Abstract
Drug-induced liver injury (DILI) continues to be a major cause of drug attrition and restrictive labeling. Given the importance of farnesoid X receptor (FXR) in bile acid homeostasis, drug-related FXR antagonism may be an important mechanism of DILI. However, a comprehensive assessment of this phenomenon broadly in the context of DILI is lacking. As such, we used an orthogonal approach comprising a FXR target gene assay in primary human hepatocytes and a commercially available FXR reporter assay to investigate the potential FXR antagonistic effects of an extensive test set of 159 compounds with and without association with clinical DILI. Data were omitted from analysis based on the presence of cytotoxicity to minimize false positive assay signals and other complications in data interpretation. Based on the experimental approaches employed and corresponding data, the prevalence of FXR antagonism was relatively low across this broad DILI test set, with 16–24% prevalence based on individual assay results or combined signals in both assays. Moreover, FXR antagonism was not highly predictive for identifying clinically relevant hepatotoxicants retrospectively, where FXR antagonist classification alone had minimal to moderate predictive value as represented by positive and negative likelihood ratios of 2.24–3.84 and 0.72–0.85, respectively. The predictivity did not increase significantly when considering only compounds with high clinical exposure (maximal or efficacious plasma exposures > 1.0 μM). In contrast, modest gains in predictive value of FXR antagonism were observed considering compounds that also inhibit bile salt export pump. In addition, we have identified novel FXR antagonistic effects of well-studied hepatotoxic drugs, including bosentan, tolcapone and ritonavir. In conclusion, this work represents a comprehensive evaluation of FXR antagonism in the context of DILI, including its overall predictivity and challenges associated with detecting this phenomenon in vitro.
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References
Aleo MD, Luo Y, Swiss R, Bonin PD, Potter DM, Will Y (2014) Human drug-induced liver injury severity is highly associated with dual inhibition of liver mitochondrial function and bile salt export pump. Hepatology 60(3):1015–1022. https://doi.org/10.1002/hep.27206
Aleo MD, Shah F, Allen S et al (2019) Moving beyond binary predictions of human drug-induced liver injury (DILI) towards contrasting relative risk potential. Chem Res Toxicol. https://doi.org/10.1021/acs.chemrestox.9b00262
Aleo MD, Shah F, He K, Bonin PD, Rodrigues AD (2017) Evaluating the role of multidrug resistance protein 3 (MDR3) inhibition in predicting drug-induced liver injury using 125 pharmaceuticals. Chem Res Toxicol 30(5):1219–1229. https://doi.org/10.1021/acs.chemrestox.7b00048
Altman DG, Bland JM (1994) Diagnostic tests 2: predictive values. BMJ 309(6947):102. https://doi.org/10.1136/bmj.309.6947.102
Antherieu S, Chesne C, Li R et al (2010) Stable expression, activity, and inducibility of cytochromes P450 in differentiated HepaRG cells. Drug Metab Dispos 38(3):516–525. https://doi.org/10.1124/dmd.109.030197
Ballet F (2016) FXR: big fish or small fry for drug-induced liver injury? Clin Res Hepatol Gastroenterol 40(1):6–8. https://doi.org/10.1016/j.clinre.2015.11.008
Benabou R, Waters C (2003) Hepatotoxic profile of catechol-O-methyltransferase inhibitors in Parkinson's disease. Expert Opin Drug Saf 2(3):263–267. https://doi.org/10.1517/14740338.2.3.263
Chan R, Benet LZ (2018) Measures of BSEP inhibition in vitro are not useful predictors of DILI. Toxicol Sci 162(2):499–508. https://doi.org/10.1093/toxsci/kfx284
Chen M, Borlak J, Tong W (2013) High lipophilicity and high daily dose of oral medications are associated with significant risk for drug-induced liver injury. Hepatology 58(1):388–396. https://doi.org/10.1002/hep.26208
Chen M, Suzuki A, Thakkar S, Yu K, Hu C, Tong W (2016) DILIrank: the largest reference drug list ranked by the risk for developing drug-induced liver injury in humans. Drug Discov Today 21(4):648–653. https://doi.org/10.1016/j.drudis.2016.02.015
Dambach DM (2014) Drug-induced Hepatotoxicity: Advances in Preclinical Predictive Strategies and Tools. In: Wang J, Urban L (eds) Predictive ADMET integrative approaches in drug discovery and development. Wiley, Hoboken, pp 433–465
Dawson S, Stahl S, Paul N, Barber J, Kenna JG (2012) In vitro inhibition of the bile salt export pump correlates with risk of cholestatic drug-induced liver injury in humans. Drug Metab Dispos 40(1):130–138. https://doi.org/10.1124/dmd.111.040758
