tissue was mounted in Ussing chambers (to measure electrophysiological parameters) and maintained under voltage clamp conditions at 37°C. SCC, the short circuit current, is a measure of net transepithelial, electrogenic ion transport. Panel B shows the specific sidedness of the agonists/inhibitors applied to the cyst epithelia. Effectors were added to the bathing media to achieve final concentrations of: amiloride, 10 μM; forskolin, 5 μM; NPPB, 100 μM. These data show that an agent that increases cAMP (forskolin) increases ion flux. The inhibitors indicate that the stimulated ion flux is not Na⁺ absorption (amiloride) but, rather Cl secretion through CFTR (NPPB).

Figure 3. To determine the effect of PP ARγ agonists on transepithelial ion flux, polarized MDCK-C7 renal cells were treated serosally with vehicle (DMSO), farglitazar (GI2570) (lμM) (Panels A and B) or pioglitazone (lμM) (Panels B and C) for 24 hrs, assembled into Ussing chambers and stimulated with vasopressin (vaso) (lOOmU/mL) at time = 0 min. Amiloride (amil, 1x10- M) was added 30 min. later. The entire duration of the electrophysiological study is shown on the left of each panel. An expanded time scale emphasizing chloride secretion is shown on the right of each panel. The magnitude of chloride secretion is shown in the insert in the left panels. Bars and symbols represent means ± SEM of 23 experiments for GI2570 and 81 experiments for pioglitazone (pio) where *p < 0.0002 as determined by unpaired Student's t-test.

Figure 4. To determine the dose-response relationships for PP ARγ agonist inhibition of Cl- secretion, polarized MDCK-C7 cells were treated for 24 hrs on the serosal side with varying concentrations of full PP ARγ agonists, GI2570, GW7845, pioglitazone and rosiglitazone (Panel A) or partial PP ARγ agonists GW5266 and GSK501A (Panel B), assembled into Ussing chambers, and subsequently stimulated with vasopressin (lOOmU/mL). The magnitudes of chloride secretion in treated cells were compared to, and expressed as a percent of, that in vehicle-treated cells. Symbols represent means ± SEM and the n for each concentration of each agonist was between 4-12. Panel C: Polarized MDCK- C7 cells were solubilized and proteins were separated by SDS-PAGE and transferred to PVDF in preparation for immunoblotting. PVDFs were cut in half and probed for Na⁺/K⁺ ATPase α-1 subunit or PPARγ. Na⁺/K⁺ ATPase α-1 subunit served as a loading control. It has been discovered herein that MDCK-C7 cells possess PPAR gamma. The expected molecular weights of the proteins were detected (Na⁺/K⁺ ATPase α-1 subunit, 113kDa; PPARγ, 55 kDa).

Figure 5. Determination of whether the PPARγ agonist effect on Cl- flux is due to an action on the apical or the basolateral membrane of polarized MDCK-C7 cells.

Panels A and B: Polarized MDCK-C7 cells were assembled into Ussing chambers and treated serosally with (line plot 2 and 3) or without (line plot 1) nystatin (280 U/mL) for 30 min. prior to vasopressin (100 mU/mL) stimulation. All cultures were treated apically with amiloride (10^-5 M) 10 min. prior to stimulation to block Na⁺ reabsorption via ENaC. Vasopressin was added at time = 0 min. The culture denoted in line plot 1 was bathed in symmetrical Ringers solution ([Cl ] = 150 mM on both apical and serosal sides). The nystatin-treated culture denoted in line plot 2 was bathed in asymmetrical Ringers solution

The nystatin-treated culture in line plot 3 was bathed in symmetrical Ringers solution ([Cl-] = 150 mM on both apical and serosal sides). In line plot 3, the absence of any increases in SCC upon vasopressin stimulation verifies the efficacy of nystatin in permeabilization of the basolateral membranes. Panels B, C, and D: Polarized MDCK-C7 cells were treated serosally with DMSO (vehicle, black circles), GI2570 (1 μM, white circles) or pioglitazone (1 μM, gray triangles) for 24 hrs. While in Ussing chambers, cells were treated serosally with (Panel C) or without nystatin (Panel B) for 30 min prior to stimulation. Cultures in panel B were bathed in symmetrical Ringers solution ([Cl ] = 150 mM). Cultures in panel C were bathed in asymmetrical Ringers solution ([Cl- ]a_picai= 15 mM, [Cr]_serosai = 150 mM). All cultures were treated apically with amiloride 10 min. prior to stimulation to block Na⁺ reabsorption via ENaC. Panel D: The magnitude of vasopressin-stimulated Cl- transport in agonist-treated cultures was normalized to their respective (permeabilized or non-permeabilized) vehicle-treated cultures and expressed as a percent. Bar graphs represent the mean ± SEM of 4 experiments for GI2570 and 5 experiments for pioglitazone, where *p < 0.02 compared to the vehicle-treated as determined by a one-way ANOVA followed by Tukey's post-hoc test. Non-permeabilized cultures treated with either agonist were not statistically different from permeabilized cultures. Thus, the effect of the PPARγ agonists are manifested via an effect on the apical membrane, the site of the CFTR localization.

Figure 6. Polarized MDCK-C7 cells were treated serosally for 24 hrs with vehicle (DMSO) (a), GI2570 (lμM) (b) or pioglitazone (lμM) (c). Cells were prepared for nucleotide extraction and HPLC, and normalized to the amount of protein in each sample. Bars represent means ± SEM of 3 experiments containing three replicates each. No significant changes were detected between vehicle and treated cells. ATP, adenosine triphosphate; ADP, adenine diphosphate; AMP, adenosine monophosphate; NAD, nicotinamide adenine dinucleotide; GTP, guanosine triphosphate; GDP, guanosine diphosphate; TAN, total adenine nucleotide. These data show that PPARγ agonist inhibition of Cl- secretion is not due to changes in these nucleotide levels.

DETAILED DESCRIPTION

In one illustrative embodiment, compositions and methods are described herein for treating liver diseases that may arise from dysregulation, dysfunction, or imbalance of salt and/or water homeostasis of the hepatic biliary system. Illustrative diseases include, but are not limited to, autosomal polycystic liver disease, liver manifestations of polycystic kidney and other, more rare diseases such as Meckel-Gruber Syndrome, Bardet-Biedl Syndrome and Nephronophthisis that present with fibrocystic formation in the liver. The compositions and methods include the administration of the peroxisome proliferator- activated receptors gamma (PPARγ) and/or mixed peroxisome proliferator- activated receptors modulators, such as alpha-gamma (PPARα/γ) modulators. In another embodiment, the PPARγ modulators are modulators of human PPARγ. In another embodiment, the patient being treated is a mammal. In another embodiment, the patient being treated is a human. In another embodiment, the PPARγ modulators are agonists. Illustrative PPARγ agonists include thiazolidinediones (also referred to as glitazones).

In another embodiment, methods and compositions are described herein for treating or ameliorating the effects of a liver disease in a patient in need of relief from the liver disease, the method comprising the step of administering to the patient a therapeutically effective amount of a PPAR gamma modulator, or a pharmaceutically acceptable salt thereof.

In another embodiment, the methods and compositions described in the preceding embodiment wherein the liver disease arises from salt and/or water homeostasis dysfunction are described. In another embodiment, the methods and compositions described above wherein the liver disease results from dysregulation, dysfunction, or imbalance of salt and/or water homeostasis of the hepatic biliary system are described. In another embodiment, the methods and composition described in the preceding embodiments wherein the liver disease is associated with transepithelial ion flux dysfunction are described. In yet another embodiment, methods and compositions described in the preceding embodiments wherein the liver disease is polycystic liver disease are described.

In another embodiment, methods and compositions for treating liver disease wherein the liver disease is chronic hepatic liver cyst formation or liver fibrosis are described. In another embodiment of the methods and compositions described above the liver disease includes liver fibrosis or periportal fibrosis, or a combination thereof are described. In another embodiment, the method and compositions described above wherein the In another embodiment, the methods and compositions described above wherein the liver disease is autosomal polycystic liver disease or the liver disease is polycystic liver disease that is comorbid with polycystic kidney disease are described. In other embodiments of the methods and composition described herein the liver disease is Meckel-Gruber Syndrome, Bardet-Biedl Syndrome, or Nephronophthisis are described. In another embodiment, the methods and compositions described in the preceding embodiments wherein the liver disease is liver cysts in the bile duct are described.

