Time to say goodbye to the drug or the model? – Why do drugs fail to live up to their promise in bile duct ligated mice?
Authors: Peter Fickert
Journal: Journal of Hepatology (2013)

Deleterious effect of oltipraz on extrahepatic cholestasis in bile duct-ligated miceJournal of HepatologyVol. 60Issue 1PreviewOltipraz (4-methyl-5(pyrazinyl-2)-1-2-dithiole-3-thione), a promising cancer preventive agent, has an antioxidative activity and ability to enhance glutathione biosynthesis, phase II detoxification enzymes and multidrug resistance-associated protein-mediated efflux transporters. Oltipraz can protect against hepatotoxicity caused by carbon tetrachloride, acetaminophen and alpha-naphthylisothiocyanate. Whether oltipraz has hepato-protective effects on obstructive cholestasis is unknown. Full-Text PDF For decades there was no effective drug treatment for patients with cholestatic liver disease apart from ursodeoxycholic acid in primary biliary cirrhosis [[1]EASL Clinical Practice Guidelines Management of cholestatic liver diseases.J Hepatol. 2009; 51: 237-267Abstract Full Text Full Text PDF PubMed Scopus (1298) Google Scholar]. However, with new insights into the molecular mechanisms of bile formation including the elucidation of the nature, localization, function, and transcriptional regulation of the major transport proteins and better understanding of the pathophysiology of specific cholestatic liver diseases, a new era of drug therapy in cholestasis and associated complications has dawned [2Boyer J.L. Bile formation and secretion.Compr Physiol. 2013; 3: 1035-1078Crossref PubMed Scopus (439) Google Scholar, 3Carey E.J. Lindor K.D. Current pharmacotherapy for cholestatic liver disease.Expert Opin Pharmacother. 2012; 13: 2473-2484Crossref PubMed Scopus (33) Google Scholar]. The attendant optimism is, at least in part, based on the development of novel “designer” drugs that are specific ligands to defined nuclear receptors regulating hepatobiliary metabolism and excretion of biliary compounds, in particular of bile acids. These drugs will significantly increase the pharmacological options available in this particular situation and are also likely to have far-reaching medical implications in other, much more prevalent conditions such as the metabolic syndrome or non-alcoholic steatohepatitis [[4]Halilbasic E. Baghdasaryan A. Trauner M. Nuclear receptors as drug targets in cholestatic liver diseases.Clin Liver Dis. 2013; 17: 161-189Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar]. Eventually, therapeutic nihilism in cholestatic liver diseases, especially in cholangiopathies, should become a thing of the past. As the term cholestasis, meaning stasis of bile flow, already indicates, we still consider secretory failure of the liver at the level of hepatocytes or cholangiocytes as the pathophysiological hallmark of cholestatic liver diseases. It may lead to cell injury, inflammation, the biliary type of liver fibrosis with portal hypertension, and finally may cause end-stage liver disease and liver failure. A variety of factors, some unidentified, may account for these complicated processes. Among them are retained potentially toxic cholephils such as bile acids, ROS production, cytokines/chemokines and their receptors, which may attract inflammatory cells, bile duct proliferation, activated myofibroblasts, increased biliary pressure, or dysbiosis. This complexity obviously is a major obstacle in finding useful treatment strategies. In fact it seems unrealistic to aim to design a “one pill” approach for the heterogeneous group of cholestatic liver diseases. Conceivable targets for drug therapy for these challenging patients include: (i) restoration of regular bile flow, (ii) reduction of the potential toxicity of bile with choleretic and thereby bile diluting drugs or increased phospholipid and glutathione (GSH) secretion; (iii) stimulation of alternative export of potentially toxic biliary compounds via basolateral hepatocyte transport and facilitated renal excretion via stimulated glucuronidation and sulfatation, which should be particularly helpful in hepatocellular cholestasis; (iv) reduction of inflammation; and (v) attenuation of ductular reaction along with an overwhelming wound healing process or dissolution of liver fibrosis. For part of those treatment goals their molecular targets have been identified (Fig. 1). With these complex mechanisms in mind, Weerachayphorn et al. used common bile duct-ligated (BDL) mice to study the effects of oltipraz (4-methyl-5(pyrazinyl-2)-1-2-dithiole-3-thione), a promising candidate for drug therapy in cholestatic liver diseases with potentially beneficial pleotropic effects [[5]Weerachayaphorn J. Luo Y. Mennone A. Soroka S.J. Harry K. Boyer J.L. Deleterious effect of oltipraz on extrahepatic cholestasis in bile duct-ligated mice.J Hepatol. 