Deeks JJ, Altman DG (2004) Diagnostic tests 4: likelihood ratios. BMJ 329(7458):168–169. https://doi.org/10.1136/bmj.329.7458.168
Dussault I, Beard R, Lin M et al (2003) Identification of gene-selective modulators of the bile acid receptor FXR. J Biol Chem 278(9):7027–7033. https://doi.org/10.1074/jbc.M209863200
Fattinger K, Funk C, Pantze M et al (2001) The endothelin antagonist bosentan inhibits the canalicular bile salt export pump: a potential mechanism for hepatic adverse reactions. Clin Pharmacol Ther 69(4):223–231. https://doi.org/10.1067/mcp.2001.114667
Feng B, Xu JJ, Bi YA et al (2009) Role of hepatic transporters in the disposition and hepatotoxicity of a HER2 tyrosine kinase inhibitor CP-724,714. Toxicol Sci 108(2):492–500. https://doi.org/10.1093/toxsci/kfp033
Garzel B, Zhang L, Huang SM, Wang H (2019) A change in bile flow: looking beyond transporter inhibition in the development of drug-induced cholestasis. Curr Drug Metab 20(8):621–632. https://doi.org/10.2174/1389200220666190709170256
Griffin LM, Watkins PB, Perry CH, St Claire RL 3rd, Brouwer KL (2013) Combination lopinavir and ritonavir alter exogenous and endogenous bile acid disposition in sandwich-cultured rat hepatocytes. Drug Metab Dispos 41(1):188–196. https://doi.org/10.1124/dmd.112.047225
Guo F, Letrent SP, Munster PN et al (2008) Pharmacokinetics of a HER2 tyrosine kinase inhibitor CP-724,714 in patients with advanced malignant HER2 positive solid tumors: correlations with clinical characteristics and safety. Cancer Chemother Pharmacol 62(1):97–109. https://doi.org/10.1007/s00280-007-0579-4
Han CY (2018) Update on FXR biology: promising therapeutic target? Int J Mol Sci 19(7):2069. https://doi.org/10.3390/ijms19072069
Heredi-Szabo K, Kis E, Krajcsi P (2012) The vesicular transport assay: validated in vitro methods to study drug-mediated inhibition of canalicular efflux transporters ABCB11/BSEP and ABCC2/MRP2. Curr Protoc Toxicol. https://doi.org/10.1002/0471140856.tx2304s54
Hillgren KM, Keppler D, Zur AA et al (2013) Emerging transporters of clinical importance: an update from the International Transporter Consortium. Clin Pharmacol Ther 94(1):52–63. https://doi.org/10.1038/clpt.2013.74
Hoofnagle JH, Serrano J, Knoben JE, Navarro VJ (2013) LiverTox: a website on drug-induced liver injury. Hepatology 57(3):873–874. https://doi.org/10.1002/hep.26175
Hsu CW, Zhao J, Xia M (2016) Transactivation and coactivator recruitment assays for measuring farnesoid X receptor activity. Methods Mol Biol 1473:43–53. https://doi.org/10.1007/978-1-4939-6346-1_5
Jackson JP, Freeman KM, St. Claire RL, Black CB, Brouwer KR (2018) Cholestatic drug induced liver injury: a function of bile salt export pump inhibition and farnesoid X receptor antagonism. Appl In Vitro Toxicol 4(3):265–279. https://doi.org/10.1089/aivt.2018.0011
Keitel V, Droge C, Haussinger D (2019) Targeting FXR in cholestasis. Handb Exp Pharmacol 256:299–324. https://doi.org/10.1007/164_2019_231
Kemper JK (2011) Regulation of FXR transcriptional activity in health and disease: emerging roles of FXR cofactors and post-translational modifications. Biochim Biophys Acta 1812(8):842–850. https://doi.org/10.1016/j.bbadis.2010.11.011
Kenna JG, Stahl SH, Eakins JA et al (2015) Multiple compound-related adverse properties contribute to liver injury caused by endothelin receptor antagonists. J Pharmacol Exp Ther 352(2):281–290. https://doi.org/10.1124/jpet.114.220491
Kenna JG, Taskar KS, Battista C et al (2018) Can bile salt export pump inhibition testing in drug discovery and development reduce liver injury risk? An international transporter consortium perspective. Clin Pharmacol Ther 104(5):916–932. https://doi.org/10.1002/cpt.1222
Klaassen CD, Aleksunes LM (2010) Xenobiotic, bile acid, and cholesterol transporters: function and regulation. Pharmacol Rev 62(1):1–96. https://doi.org/10.1124/pr.109.002014
Kock K, Ferslew BC, Netterberg I et al (2014) Risk factors for development of cholestatic drug-induced liver injury: inhibition of hepatic basolateral bile acid transporters multidrug resistance-associated proteins 3 and 4. Drug Metab Dispos 42(4):665–674. https://doi.org/10.1124/dmd.113.054304
Kojetin DJ, Burris TP (2013) Small molecule modulation of nuclear receptor conformational dynamics: implications for function and drug discovery. Mol Pharmacol 83(1):1–8. https://doi.org/10.1124/mol.112.079285