In another embodiment, the methods and compositions described in the preceding embodiments wherein the liver disease is responsive to the inhibition of CFTR- mediated chloride secretion or wherein the liver disease is a disease of the hepatic biliary system responsive to inhibition or down regulation of the CFTR are described.

In another embodiment, the methods and compositions described in the preceding embodiments wherein the PPAR gamma modulator is capable of modulating cystic fibrosis transmembrane regulator-mediated ion secretion in liver tissue are described. In another embodiment, the methods and compositions described in the preceding embodiments wherein the PPAR gamma modulator is capable of downregulating CFTR expression in liver tissue are described. In another embodiment, the methods and composition described in the preceding embodiments wherein the therapeutically effective amount of the PPAR gamma modulator results in decreased cystic fibrosis transmembrane regulator-mediated ion secretion in liver tissue in vivo are described. In another embodiment, the methods and compositions described in the preceding embodiments wherein the PPAR gamma modulator a PPAR gamma agonist or partial agonist are described. In another embodiment, the methods and compositions of the preceding embodiments wherein the PPAR gamma modulator is a thiazolidinedione, or a pharmaceutically acceptable salt thereof are described. In another embodiment, the methods and compositions of the preceding embodiment wherein the thiazolidinedione is selected from the group consisting of rosiglitazone, pioglitazone, and analogs and derivatives thereof, and combinations of there foregoing are described.

As used herein, the term "liver disease" includes all diseases, disease states, and disorders where the liver or liver function is compromised by dysfunction and/or imbalance in salt and/or water homeostasis or balance, such as diseases, disease states, and disorders that include the presence of liver cysts. Illustrative liver diseases include, but are not limited to chronic hepatic liver fibrosis, PLD accompanying PKD, nephronophthisis (NPHP), Meckel-Gruber Syndrome, Bardet-Biedl Syndrome, and the like.

It has been discovered herein that cysts derived from the biliary system in a model of PLD respond to agents that increase cAMP with an increased Cl secretion via CFTR. The bile duct is lined with cholangiocytes, and cholangiocytes express CFTRs. It has also been discovered herein that the administration of PPAR gamma, whether pure or mixed modulators such as PPARα/γ modulators, effect inhibition of CFTR-mediated chloride secretion and/or transport. It is appreciated, based on the cAMP assay, that the effect of the PPARγ appears to be downstream of this second messenger. This finding is substantiated herein by the demonstration that constitutive activation of adenylyl cyclase by forskolin did not reverse the observed inhibition of chloride secretion. It is further appreciated that the effect of PPARγ modulators on CFTR activity also appears to be independent of direct modulation of PKA, basolateral transport proteins and adenosine nucleotides.

As used herein, the term "PPAR gamma modulator" generally refers to any agent that regulates the activity of any member of the PPAR gamma pathway. Illustratively, such a modulator may be an agonist, partial agonist, inverse agonist, antagonist, and the like. Illustratively, the results of the modulation include, but are not limited to, decrease in expression of the cystic fibrosis transmembrane regulator. It is to be understood that the modulators described herein may act upstream or downstream of PPAR gamma receptor function, and/or upstream or downstream of endogenous ligands of PPAR gamma receptors. It is also to be understood that PPAR gamma modulators useful in the inventions described herein may have mixed receptor activity. Accordingly, the term PPAR gamma modulator also refers to those compounds that also may have PPAR alpha or other receptor activity. It is to be understood that in each of the foregoing, pharmaceutically acceptable salts of PPARγ and/or PPARα/γ modulators may be included in the methods described herein. In addition, it is appreciated that PPARγ and/or PPARα/γ modulators (PPAR modulators) may be used in a wide variety of hydrate, solvate, and morphological forms, including various crystal and non- crystal forms, and mixtures thereof. Accordingly, as used herein, the terms PPARγ and/or PPARα/γ modulators individually and collectively include the individual compounds, pharmaceutically acceptable salts, hydrates, solvates, and the various morphological forms. Pharmaceutically acceptable salts of the compounds used in the methods described herein include the acid addition and base salts thereof. In another embodiment, the methods and compositions described herein include one or more PPAR gamma agonists and/or partial agonists.

Illustrative PPAR gamma moculators include thiazolidinediones. Illustrative thiazolidinediones include rosiglitazone (specific formulations of which are also known as AVANDIA) and analogs and derivatives thereof, pioglitazone (specific formulations of which are also known as ACTOS) and analogs and derivatives thereof, troglitazone (specific formulations of which are also known as REZULIN) and analogs and derivatives thereof, farglitazar (GI2570) and analogs and derivatives thereof, MCC-555 and analogs and derivatives thereof, rivoglitazone and analogs and derivatives thereof, ciglitazone and analogs and derivatives thereof, and combinations thereof. Illustrative PPARα/γ modulators include muraglitazar and analogs and derivatives thereof, tesaglitazar and analogs and derivatives thereof, aleglitazar and analogs and derivatives thereof, and the like.

In another embodiment, the compositions and methods described herein include PPAR gamma modulators is described in European Patent 306228. The foregoing publication, and each publication cited herein, is incorporated herein by reference. In another embodiment, the PPAR gamma modulator is 5-[4-[2-(N-methyl-N-(2- pyridyl)amino)ethoxy]benzyl]thiazolidine-2,4-dione (rosiglitazone), including salts thereof, such as the maleate salt described in WO94/05659. In another embodiment, the PPAR gamma modulator is described in European Patent Applications, Publication Numbers: 0008203, 0139421, 0032128, 0428312, 0489663, 0155845, 0257781, 0208420, 0177353, 0319189, 0332331, 0332332, 0528734, 0508740; International Patent Application,

Publication Numbers 92/18501, 93/02079, 93/22445 and U.S. Pat. Nos. 5,104,888 and 5,478,852. In another embodiment, the PPAR gamma modulator is 5-[4-[2-(5-ethyl-2- pyridyl)ethoxy] benzyl] thiazolidine-2,4-dione (pioglitazone) , 5 - [4- [ ( 1 - methylcyclohexyl)methoxy]benzyl]thiazolidine-2,4-dione (ciglitazone), 5[[4-[(3,4-dihydro-6- hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-yl)met- hydroxy]phenyl]methyl]-2,4- thiazolidinedione (troglitazone) and 5-[(2-enzyl-2,3-dihydrobenzopyran)-5- ylmethyl)thiazolidine-2,4-dione (englitazone) .

In another embodiment, the PPAR gamma agonist is not a thiazolidinedione but instead 0- and N- substituted derivatives of tyrosine. Illustrative PPAR gamma agonists are described in U.S. Pat. No. 6,294,580. In another embodiment, the PPAR gamma agonist is N-(2-benzoylphenyl)-O-[2-(5-m ethyl-2-phenyl-4-oxazolyl)ethyl]-L -tyrosine, 2(S)-(2- benzoyl-phenylamino }-3-{4-[2-5-methyl-2-phenyl-oxazol-4-yl)-ethoxy]phenyl}-propionic acid, (farglitazar), or GI262570. Farglitazar may be prepared by conventional methods, or as described in US Patent Applications Publication No. 20080113996.

In another embodiment, the PPAR gamma agonist is described in WO 02/062774, WO 02/30895, WO 00/08002, WO 02/059098, WO 03/074495.

In another embodiment, the PPAR gamma agonist is farglitazar, farglitazar sodium salt, rosiglitazone, rosiglitazone maleate salt, 2-({4-[({4-({4-[4-(ethyloxy)phenyl]-1- piperazinyl)methyl)-2-[4- trifluoromethyl)phenyl]-1,3-thiazol-5-yl}methyl)thio]phenyl}oxy)- 2-methyl-propanoic acid, ( { 2-ethyl-4- [ ( { 4- ( { 4- [4- (methyloxy)phenyl] - 1 -piperazinyl j methy- l)-2-[4-(trifluoromethyl)phenyl]-1,3-thiazol-5-yl}methyl)thio]phenyl}oxy)a- cetic acid, 2-{4- [{2-[2-fluoro-4-(trifluoromethyl)phenyl]-4-methyl-1,3-thi- azol-5-yl}methyl)sulfanyl]-2- methylphenoxy}-2-methylpropanoic acid. In this embodiment, the compounds may be prepared by conventional methods, or alternatively as described in WO 02/059098 and WO 02/062774.

