1521-0103/352/2/281–290$25.00 http://dx.doi.org/10.1124/jpet.114.220491 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 352:281–290, February 2015 Copyright ª 2014 by The American Society for Pharmacology and Experimental Therapeutics Multiple Compound-Related Adverse Properties Contribute to Liver Injury Caused by Endothelin Receptor Antagonists s J. Gerry Kenna, Simone H. Stahl, Julie A. Eakins, Alison J. Foster, Linda C. Andersson, Jonas Bergare, Martin Billger, Marie Elebring, Charles S. Elmore, and Richard A. Thompson Drug Safety Consultant, Macclesfield, Cheshire, United Kingdom (J.G.K.); DMPK (S.H.S.), Discovery Safety (J.A.E.), and Translational Safety (A.J.F.), Drug Safety and Metabolism, AstraZeneca R&D Alderley Park, Macclesfield, Cheshire, United Kingdom; DMPK (L.C.A., J.B., C.S.E.), Regulatory Safety (M.B.), Drug Safety and Metabolism, AstraZeneca R&D Mölndal, Mölndal, Sweden; and DMPK, Cardiovascular and Metabolic Diseases (M.E.), and Respiratory, Inflammation, and Autoimmunity (R.A.T.), iMED AstraZeneca R&D Mölndal, Mölndal, Sweden Received October 9, 2014; accepted November 26, 2014 ABSTRACT Drug-induced liver injury has been observed in patients treated with the endothelin receptor antagonists sitaxentan and bosentan, but not following treatment with ambrisentan. The aim of our studies was to assess the possible role of multiple contributory mechanisms in this clinically relevant toxicity. Inhibition of the bile salt export pump (BSEP) and multidrug resistance-associated protein 2 was quantified using membrane vesicle assays. Inhibition of mitochondrial respiration in human liver–derived HuH-7 cells was determined using a Seahorse XF e96 analyzer. Cytochrome P450 (P450)–independent and P450-mediated cell toxicity was assessed using transfected SV40-T-antigen–immortalized human liver epithelial (THLE) cell lines. Exposure-adjusted assay ratios were calculated by dividing the maximum human drug plasma concentrations by the IC 50 or EC 50 values obtained in vitro. Covalent binding (CVB) of radiolabeled drugs to human hepatocytes was quantified, and CVB body burdens were calculated by adjusting CVB values for fractional drug turnover in vitro and daily therapeutic dose. Sitaxentan exhibited positive exposure-adjusted signals in all five in vitro assays and a high CVB body burden. Bosentan exhibited a positive exposure-adjusted signal in one assay (BSEP inhibition) and a moderate CVB body burden. Ambrisentan exhibited no positive exposure-adjusted assay signals and a low CVB body burden. These data indicate that multiple mecha- nisms contribute to the rare, but potentially severe liver injury caused by sitaxentan in humans; provide a plausible rationale for the markedly lower propensity of bosentan to cause liver injury; and highlight the relative safety of ambrisentan. Introduction Numerous different drugs may cause liver injury in humans, which occurs infrequently in certain susceptible patients (i.e., is idiosyncratic), and is not evident from safety studies un- dertaken in animals. The consequences of drug-induced liver injury (DILI) range from mild and asymptomatic liver injury, which results in elevated plasma levels of alanine aminotrans- ferase (ALT) and other enzymes released from damaged liver cells, to clinically concerning liver dysfunction that, in the most severe instances, may result in acute liver failure (Yuan and Kaplowitz, 2013). Consequently, DILI is a leading cause of withdrawal of licensed drugs, failed drug registration, and human ill health (Lasser et al., 2002; Yuan and Kaplowitz, 2013). The underlying mechanisms are complex and include both drug-related adverse properties, which explain why some drugs cause DILI, whereas others do not, and patient-related factors that influence individual susceptibility (Thompson et al., 2011; Yuan and Kaplowitz, 2013). Drug-related adverse properties that have been implicated in the development of DILI include covalent and noncovalent modification of cellular macromolecules caused by electrophilic reactive metabolites (Park et al., 2011), intrinsic cell cytotoxicity (Gustafsson et al., 2014), impaired mitochondrial function (Nadanaciva and Will, 2011), inhibition of hepatocyte bile salt export pump (BSEP) activity (Dawson et al., 2012), stimulation of innate and adaptive immune responses (Yuan and Kaplowitz, 2013), and relatively high in vivo drug dose and exposure (Lammert et al., 2008). Patient-related susceptibility is complex and includes dx.doi.org/10.1124/jpet.114.220491. s This article has supplemental material available at jpet.aspetjournals.org. ABBREVIATIONS: ALT, alanine aminotransferase; BSEP, bile salt export pump; BSF208075, (2S)-2-[(4,6-dimethylpyrimidin-2-yl)oxy]-3-methoxy- 3,3-diphenylpropanoic acid; BSO, buthionine sulfoximine; CDF, 5(6)-carboxy-29,79-dichlorofluorescein; CVB, covalent binding; DILI, drug-induced liver injury; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethylsulfoxide; ECAR, extracellular acidification rate; ERA, endothelin receptor antagonist; FBS, fetal bovine serum; FCCP, carbonyl-cyanide-4-(trifluoromethoxy)phenylhydrazone; FDA, Food and Drug Administration; GSH, glutathione; HPLC, high-performance liquid chromatography; MRP2, multidrug resistance-associated protein 2; MRR, maximal respiration rate; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; OCR, oxygen consumption rate; P450, cytochrome P450; PMFR, Basal Pasadena Foundation for Medical Research; Ro 47-0203, 4-tert-butyl-N-[6-(2-hydroxy-ethoxy)-5-(2-methoxy-phenoxy)-2,29- bipyrimidin-4-yl]-benzenesulfonamide; Rot/AA, rotenone plus antimycin A; TBC11251, N-(4-chloro-3-methyl-1,2-oxazol-5-yl)-2-[2-(6-methyl-2H- 1,3-benzodioxol-5-yl)acetyl]thiophene-3-sulfonamide. 281 http://jpet.aspetjournals.org/content/suppl/2014/12/02/jpet.114.220491.DC1.html Supplemental material to this article can be found at: at ASPET Journals on February 3, 2016 jpet.aspetjournals.org Downloaded from