2014; 60: 160-166Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar]. The molecular effects among others include the induction of phase II detoxifying enzymes, UDP-glucuronyltransferase and glutathione-S-transferase as well as the stimulation of bile acid excretion transporters Bsep, Mrp2, Mrp3, and Mrp4 (Fig. 1) [[6]Brooks 3rd, S.C. Brooks J.S. Lee W.H. Lee M.G. Kim S.G. Therapeutic potential of dithiolethiones for hepatic diseases.Pharmacol Ther. 2009; 124: 31-43Crossref PubMed Scopus (24) Google Scholar]. These orchestrated effects were anticipated to reduce hepatocyte oxidative stress by limiting bile acid toxicity and to facilitate canalicular as well as adaptive basolateral efflux of accumulating bile acids and bilirubin and therefore to ameliorate cholestatic liver injury. Thus, there was a clear molecular rationale to test oltipraz in a model of cholestatic liver disease. For their experiments Weerachayphorn et al. chose a preventive protocol and rather short duration of BDL. They treated mice with oltipraz (150 mg/kg b.w.) 5 days prior to BDL and 3 days thereafter. Contradictory to the authors’ expectations, oltipraz treatment in BDL mice led to aggravation of liver injury with extensive necroinflammation with significantly increased extent of bile infarcts affecting more than 20% of the liver parenchyma. TUNEL-positive hepatocytes were not increased in oltipraz-treated BDL mice, suggesting that necrosis rather than apoptosis represented the main type of cell death. This is in line with previous observations in BDL mice and rats [7Fickert P. Trauner M. Fuchsbichler A. Zollner G. Wagner M. Marschall H.U. et al.Oncosis represents the main type of cell death in mouse models of cholestasis.J Hepatol. 2005; 42: 378-385Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 8Gujral J.S. Liu J. Farhood A. Jaeschke H. Reduced oncotic necrosis in Fas receptor-deficient C57BL/6J-lpr mice after bile duct ligation.Hepatology. 2004; 40: 998-1007Crossref PubMed Scopus (92) Google Scholar, 9Schoemaker M.H. Gommans W.M. Conde de la Rosa L. Homan M. Klok P. Trautwein C. Resistance of rat hepatocytes against bile acid-induced apoptosis in cholestatic liver injury is due to nuclear factor-kappa B activation.J Hepatol. 2003; 39: 153-161Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar]. The authors were not able to study later time points after BDL because the oltipraz-treated mice died quickly following BDL. This is an interesting parallel to the findings in UDCA-treated BDL mice [[10]Fickert P. Zollner G. Fuchsbichler A. Stumptner C. Weiglein A.H. Lammert F. et al.Ursodeoxycholic acid aggravates bile infarcts in bile duct-ligated and Mdr2 knockout mice via disruption of cholangioles.Gastroenterology. 2002; 123: 1238-1251Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar]. Increased bile flow and biliary pressure, as induced by UDCA, was previously shown to represent a pivotal trigger for the specific type of liver injury including characteristic bile infarcts in BDL mice [10Fickert P. Zollner G. Fuchsbichler A. Stumptner C. Weiglein A.H. Lammert F. et al.Ursodeoxycholic acid aggravates bile infarcts in bile duct-ligated and Mdr2 knockout mice via disruption of cholangioles.Gastroenterology. 2002; 123: 1238-1251Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 11Wagner M. Fickert P. Zollner G. Fuchsbichler A. Silbert D. Tsybrovskyy O. et al.Role of farnesoid X receptor in determining hepatic ABC transporter expression and liver injury in bile duct-ligated mice.Gastroenterology. 2003; 125: 825-838Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, 12Wiener S.M. Hoyt Jr., R.F. Deleonardis J.R. Clevenger R.R. Jeffries K.R. Nagashima K. et al.Manometric changes during retrograde biliary infusion in mice.Am J Physiol Gastrointest Liver Physiol. 2000; 279: G49-G66PubMed Google Scholar]. The authors therefore determined bile flow in response to olpitraz and they indeed found a substantial increase in bile acid-independent bile flow primarily attributable to increased biliary GSH secretion. Moreover, olpitraz enhanced hepatic GSH content and stimulated Mrp4 expression as suggested. In contrast, there were no differences in Mrp2, Bsep, and Mrp3 expression compared to controls. Based on these findings the authors conclude that olpitraz increased liver injury in BDL mice primarily due to increased bile acid-independent GSH-mediated bile flow leading to a critical biliary pressure, so supporting the concept that increased flow rates in an obstructed biliary tree may be detrimental [[10]Fickert P. Zollner G. Fuchsbichler A. Stumptner C. Weiglein A.H. Lammert F. et al.Ursodeoxycholic acid aggravates bile infarcts in bile duct-ligated and Mdr2 knockout mice via disruption of cholangioles.Gastroenterology. 