In another illustrative embodiment, treatment of a liver disease by administering one or more of rosiglitazone, pioglitazone, and/or analogs and derivatives thereof having the following formula

wherein Ar is an optionally substituted aryl or heteroaryl; and X is a bond, an optionally substituted alkylene, or NR¹, where R¹ is hydrogen or alkyl is described.

In another embodiment, treatment of a liver disease by administering one or more of ciglitazone, troglitazone, rivoglitazone, and/or analogs and derivatives thereof having the formula wherein Q is selected from the group consisting of cycloalkyl, benzocycloalkyl, heterocycloalkyl, benzoheterocycloalkyl, aryl, and heteroaryl, each of which is optionally substituted is described.

In another embodiment, treatment of a liver disease by administering one or more of englitazone, and/or analogs and derivatives thereof having the formula

wherein X¹ is O, NR¹ or CH₂; and Ar is optionally substituted aryl or heteroaryl is described.

As used herein, references to suitable acid addition salts, include but are not limited to, those formed from acids which form non-toxic salts. Illustrative examples include the acetate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulphate/sulphate, borate, camsylate, citrate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulphate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, saccharate, stearate, succinate, tartrate, tosylate and trifluoroacetate salts.

As used herein, references to suitable base salts, include but are not limited to, those formed from bases which form non-toxic salts. Illustrative examples include the aluminium, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts.

As used herein, references to hemisalts of acids and bases, include but are not limited to, those formed from, for example, hemisulphate and hemicalcium salts.

In another embodiment, pharmaceutical dosage forms of and methods of administration of the compounds are described herein. PPAR modulators useful for the treatment of liver disease can be prepared and administered in a wide variety of oral and parenteral dosage forms, utilizing art-recognized products and procedures. See generally,

Remington: The Science and Practice of Pharmacy, (21^st ed., 2005). In another embodiment, pharmaceutical compositions containing the PPAR modulator may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparation.

Formulations for oral use include tablets which contain the PPAR modulator in admixture with non-toxic pharmaceutically acceptable excipients. These excipients may be, for example, inert diluents, such as calcium carbonate, sodium chloride, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, maize starch, or alginic acid; binding agents, for example, starch, gelatin or acacia, and lubricating agents, for example, magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed.

Formulations for oral use may also be presented as hard gelatin capsules wherein the PPAR modulator is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate, or kaolin, or as soft gelatin capsules wherein the PPAR modulator is mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil.

Aqueous suspensions usually contain the active materials in admixture with appropriate excipients. Such excipients are suspending agents, for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents which may be a naturally- occurring phosphatide, for example, lecithin; a condensation product of an alkylene oxide with a fatty acid, for example, polyoxyethylene stearate; a condensation product of ethylene oxide with a long chain aliphatic alcohol, for example, heptadecaethyleneoxycetanol; a condensation product of ethylene oxide with a partial ester derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate; or a condensation product of ethylene oxide with a partial ester derived from fatty acids and hexitol anhydrides, for example, polyoxyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example, ethyl, n-propyl, or p- hydroxybenzoate; one or more coloring agents; one or more flavoring agents; and one or more sweetening agents such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the PPAR modulator in a vegetable oil, for example, arachis oil, olive oil, sesame oil, or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example, beeswax, hard paraffin, or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid. Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the PPAR modulator in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example, sweetening, flavoring, and coloring agents, may also be present.

Pharmaceutical compositions of PPAR modulators useful for the treatment of liver disease may also be in the form of oil-in- water emulsions. The oily phase may be a vegetable oil, for example, olive oil or arachis oils, or a mineral oil, for example, liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally- occurring gums, for example, gum acacia or gum tragacanth; naturally- occurring phosphatides, for example, soybean lecithin; and esters including partial esters derived from fatty acids and hexitol anhydrides, for example, sorbitan mono-oleate, and condensation products of the said partial esters with ethylene oxide, for example, polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavoring agents. Syrups and elixirs may be formulated with sweetening agents, for example glycerol, sorbitol, or sucrose. Such formulations may also contain a demulcent, a preservative, flavoring agents, and coloring agents. The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent. Among the acceptable vehicles and solvents that may be employed are water, 1,3- butanediol, 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 diglycerides. Fatty acids such as oleic acid also find use in the preparation of injectibles.

In one embodiment, PPAR modulators useful for the treatment of liver disease may be administered directly into the blood stream, into muscle, or into an internal organ. Suitable routes for such parenteral administration include intravenous, intraarterial, intraperitoneal, intramuscular, and subcutaneous delivery. Suitable means for parenteral administration include needle injectors (including microneedles), needle-free injectors, and infusion techniques.

Parenteral formulations are typically aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents (preferably at a pH of from 3 to 9), but, for some applications, they may be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water.

The preparation of parenteral formulations under sterile conditions, for example, by lyophilisation, may readily be accomplished using standard pharmaceutical techniques well known to those skilled in the art.

In another embodiment, the solubility of a PPAR modulator used in the preparation of a parenteral formulation may be increased by the use of appropriate formulation techniques, such as the incorporation of solubility-enhancing agents. Formulations for parenteral administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed, sustained, pulsed, controlled, targeted, and programmed release formulations. Thus, a PPAR modulator may be formulated as a solid, semi- solid, or thixotropic liquid for administration as an implanted depot providing modified release of the PPAR modulator. Examples of such formulations include drug-coated stents and poly(dl-lactic-coglycolic)acid (PGLA) microspheres.

The term "therapeutically effective amount" as used herein, refers to that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes alleviation of the symptoms of the disease or disorder being treated. In one aspect, the therapeutically effective amount is that which may treat or alleviate the disease or symptoms of the disease at a reasonable benefit/risk ratio applicable to any medical treatment. However, it is to be understood that the total daily usage of the compounds and compositions described herein may be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically-effective dose level for any particular patient will depend upon a variety of factors, including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, gender and diet of the patient: the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidentally with the specific compound employed; and like factors well known in the medical arts.

In another embodiment, the therapeutically effective amount is capable of treating polycystic liver disease but lower than that capable of treating diabetes mellitus type 2, also referred to as type 2 diabetes, or non-insulin dependent diabetes mellitus. The data shown in TABLE 1 demonstrate that low doses of PPAR gamma compounds are highly active in treating liver diseases. For example, several compounds exhibited IC50 values that are more than 10-fold lower that the corresponding EC₅₀ value for receptor transactivation. Illustratively, such doses are lower or substantially lower that those doses or established for treating diabetes mellitus type 2. Illustrative per day doses of pioglitazone that are included in the methods and compositions described herein include about 5 to about 15 mg/day, about 2 to about 5 mg/day, about 1 to about 3 mg/day, and about 0.5 to about 1.5 mg/day. Illustrative per day doses of rosiglitazone that are included in the methods and compositions described herein include about 2 to about 4 mg/day, about 1 to about 2 mg/day, about 0.5 to about 1 mg/day, and about 0.25 to about 0.5 mg/day.

Effective doses of the present compounds depend on many factors, including the indication being treated, the route of administration, and the overall condition of the patient. For oral administration, for example, effective doses of the present compounds can range from about 0.001 mg/kg to about 50 mg/kg, from about 0.005 mg/kg to about 25 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, from about 5.0 mg/kg to about 25 mg/kg, or from about 10.0 mg/kg to about 50 mg/kg. Effective parenteral doses can range from about 0.01 mg/kg to about 10 mg/kg, from about 0.05 mg/kg to about 5 mg/kg, from about 0.1 mg/kg to about 5 mg/kg, from about 1.0 mg/kg to about 10 mg/kg, from about 1.0 mg/kg to about 5.0 mg/kg, or from about 1.0 mg/kg to about 10 mg/kg. In general, treatment regimens utilizing compounds in accordance with the present invention comprise administration of from about 1 mg to about 500 mg of the compounds of this invention per day in multiple doses or in a single dose. In another embodiment, the methods described herein are for treating liver diseases of mammals. As used herein, mammals include, but are not limited to, humans, horses, cows, pigs, dogs, cats, and the like. In another embodiment, the patient is a human.

It is understood that in both the kidney and liver bile ducts, the cysts are lined with epithelial cells, and that secretion of ions and fluid by the epithelial cells contribute to cyst expansion in renal cells (Torres, 2006; Torres, 2007). CFTR (Cystic Fibrosis Transmembrane Regulator) is expressed in the kidney (Crawford, L, et al., Proceedings of the National Academy of Sciences of the United States of America 88, 9262-9266, 1991; Morales, M.M., et al., The American journal of physiology 270, F1038-1048, 1996) where it is able to transport chloride into and out of the cell depending on the electrochemical driving force across the cellular membrane and this CFTR-mediated Cl- transport has been shown to be involved in the formation of the cysts in the kidney tissue (Ye M., et al., N Engl J Med 329:310-313, 24, 1993; Grantham JJ., et al., J Clin Invest 95:195-202, 1995; Mangoo-Karim R., et al., Am J Physiol 269:F381-388, 1995). Without being bound by theory, it is believed that the cystic fibrosis transmembrane regulator (CFTR), an ion transport protein, plays a primary role in electrolyte and fluid secretion into the cyst in hepatic biliary cystic disease. In particular, it is believed that the inhibitor studies and electrophysiological analyses described herein indicate that the CFTR, located in the apical membrane, may be the ultimate chloride channel responsible for the secretion into the hepatic biliary cysts. Described herein are electrophysiological studies of the CFTR showing that it is active in the cholangiocyte-lined cysts of the liver bile duct and it moves chloride in a secretory direction.

The potential importance of PPARγ agonists in modulating CFTR is also described herein. Twenty-four hour incubations with a variety of PPARγ agonists show a concentration response for agonist inhibition of vasopressin- stimulated anion transport with IC₅₀ values that are similar to the EC₅₀ values for receptor trans-activation (TABLE 1). As mentioned previously, each agonist binds to PPARγ with high affinity; however, each activates the receptor differently. It has been observed herein that while the rank order of agonist efficacy is the same for inhibition of anion transport and receptor transactivation, the chloride inhibitory action is left-shifted by comparison with the receptor transactivation.

* Willson TM, et al , J Med Chem, 43 527-550, 2000 In addition, it is appreciated that the effect of PPARγ agonists appears to be manifested directly on apical transport proteins Also observed with PPARγ agonist treatment is the substantial decrease in CFTR mRNA, as shown in the following Table

Values are means SE of 16-18 experiments. The values represent the number of copies/ 10 ng input RNA. All primer/probe gene sets were validated for linearity and slope using a pool of cDNA from multiple tissues. Polarized Madin-Darby canine kidney (MDCK)-C7 cells were treated with vehicle (DMSO), GI2570 (1 μM), or pioglitazone (1 μM) for 24 h on the serosal side and prepared for quantitative PCR. PPARγ, peroxisome proliferator-activated receptor-γ; AQP2, aquaporin-2; ENaC, epithelial Na channel;. The copy number of each gene was normalized to the geometric mean of 3 housekeeping gene count numbers (β-actin, GAPDH, and cyclophilin). The genes that were significantly changed compared with vehicle treatment are shown in bold and denoted by asterisks. The direction of change is indicated by the arrows (up, increase; down, decrease; -, no change). P values were calculated relative to vehicle-treated cells by Student's Mest.

Without being bound by theory, it is believed that this decrease is consistent with the functional data shown in Figure 4 and suggests a heretofore unappreciated role for chloride transport in renal-driven salt and water homeostatic mechanisms.

As described herein, effects of the PPARγ agonists on vasopressin-stimulated ion transport were demonstrated in the MDCK-C7 cell line, a high resistance subclone of the Madin-Darby Canine Kidney (MDCK) cell line. The C7 subclone exhibits the characteristics of the principal cell type including high transepithelial resistances (> 1000 Ω«cm²) and natriferic (salt retaining) responses to various hormones (Blazer- Yost, et al., Pflugers Arch 432, 685-691(1996); Gekle, M., et al., Pflugers Arch 428, 157-162, 1994; Lahr, T.F., et al., Pflugers Arch 439, 610-617, 2000). Under short-circuit conditions, the high resistance monolayer formed by this cell line has a tri-phasic response to vasopressin stimulation beginning with an immediate and rapid anion secretory event via CFTR. This transient transport event is followed by more delayed K⁺ and Na⁺ reabsorptive ion fluxes (Lahr, 2000) (Figure 3). These cells also express PPARγ (Figure 4, panel C). It has been discovered herein that the chloride secretory response to vasopressin, a hormone involved in fluid retention, combined with the distal site of origin, make the MDCK-C7 cell line a useful model to examine potential effects of PPARγ ligands on chloride transport.

Figure 3 shows that a 24 hour incubation with two chemically different PPARγ agonists, GI2570 and pioglitazone, inhibit the magnitude of vasopressin stimulated chloride secretion (GI2570 by 66.0 ± 3.9% and pioglitazone by 39.1 ± 7.9%). Both agents also inhibit amiloride-sensitive current (GI2570 by 56.8 ± 2.4% and pioglitazone by 39.8 ± 6.2%).

To examine the concentration-response relationships of agonist-mediated inhibition of chloride secretion, MDCK-C7 cultures were pre-incubated with the compounds for 24 hours. Long-term incubations within pharmacologically relevant concentrations of each agonist inhibited vasopressin-stimulated anion secretion. The concentration response relationships for inhibition of anion secretion of various PPARγ agonists are shown in Figure 4, panels A and B. The IC₅₀ for inhibition of anion secretion follows the rank order of the EC₅₀ at which each agonist is able to transactivate PPARγ in vitro (TABLE 1). Each of the compounds used in these experiments is a potent and selective activator of PPARγ in vitro. The compounds shown in Figure 4, panel A are considered selective, full PPARγ agonists. While the compounds used in Figure 4, panel B display potent and selective binding to PPARγ, they are considered partial agonists. Even so, in this assay of inhibiting vasopressin stimulated anion secretion, both the full and partial agonists had a maximal effect in inhibiting vasopressin stimulated anion secretion by 60% or greater.

Without being bound by theory, because PPARγ is a nuclear transcription factor, it is appreciated that the effects observed with treatment would likely be genomic rather than immediate. The time-course of the inhibitory effects on anion secretion were examined using maximal (1 μM) concentrations of pioglitazone and GI2570. At this concentration, the effect on chloride secretion is manifested only after 12 hours of GI2570 incubation and 24 hours of pioglitazone incubation.

It is appreciated that PPARγ agonists block an apically located chloride conductance. The action of vasopressin in renal principal cells is mediated via the V2 receptor located on the basolateral membrane. Binding of vasopressin to its receptor on the basolateral membrane results in activation of adenylyl cyclase (AC), subsequent increase in production of cAMP, activation of cAMP-dependent protein kinase A (PKA) and the stimulatory phosphorylation of CFTR resident in the plasma membrane. (Carroll, T.P., et al., Cell Physiol Biochem 3, 388-399, 1993). It is appreciated that this pathway is also known to stimulate the insertion of ENaC and aquaporin 2 (AQP2) into the apical plasma membrane. Sodium ionreabsorption, chloride secretion and water reabsorption through CFTR, ENaC and AQPs, respectively, are activated

The role of basolateral membrane conductances could be important, therefore that membrane was "eliminated" with nystatin, a polyene compound which permeabilizes sterol-containing membranes to small, monovalent ions including Na⁺, K⁺ and Cl- (HoIz, R., et al., B. J Gen Physiol 56, 125-145, 1970). Without being bound by theory, it is believed that if the inhibition of chloride transport is evident after basolateral membrane permeabilization, the ion transport element responsible must be located beyond the basolateral membrane. On the other hand, if permeabilization of the basolateral membrane rescues agonist-mediated decreases in chloride secretion, the transport protein(s) responsible must be located on the basolateral membrane.