2002; 123: 1238-1251Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar]. They conclude that olpitraz should be avoided in patients whose cholestasis is due to or associated with bile duct obstruction. Does this mean the end for olpitraz as an anticholestatic agent? Before drawing such a definite conclusion mouse models of cholestatic liver diseases should be reviewed carefully [[13]Fickert P. Pollheimer M.J. Österreicher C.H. Trauner M. Animal models of cholestasis in animal models for the study of human diseases. Academic Press, 2013Google Scholar]. First we should keep in mind that mice and humans differ substantially in major determinants of cholestatic liver injury such as bile acid composition and the relative amount of bile acid-dependent/independent bile flow. In addition, the question must be raised as to which specific aspects of the BDL mouse model are relevant in relation to human cholestatic liver diseases other than simple bile duct obstruction due to stone or tumor. Since its first comprehensive description in the early 20th century, BDL has significantly helped us to understand bile formation and secretion as well as lipid metabolism and associated regulatory mechanisms. With regard to the pathophysiology of cholestatic liver diseases, BDL is particularly suitable to study the consequences of cholestasis due to total bile duct obstruction with a characteristic liver phenotype comprising bile infarcts (reflecting foci of lytic necrosis of hepatocytes due to regurgitated bile with millimolar concentration of bile acids out of disrupted canals of Herring), ductular reaction and biliary fibrosis. Since the pathological changes are definitive, the cholestatic phenotype in BDL mice is hard to overcome pharmacologically, as outlined recently in more detail elsewhere [[13]Fickert P. Pollheimer M.J. Österreicher C.H. Trauner M. Animal models of cholestasis in animal models for the study of human diseases. Academic Press, 2013Google Scholar] (Fig. 2). Therefore, the BDL model is not well suited for treatment studies, especially in the case of choleretic drugs, which will always carry the risk of worsening bile infarcts due to high biliary pressure. It so would be premature to dismiss oltipraz for the treatment of cholestasic liver disease in general solely based on negative findings in BDL mice. One should also test alternative models such as Abcb4−/− mice or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-fed mice modeling sclerosing cholangitis with biliary fibrosis, since these have different pathogenesis and show a more diverse degree and dynamic of cholestasis than do BDL mice [[14]Pollheimer M.J. Trauner M. Fickert P. Will we ever model PSC? – “It’s hard to be a PSC model!”.Clin Res Hepatol Gastroenterol. 2011; 35: 792-804Crossref PubMed Scopus (53) Google Scholar]. Testing promising drug candidates in different experimental models of cholestasis could give important answers, e.g., as to whether oltipraz could be beneficial at different stages of cholestatic liver injury (i.e., early vs. late disease). In a broader context we should discuss whether testing a potential novel drug for cholestasis and cholestatic liver disease should always be done in different models in parallel as it has become standard for experimental liver fibrosis (e.g., combinations of CCl4-intoxicated, BDL, DDC-fed, porcine serum-treated mice). Nevertheless, BDL mice still represent an interesting model for several open questions in the pathophysiology of cholestasis: (i) Why do BDL mice not develop bile plugs whereas this is a characteristic finding in obstructive cholestasis in humans? (ii) Do mice develop cholate stasis like humans and what does this characteristic morphological sign mean? (iii) How do regenerating hepatocytes find their access to bile ducts? (iv) Why do bile ducts proliferate from portal field to portal field? Not to forget that BDL rodents, which have the highest degree of cholestasis within models of cholestatic liver disease, may be particularly suited to study the pathobiology of important extrahepatic problems such as adrenal insufficiency and acute kidney injury [15McNeilly A.D. Macfarlane D.P. O’Flaherty E. Livingstone D.E. Mitić T. McConnell K.M. et al.Bile acids modulate glucocorticoid metabolism and the hypothalamic-pituitary-adrenal axis in obstructive jaundice.J Hepatol. 2010; 52: 705-711Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 16Fickert P. Krones E. Pollheimer M.J. Thueringer A. Moustafa T. Silbert D. et al.Bile acids trigger cholemic nephropathy in common Bile-Duct-ligated mice.Hepatology. 2013; https://doi.org/10.1002/hep.26599Crossref PubMed Scopus (102) Google Scholar]. Weerachayaphorn et al. again teach us caution with choleretic drugs in cholestatic liver diseases with an obstructive component, as is the case in several cholangiopathies (e.g., PSC, SSC, SSC-CIP). The current study also reminds us that the time point for starting a drug always matters greatly in cholestatic liver diseases. Probably there is a point of no return, especially in the case of cholangiopathies, where drugs may potentially be harmful. This may come into play especially for choleretic drugs when one falls below a critical number of functional secretory units of the liver (probably comparable to the nephron in kidney) and there is no emergency exit for bile. The “ideal drug (cocktail?) for cholestasis” will in addition not only increase bile flow and restore excretory liver function or enhance alternative excretory routes, but also in parallel resolve the underlying defect in the liver. Since this has not yet been completely evaluated, the case of oltipraz for cholestatic liver disease may still be open. The author declared that he does not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.