An illustrative example demonstrating selective membrane permeabilization is shown in Figure 5, panel A. Figure 5, panels B-D, also shows that agonist-mediated inhibition of chloride secretion is still evident after serosal membrane permeabilization, indicating that the event mediating the changes in ion transport lies predominately within a non-diffusible intracellular component or within the apical membrane. The similar experiment was performed using forskolin to attain a constitutive and maximal activation of adenylyl cyclase (Moran, W.M., et al., Am J Physiol 260, C824-831, 1991). Inhibition of chloride secretion persisted after basolateral membrane permeabilization and stimulation with forskolin.

The effects on intracellular mediators of vasopressin signaling were also evaluated. In MDCK-C7 cells, there is a concentration dependent correlation between the magnitude of vasopressin-stimulated cAMP production and the magnitude of chloride secretion suggesting that a PPARγ agonist-induced effect on cAMP concentrations could alter CFTR activity. In addition, it was previously shown in intestinal epithelia that troglitazone lowered cellular cAMP (Hosokawa, 1999). Vasopressin stimulation resulted in a substantial increase in cAMP levels in both vehicle and agonist-treated cultures. However, it was observed herein that neither pioglitazone nor GI2570 significantly altered the magnitude of vasopressin-stimulated cAMP production as compared to vehicle-treated cells (TABLE 5).

The elimination of cAMP modulation therefore suggests that proteins downstream of this second messenger, such as PKA, are likely to be impacted by these agents. PKA consists of a heterotetramer (two regulatory and two catalytic subunits).

Binding of cAMP releases the catalytic subunits from the inhibitory control of the regulatory subunits. Although production of cAMP does not activate PKA by direct phosphorylation, the phosphorylation of the catalytic subunits at Thr ⁹⁷ is ultimately required for the biological function of the enzyme (Taylor, S. S., et al., Annu Rev Biochem 59, 971-1005, 1990), and PPARγ ligands may alter this property. In order to determine the role of PKA in agonist- mediated chloride secretion inhibition, MDCK-C7 cells were challenged with pioglitazone and probed by Western blotting for PKA C and phospho-PKA expression. Neither

pioglitazone nor GI2570 had an effect on the amount of either PKA C or pPKA

In addition to phosphorylation by PKA, the adenine nucleotides, including adenine diphosphate (ADP) and adenine triphosphate (ATP), are capable of regulating CFTR activity (Anderson, M.P., et al., Science 257, 1701-1704, 1992). It is also established that troglitazone and rosiglitazone can alter the total adenine nucleotide (TAN) pool in some cells (Fryer, L.G., et al., J Biol Chem 277, 25226-25232, 2002). Accordingly, it is appreciated herein that TAN may be an avenue by which PPARγ agonists alter CFTR-driven chloride secretion in MDCK-C7 cells. Cellular levels of adenine monophosphate (AMP), ADP, ATP, nicotinamide adenine dinucleotide (NAD), guanine diphosphate (GDP), guanine triphosphate (GTP) and TAN in MDCK-C7 cells treated with either GI2570 or pioglitazone are shown in Figure 6. There were no changes in any of the aforementioned nucleotide levels compared to vehicle-treated cells, indicating, without being bound by theory, that cellular nucleotide levels are not the underlying mechanism for decreases in chloride secretion. From these studies it is concluded that the effect of the PPARγ agonists on Cl- secretion in the MDCK-C7 cell line is manifested by a direct effect on the activity and/or amount of CFTR in the apical membrane.

Ion transport properties of native hepatic biliary cyst epithelia were also measured. A surrogate model of both PKD and PLD is the B ALB/c-cpk/cpk mouse, which dies of renal disease before maturity. In contrast, the heterozygous BALB/c-cpk/+ animals live to breeding age and after 12-18 months express biliary hepatic cysts which are similar to those found in human PKD and ADPKD patients (Ricker J. L., et al., J Am Soc Nephrol 11: 1837-1847, 2000). In particular, older heterozygous mice (12-18 months) develop intra- hepatic bile duct cysts which are phenotypically similar to the multiple, large, hepatic biliary cysts observed in human ADPKD (Ricker, 2000). The phenotypic presentation of the liver cysts in this model is dominated by extracellular fibrosis, particularly in the tissues immediately surrounding the cysts. The results herein described are similar to Hou et al., who reported extracellular fibrosis surrounding the renal cysts in C57BL/6J model of PKD (Hou, 2002). Proliferative changes may be enhanced by the presence of proto-oncogenes in PKD (Ye, 1993; Harding MA, et al., Kidney Int 41:317-325, 1992). Described herein are methods for analyzing ciliary structure and transepithelial ion flux during hepatic cyst development in the B ALB/c cpk mouse model of PKD, including isolation of hepatic bile duct cysts and the use of electrophysiological techniques to characterize the ion transport properties of the freshly isolated cyst wall.

Electrophysiological experiments were performed on isolated cyst wall epithelia from large biliary liver cysts of a mouse model of PKD/PLD (Figure 2). The cholangiocytes which line the cysts showed electrogenic ion secretion in response to forskolin, an agent known to increase intracellular cAMP. The first panel (Figure 2A) illustrates a response when forskolin was added to both the luminal and serosal sides of the epithelia. The increase in SCC represents either an increase in cationic movement in an absorptive direction (luminal to basal) or anionic movement in a secretory direction (basal to luminal). In contrast to most other epithelial cells where adenylyl cyclase (the protein target activated by forskolin) is associated with receptors on the basolateral membrane, forskolin was ineffective when added to the serosal face of the monolayer but was always effective when added to the apical side (Figure 2B, representative of 5 out of 5 experiments).

Inhibitors of ion channels previously allied with transport in renal cysts were used to determine the nature of the SCC. Ion transport was not inhibited by amiloride, a specific ENaC blocker, indicating that Na⁺ reabsorption does not contribute to basal or forskolin stimulated ion transport. 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), a chloride channel blocker, inhibited trans-epithelial transport but only when added to the apical side of the epithelial sheet indicating that in the cholangiocytes, like the renal cells, the anion channels are in the apical membrane. Based on data suggesting a role for CFTR in renal cysto genesis, it described herein that the chloride channel is likely involved in the biliary cysts is also CFTR. The two experiments shown in Figure 2 are representative of 5 such experiments. In all 5 experiments, the absence of amiloride sensitivity was consistent. The figures shown were selected to illustrate the variability in basal transport rates (from 7 - 40 μA/cm2) as well as the differences in the percentage of the final current which was inhibited by NPPB.

As described herein, sheets of liver cyst wall can be successfully isolated from heterozygous animals and used in electrophysiological studies to examine electrogenic transepithelial transport characteristics. The cholangiocytes which line the biliary duct cysts are responsible for the secretion of ions and fluid into the cyst cavity. Therefore, a characterization of the ion transport proteins and an elucidation of some of the intracellular biochemical pathways controlling the activity of the channels forms a background for understanding cyst development and maintenance. The basal level of electrogenic ion flux in the isolated cholangiocyte-lined tissues showed considerable variation. Without being bound by theory, it is suggested that the variation is unlikely to be due to the size of the cysts since only the largest of these could be used for electrophysiological experiments.

In contrast to cultured epithelia from cyst walls (Doctor, 2007), the freshly isolated cystic epithelia were unresponsive to amiloride, a specific inhibitor of the epithelial Na+ channel, ENaC. Without being bound by theory, it is believed herein that these results suggest that electrogenic Na+ reabsorption does not contribute to basal or forskolin stimulated ion transport ex vivo and in vivo. Instead, it is believed herein that the differences between the amiloride sensitivity in the cultured cells versus the freshly isolated tissue may be explained, in part, by the conditions necessary to grow cells in primary culture. In particular, these conditions generally include culture media with high levels of steroids (dexamethasone), growth factors (epidermal growth factor), pituitary extract, triiodothyrodine, insulin and forskolin - all factors that are known to regulate ENaC synthesis and/or activity.

NPPB inhibits transepithelial transport but only when added to the apical side of the epithelial sheet. The inhibition on the apical side is consistent with the expected apical localization of the CFTR chloride channel where it can secrete anions into the cyst lumen. However, NPPB inhibits multiple transport proteins and is often used as an inhibitor of CFTR. Forskolin, an agent that activates adenylyl cyclase and thereby increases intracellular cAMP concentrations, increases net electrogenic transport with a directionality that is consistent with an increase in either cation absorption or anion secretion. Without being bound by theory, it is believed that because the process is inhibited by NPPB, but not amiloride, forskolin may be causing an increase in chloride secretion; however, forskolin is effective when added to the apical membrane. In many epithelial cells, adenylyl cyclase is localized to the serosal compartment where it is in contact with peptide hormone receptors resident in the basolateral membrane. For this reason, forskolin is usually effective from the serosal bathing media, not the apical. The results described herein are consistent with the recent observation by Masyuk et al. that adenylyl cyclase is found in cholangiocyte primary cilia on the apical surface (Masyuk, 2006). As such, this enzyme may be a target for agents that increase anion secretion (Nichols M.T., et al., Hepatology 40:836-846, 2004).

Alternatively, the supporting, fibrous membranes surrounding the basolateral surface of the epithelial monolayers may form a barrier for the diffusion of the forskolin.

Described herein are the ion transport characteristics of freshly isolated bile duct cystic epithelia. It is appreciated that the results indicate similarity between the biliary and renal cystic tissue. However, it is appreciated that the absence of vasopressin, V2, receptors in cholangiocytes means that, unlike the renal principal cells, antidiuretic hormone (ADH; vasopressin) will not be an endogenous ligand stimulating increases in intracellular cAMP. Thus, the V2 receptor antagonists are not likely be effective for treating the hepatic manifestations of PKD. However, in the PCK rat model of ARPKD, octreotide, an analogue of somatostatin appears to be effective in inhibiting cAMP levels and reducing cyst growth (Masyuk T.V., et al., Gastroenterology 132:1104-1116, 2007). Without being bound by theory, it is believed that the data provided herein are consistent with this finding and provide additional support for the proposed mechanism of drug action.

As described herein, ex vivo tissue has been used to demonstrate ion secretory flux which is consistent with cyst enlargement via compensatory fluid movement. It is also appreciated that the observation that activation of adenylyl cyclase is a stimulus for ion flux suggests that the components of the cyst fluid may contribute to cyst enlargement. Notably Cl secretion via CFTR is likely a driving force in cyst growth and expansion. Based on the findings presented herein, the PPARγ agonists inhibit CFTR activity and/or expression, and therefore these drugs are likely to be effective agents in the treatment of polycystic liver disease.

The methods described herein for treating or ameliorating one or more effects of a liver disease, such as a polycystic liver disease, using a modulator of PPAR gamma may be based upon animal models, such as murine models. It is understood that for example PLD in humans is characterized by a loss of function, and/or the development of symptoms, each of which may be elicited in animals, such as mice (see, e.g. Muchatuta et al. EMB 2009). In particular the mouse cpk/+ model may be used to evaluate the methods of treatment and the pharmaceutical compositions described herein to determine the therapeutically effective amounts described herein.

EXAMPLES

GENERAL. Nystatin, vasopressin, amiloride, and protease inhibitor cocktail were obtained from Sigma Aldrich (St. Louis, MO). PP ARγ modulators were from

GlaxoSmithKline (Research Triangle Park, NC). Primary antibodies included rabbit anti- PP ARγ (Affinity BioReagents; Golden, CO) used at a 1:3000 dilution, rabbit anti-PKA C and rabbit anti-pPKA^thr197 C (Cell Signaling Technology; Beverly, MA) used at a 1: 1000 dilution, and mouse anti-Na⁺/K⁺ ATPase α-1 used at a 1:10000 dilution. The secondary antibodies for Western blotting protocols were anti-rabbit/mouse IgG conjugated to horseradish peroxidase (Upstate Inc.; Charlottesville, VA) used at a 1: 50000 dilution.

MOUSE MODEL. The cpk gene is expressed primarily in liver and kidney and encodes the protein 'cystin'. BALB/c mice homozygous for a cpk mutation (BALB/c- cpk/cpk) rapidly develop polycystic kidney disease with the expression of a multi-organ phenotype and die within 2-4 weeks (Ricker, 2000). Heterozygous animals (BALB/c-cpk/+) have a relatively normal life span and can be used for breeding. As heterozygote mice age, they begin to develop liver cysts which are phenotypically similar to hepatic biliary cysts observed in human ADPKD. The cysts range in size from microscopic to the large fluid filled masses. At approximately 12-18 months of age BALB/c-cpk/+ mice (male/female) exhibit abdominal distention as a result of the expansion of the hepatic cysts. There are multiple cysts of varying sizes in the hepatic biliary tree. The mice were bred at the Indiana University School of Medicine laboratory animal resource center and used under protocols approved by IACUC.

RAT PLD MODEL. The PCK rat model was used because the genetic mutation in this animal is orthologous to that found in human ARPKD. These animals express many of the characteristics of human ADPKD (Lager DJ, et al., Kidney Internat 59: 126-136, 2001; Harris, Curr Opin Nephrol Hypertens 11:309-314, 2002). The animals carrying this mutation present with both kidney and liver fibrocystic disease and these animals live long enough to facilitate long-term treatment protocols (Gattone et al.,2003; Torres et al., 2004; Masyuk et al., 2007). Female animals show more severe liver disease than male animals.

EFFECTS OF ADMINISTERING PPARγ MODULATORS. Female PCK rats were fed control or PPARγ agonist-supplemented diets starting after weaning (4 weeks of age) and continuing until the animals were 18 weeks of age. Previous rodent studies utilizing pioglitazone have shown that a diet containing 25 mg pioglitazone/kg body weight is well tolerated and, in mice, results in a serum pioglitazone concentration of 15 M (Artunc F, et al., Pflugers Arch Eur J Physiol 456:425-436, 2008). This serum concentration is an approximately 10-fold higher concentration than is necessary to maximally inhibit Cl- secretion in a tissue culture model system (Nofziger et al., 2009). The animals were fed a dose of 20 mg/kg body weight. Overall, the animals appeared healthy throughout the study with no noticeable differences in general health between the animals on the control or pioglitazone-supplemented diets. Observations include:

1) Overall animal health and survival;

2) Kidney and liver weights as a function of overall body weight;

3) Bilirubin;

4) Plasma electrolytes;

5) liver enzymes;

6) blood urea nitrogen (BUN)

At 18 weeks of age, treated female PCK rats were evaluated for renal and liver cyst growth and pathology including serum analysis and histopathology (data expressed as mean 1 SEM). Treatment decreased the renal cystic enlargement, either expressed as total weight (decreased from 4.79 i_0.12, n = 6 in control cystic rats to 3.87 i_0.30, n = 5, in pioglitazone treated rats) or as a percentage of body weight (decreased from 1.53% ±_0.09, to 1.241 0.06). This appeared to be due to a reduction in cysts evidenced by a decrease in renal cyst volume density (Vv as a %, from 14.0% 1 1.3 to 7.8% 1 2.0) and total cyst volume (from 0.67 1 .06 to 0.29 ± 0.08 mL). Similarly, liver weight decreased either as total weight (from 21.28 ± 0.83, to 15.89 ± 0.82 grams) or expressed as a % of body weight (from 6.86% ± 0.66, to 5.11% 1 0.16). These decreases were statistically signficant in both organs ( p < 0.05). There was no effect of treatment on body weight (see TABLES 2 and 3). Female PCK rats are not azotemic at 18 weeks and the BUN level was not affected by the pioglitazone treatment (see TABLE 4).

The values given are averages 1 Standard Error Measurement for 6 animals on the control diet and 5 animals on the pioglitazone-supplemented diet. BW = body weight in grams; LW = liver weight in grams; LW as a % of BW - liver weight as a percentage of total body weight; KW = total kidney weight in grams; KW as a % of BW = total kidney weight as a percentage of total body weight; n = number of animals tested. P values are for the comparison of Control versus Pioglitazone diets by Students T-test. P less than 0.05 is considered si nificant. NS = not si nificant.

SEM = Standard Error Measurement; Control = PCK rats on the control diet; PIO = PCK rats on a diet containing 20 mg/kg body weight pioglitazone; n = number of animals; P values greater than 0.05 are considered non-significant (NS).

ELECTROPHYSIOLOGICAL ANALYSIS OF ION TRANSPORT IN ISOLATED LIVER CYST EPITHELIA. Normal and cystic BALB/c mice (-18 months of age) were euthanized (150 mg pentobarbital/Kg body weight) and perfused with phosphate buffered saline (PBS). Large liver cysts from BALB/c-cpk/+ mice were dissected free from surrounding hepatic tissue, opened by peripheral cuts to form a single sheet and mounted in a modified Ussing chamber. Cysts used for electrophysiological measurements had tissue sheets large enough to obtain a circular area of at least 1.3 cm in diameter. During the electrophysiological experiments, the cystic tissue was bathed in serum-free Dulbecco's modified Eagles medium/F12 media (DMEM) (In vitro gen, Grand Island, NY), supplemented with 2.4mg/L sodium bicarbonate (Fisher Scientific, Fair Lawn, NJ), and 2mM glutamine. The media was maintained at 37°C, with gentle circulation 8 provided by a 5% CO2-95% 02 gas lift. Electrodes inserted into the bathing media on either side of the tissue allowed for the measurement of the spontaneous transepithelial potential difference (PD). Using a current/voltage clamp, the PD was clamped to zero and the resulting short circuit current (SCC), a measure of net transepithelial ion transport, was recorded continuously. The epithelial layers were monitored until a steady basal current was obtained (20- 60 minutes). Effectors (from IOOOX stock) were added to the apical and/or serosal bathing medium as indicated in the figures and the ion transport responses of the tissue were measured. Agonist/inhibitors and final concentrations: forskolin, 5 μM, is an activator of adenylyl cyclase; 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), 100 μM, is a chloride channel blocker; and amiloride, 10 μM, is a specific inhibitor of ENaC. LIGHT AND ELECTRON MICROSCOPY (EM). Cysts were excised from the livers of the animals, immediately placed in EM fixative (2% glutaraldehyde, 2% paraformaldehyde, 10OmM phosphate buffer, pH 7.4) and fixed overnight at room temperature. The next day cysts were cut into smaller pieces and returned to the EM fixative before embedment in Epon812 resin for evaluation by light and electron microscopy. Light microscopy of lμm thick sections was used to analyze structural differences and similarities between normal and cystic tissue epithelia, and to provide an overview of the area to be viewed using electron microscopy. Electron microscopy was performed to analyze the structural differences between the cystic epithelial cells in contrast to normal bile duct cholangiocytes. For transmission electron microscopy (TEM), tissues were post-fixed in 1% osmium tetroxide for 1 h, rinsed, and dehydrated in graded ethanol solutions and propylene oxide 9 resin of 1:3, 1: 1 and 3: 1 and left overnight before being embedded in Epon812 and allowed to polymerize at 60 °C for 24 hrs. Semi-thick (lμm) resin sections were cut and stained with Toluidine blue and visualized with a light microscope. Thin sections (60nm/ 80nm) were cut, stained with uranyl acetate and lead citrate and visualized on a Tecnai G12 TEM (FEI, Hillsboro, Oregon).

Samples to be visualized using scanning electron microscope (SEM) were rinsed in PBS, incubated in 1% osmium tetroxide for 1 hr, rinsed in distilled water, dehydrated in graded ethanol solutions and then dried using a Tousimis Samdri 790 critical point drier, with liquid CO2 as the transitional fluid. After drying, samples were mounted onto stubs, coated with a gold/palladium (Au/Pd) by a Polaron direct sputter coater and viewed with a JEOL JSM 6390 scanning EM (JEOL USA Inc, Peabody MA). Micrographs were taken at appropriate magnifications. Cilia length was determined from the SEM micrographs with the aid of Scandium program (Sift Imaging Systems, Lakewood, CO). 10

CELL CULTURE. MDCK-C7 cells were grown at 37°C in a humidified incubator gassed with 5% CO₂. Culture media consisted of DMEM/F12 base media supplemented with 5% fetal bovine serum (ICN Biochemicals Inc), 25 U/mL penicillin, 25 mg/mL streptomycin (Invitrogen; Carlsbad, CA), and 12 mg/L ciprofloxacin (Voigt Global Distribution; Kansas City, MO). Media was replaced every two days. Cell cultures were maintained in plastic flasks until confluent and subcultured at a 1:10 dilution. For electrophysiological experiments, cells were subcultured onto permeable supports (Costar Transwells; Fisher, Chicago, IL) at a 1:3 dilution.

ELECTROPHYSIOLOGICAL ANALYSES. Short-circuit current (SCC) methodology was used to monitor net ion flux in polarized MDCK-C7 cultures. Confluent monolayers which had achieved a high resistance phenotype (> 1000 ohm.cm²) were removed from the Trans well support system and assembled into Us sing chambers. The spontaneous potential difference across the principal cell monolayer was clamped to zero, and the resulting SCC was measured. By convention, cation absorption (apical to serosal transport) or anion secretion (serosal to apical transport) is depicted an increase in SCC. During electrophysiological analyses, cell cultures were bathed with serum-free media (unless otherwise noted) maintained at 37°C. The compartment and membrane facing the lumen are defined as apical, the compartment facing the blood as serosal, and the membrane facing the blood as basolateral. A 5/95% CO₂/O₂ gas lift served to circulate the bathing media, as well as maintain oxygen and pH. Solutions of varying concentrations of PPARγ agonists were prepared at a 1000-fold excess of the desired final concentration via serial dilutions of a stock solution. The same volume of agonist/vehicle was added to each culture, regardless of concentration. Vasopressin (100 mU/mL) was added to the serosal bathing media and amiloride (10- M) was added to the apical bathing media 30 minutes after vasopressin addition. Transepithelial resistance (an indication of cellular viability) was monitored throughout the duration of each electrophysiological experiment by stimulating the cells with a 2000 μV pulse every 200 seconds. Resistance values were calculated from the resulting current deflections using Ohm's law.

To determine the IC₅₀ for PPARγ agonists, the raw data were expressed as percentage of the maximal inhibition of chloride transport and curves were fit to the data using the Hill-Slope four parameter logistic (4PL) model with an offset. This model used the equation Y = ((V_max*xⁿ)/(Kⁿ + x¹¹)) + Y2. To fit the 2570 data, the Y2 value was fixed at - 12%, the response at the lowest concentration tested, I X lO ¹² M.

PERMEABILIZATION EXPERIMENTS. Polarized MDCK-C7 cells were assembled into Ussing chambers and bathed in either physiological Cl- Ringers solution (in mM; 140 NaCl, 5 KCl, 0.36 K₂HPO₄, 0.44 KH₂PO₄, 1.3 CaCl₂, 0.5 MgCl₂, 4.2 NaHCO₃, 10 HEPES, 5 D-glucose, pH 7.2 with Tris-base) or low Cl- Ringers solution (in mM; 2.5 NaCl, 133.3 sodium gluconate, 5 potassium gluconate, 0.36 K₂HPO₄, 0.44 KH₂PO₄, 5.7 CaCl₂, 0.5 MgCl₂, 4.2 NaHCO₃, 10 HEPES, 5 D-glucose, pH 7.2 with Tris-base). The final chloride concentrations were 150 mM and 15.0 mM, respectively. Cultures treated serosally with nystatin (280 UVmL) were bathed asymmetrically (apical compartment = low Cl- Ringers, serosal compartment = physiological Cl- Ringers). All other cultures were bathed symmetrically in physiological Cl- Ringers. All cultures were treated with amiloride (10^-5 M) 10 min prior to hormonal stimulation to prevent Na⁺ transport via the epithelial Na⁺ channel.

CYCLIC AMP ASSAY. Polarized MDCK-C7 cells were treated serosally with DMSO, GI570 (lμM) or pioglitazone (lOμM) for 24 hrs, followed by stimulation with or without vasopressin (lOOmU/mL) for 10 seconds. Each culture was washed twice with 37°C Hank's Balanced Salt Solution (HBSS) and incubated for 10 min. with 1% Triton X- 100 in 0.1M HCl at 37°C. Lysates were centrifuged for 1 min. at maximum rpm to remove cellular debris. Protein concentrations and cAMP concentrations per sample were determined with the RC/DC Protein Assay (Biorad; Hercules, CA) and the Direct Cyclic AMP Enzyme Immunoassy Kit (Assay Designs Inc.; Ann Arbor, MI), respectively. Final cAMP concentrations were calculated as pmol cAMP/mg protein (see TABLE 5).

IMMUNODETECTION. Cells grown on permeable supports were washed in ice-cold, serum-free culture media and solubilized with lysis buffer (4% SDS, 10% glycerol, and 1 mM DTT in 0.05 M Tris pH 6.8). Lysates were clarified with an overnight spin at maximum speed in a microcentrifuge. Protein concentrations were determined with the RC/DC Protein Assay. Equal amounts of protein were separated by SDS-PAGE on 7.5% acrylamide gels and blotted onto Immobilon-P transfer membrane (Millipore Corp.; Bedford, MA). The membranes were blocked with 5% milk- TBS, pH 7.5 and subsequently incubated overnight at 4°C with gentle agitation with primary antibody, followed by incubation with a secondary antibody conjugated to horseradish peroxidase. Primary antibodies were diluted in 0.5% BSA-TBS, pH 7.5. Secondary antibodies were diluted in 0.5% milk-TBS, pH 7.5. The protein bands were visualized with SuperSignal West Dura enhanced chemiluminesence reagent and developed onto ClearBlue^® film (Pierce; Rockford, IL). NUCLEOTIDE EXTRACTION AND HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY. Polarized MDCK-C7 cells were washed twice with ice-cold HBSS on ice. Cells were scraped in 500 μL 0.4 M cold perchloric acid on ice and centrifuged for 3 min. at 9825 x g. A fixed volume of supernatant was removed and neutralized with 3 M K₃PO₄ and extracts were analyzed with high performance liquid chromatography (HPLC) according to the reverse-phase procedures described previously (Kalsi, K.K., et al., European journal of clinical investigation 29, 469-477, 1999). The equipment used was the Hewlett- Packard 1100 series linked to a diode array detector. The perchlorate precipitate was re- suspended in 500 μL 0.5 M NaOH and the protein content wad determined using a Bradford assay. STATISTICAL ANALYSES. In the figures, symbols and/or bar graphs are represented as means ± S. E. Differences between two or more groups in a given experiment were analyzed by a one-way ANOVA followed by Tukey' s post-hoc test using SPSS 14.0 statistical software. Unpaired Student's t-test was used to analyze experiments containing only two groups. Differences were considered significant when p < 0.02. Line and bar graphs were generated using Sigma Plot 2000 graphing software.

Claims

WHAT IS CLAIMED IS:

1. A pharmaceutical composition for treating or ameliorating the effects of a liver disease in a patient in need of relief from the liver disease, the composition comprising a therapeutically effective amount of a PPAR gamma modulator, or a pharmaceutically acceptable salt thereof, where the therapeutically effective amount is less than an amount therapeutically effective to treat type II diabetes mellitus.

2. The composition of claim 1 wherein the liver disease arises from salt and/or water homeostasis dysfunction.

3. The composition of claim 1 wherein the liver disease results from dysregulation, dysfunction, or imbalance of salt and/or water homeostasis of the hepatic biliary system.

4. The composition of claim 1 wherein the liver disease is associated with transepithelial ion flux dysfunction.

5. The composition of claim 1 wherein the liver disease is polycystic liver disease.

6. The composition of claim 1 wherein the liver disease is chronic hepatic liver cyst formation or liver fibrosis.

7. The composition of claim 1 wherein the liver disease includes liver fibrosis or periportal fibrosis, or a combination thereof.

8. The composition of claim 1 wherein the liver disease is autosomal polycystic liver disease.

9. The composition of claim 1 wherein the liver disease is polycystic liver disease that is comorbid with polycystic kidney disease.

10. The composition of claim 1 wherein the liver disease is Meckel-Gruber Syndrome.

11. The composition of claim 1 wherein the liver disease is Bardet-Biedl Syndrome.

12. The composition of claim 1 wherein the liver disease is Nephronophthisis.

13. The composition of claim 1 wherein the liver disease is liver cysts in the bile duct.

14. The composition of claim 1 wherein the liver disease is responsive to the inhibition of CFTR-mediated chloride secretion.

15. The composition of claim 1 wherein the liver disease is a disease of the hepatic biliary system responsive to inhibition or down regulation of the CFTR.

16. The composition of any one of claims 1 to 15 wherein the PPAR gamma modulator is capable of modulating cystic fibrosis transmembrane regulator-mediated ion secretion in liver tissue.

17. The composition of any one of claims 1 to 15 wherein the PPAR gamma modulator is capable of downregulating CFTR expression in liver tissue.

18. The composition of any one of claims 1 to 15 wherein the therapeutically effective amount of the PPAR gamma modulator results in decreased cystic fibrosis transmembrane regulator-mediated ion secretion in liver tissue in vivo.

19. The composition of any one of claims 1 to 15 wherein the PPAR gamma modulator is a PPAR gamma agonist or partial agonist.

20. The composition of any one of claims 1 to 15 wherein the PPAR gamma modulator is a thiazolidinedione, or a pharmaceutically acceptable salt thereof.

21. The composition of claim 20 wherein the thiazolidinedione is selected from the group consisting of rosiglitazone, pioglitazone, and analogs and derivatives thereof, and combinations of there foregoing.

22. A method for treating or ameliorating the effects of a liver disease in a patient in need of relief from the liver disease, the method comprising the step of administering to the patient a therapeutically effective amount of a PPAR gamma modulator, or a pharmaceutically acceptable salt thereof.

23. The method of claim 1 wherein the liver disease arises from salt and/or water homeostasis dysfunction.

24. The method of claim 1 wherein the liver disease results from dysregulation, dysfunction, or imbalance of salt and/or water homeostasis of the hepatic biliary system.

25. The method of claim 1 wherein the liver disease is associated with transepithelial ion flux dysfunction.

26. The method of claim 1 wherein the liver disease is polycystic liver disease.

27. The method of claim 1 wherein the liver disease is chronic hepatic liver cyst formation or liver fibrosis.

28. The method of claim 1 wherein the liver disease includes liver fibrosis or periportal fibrosis, or a combination thereof.

29. The method of claim 1 wherein the liver disease is autosomal polycystic liver disease.

30. The method of claim 1 wherein the liver disease is polycystic liver disease that is comorbid with polycystic kidney disease.

31. The method of claim 1 wherein the liver disease is Meckel-Gruber

Syndrome.

32. The method of claim 1 wherein the liver disease is Bardet-Biedl Syndrome.

33. The method of claim 1 wherein the liver disease is Nephronophthisis.

34. The method of claim 1 wherein the liver disease is liver cysts in the bile duct.

35. The method of claim 1 wherein the liver disease is responsive to the inhibition of CFTR-mediated chloride secretion.

36. The method of claim 1 wherein the liver disease is a disease of the hepatic biliary system responsive to inhibition or down regulation of the CFTR.

37. The method of any one of claims 22 to 36 wherein the PPAR gamma modulator is capable of modulating cystic fibrosis transmembrane regulator-mediated ion secretion in liver tissue.

38. The method of any one of claims 22 to 36 wherein the PPAR gamma modulator is capable of downregulating CFTR expression in liver tissue.

39. The method of any one of claims 22 to 36 wherein the therapeutically effective amount of the PPAR gamma modulator results in decreased cystic fibrosis transmembrane regulator-mediated ion secretion in liver tissue in vivo.

40. The method of any one of claims 22 to 36 wherein the PPAR gamma modulator is a PPAR gamma agonist or partial agonist.

41. The method of any one of claims 22 to 36 wherein the PPAR gamma modulator is a thiazolidinedione, or a pharmaceutically acceptable salt thereof.

42. The method of claim 41 wherein the thiazolidinedione is selected from the group consisting of rosiglitazone, pioglitazone, and analogs and derivatives thereof, and combinations of there foregoing.