LeCluyse EL (2001) Human hepatocyte culture systems for the in vitro evaluation of cytochrome P450 expression and regulation. Europ J Pharm Sci 13(4):343–368. https://doi.org/10.1016/s0928-0987(01)00135-x
Lu W, Cheng F, Jiang J et al (2015) FXR antagonism of NSAIDs contributes to drug-induced liver injury identified by systems pharmacology approach. Sci Rep 5:8114–8114. https://doi.org/10.1038/srep08114
Maloney PR, Parks DJ, Haffner CD et al (2000) Identification of a chemical tool for the orphan nuclear receptor FXR. J Med Chem 43(16):2971–2974. https://doi.org/10.1021/jm0002127
Modica S, Gadaleta RM, Moschetta A (2010) Deciphering the nuclear bile acid receptor FXR paradigm. Nucl Recept Signal 8:e005–e005. https://doi.org/10.1621/nrs.08005
Moore TW, Mayne CG, Katzenellenbogen JA (2010) Minireview: not picking pockets: nuclear receptor alternate-site modulators (NRAMs). Mol Endocrinol 24(4):683–695. https://doi.org/10.1210/me.2009-0362
Morgan RE, Trauner M, van Staden CJ et al (2010) Interference with bile salt export pump function is a susceptibility factor for human liver injury in drug development. Toxicol Sci 118(2):485–500. https://doi.org/10.1093/toxsci/kfq269
Morgan RE, van Staden CJ, Chen Y et al (2013) A multifactorial approach to hepatobiliary transporter assessment enables improved therapeutic compound development. Toxicol Sci 136(1):216–241. https://doi.org/10.1093/toxsci/kft176
Mosedale M, Watkins PB (2017) Drug-induced liver injury: advances in mechanistic understanding that will inform risk management. Clin Pharmacol Ther 101(4):469–480. https://doi.org/10.1002/cpt.564
Pedersen JM, Matsson P, Bergstrom CA et al (2013) Early identification of clinically relevant drug interactions with the human bile salt export pump (BSEP/ABCB11). Toxicol Sci 136(2):328–343. https://doi.org/10.1093/toxsci/kft197
Proctor WR, Foster AJ, Vogt J et al (2017) Utility of spherical human liver microtissues for prediction of clinical drug-induced liver injury. Arch Toxicol 91(8):2849–2863. https://doi.org/10.1007/s00204-017-2002-1
Rana P, Aleo MD, Gosink M, Will Y (2019) Evaluation of in vitro mitochondrial toxicity assays and physicochemical properties for prediction of organ toxicity using 228 pharmaceutical drugs. Chem Res Toxicol 32(1):156–167. https://doi.org/10.1021/acs.chemrestox.8b00246
Rodriguez-Antona C, Donato MT, Boobis A et al (2002) Cytochrome P450 expression in human hepatocytes and hepatoma cell lines: molecular mechanisms that determine lower expression in cultured cells. Xenobiotica 32(6):505–520. https://doi.org/10.1080/00498250210128675
Schadt S, Simon S, Kustermann S et al (2015) Minimizing DILI risk in drug discovery—a screening tool for drug candidates. Toxicol In Vitro 30(1 Pt B):429–437. https://doi.org/10.1016/j.tiv.2015.09.019
Shah F, Leung L, Barton HA et al (2015) Setting clinical exposure levels of concern for drug-induced liver injury (DILI) using mechanistic in vitro assays. Toxicol Sci 147(2):500–514. https://doi.org/10.1093/toxsci/kfv152
Shah F, Medvedev A, Wassermann AM et al (2018) The identification of pivotal transcriptional factors mediating cell responses to drugs with drug-induced liver injury liabilities. Toxicol Sci 162(1):177–188. https://doi.org/10.1093/toxsci/kfx231
Slizgi JR, Lu Y, Brouwer KR et al (2016) Inhibition of human hepatic bile acid transporters by tolvaptan and metabolites: contributing factors to drug-induced liver injury? Toxicol Sci 149(1):237–250. https://doi.org/10.1093/toxsci/kfv231
Staudinger JL, Woody S, Sun M, Cui W (2013) Nuclear-receptor-mediated regulation of drug- and bile-acid-transporter proteins in gut and liver. Drug Metab Rev 45(1):48–59. https://doi.org/10.3109/03602532.2012.748793
Sulkowski MS, Thomas DL, Chaisson RE, Moore RD (2000) Elevated liver enzymes following initiation of antiretroviral therapy. JAMA 283(19):2526–2527
Taoka H, Yokoyama Y, Morimoto K et al (2016) Role of bile acids in the regulation of the metabolic pathways. World J Diabetes 7(13):260–270. https://doi.org/10.4239/wjd.v7.i13.260
Thompson RA, Isin EM, Li Y et al (2012) In vitro approach to assess the potential for risk of idiosyncratic adverse reactions caused by candidate drugs. Chem Res Toxicol 25(8):1616–1632. https://doi.org/10.1021/tx300091x
Watkins P (2000) COMT inhibitors and liver toxicity. Neurology 55(11 Suppl 4):S51–S52 (discussion S53-6)
Watkins PB (2011) Drug safety sciences and the bottleneck in drug development. Clin Pharmacol Ther 89(6):788–790. https://doi.org/10.1038/clpt.2011.63
Williams DP, Lazic SE, Foster AJ, Semenova E, Morgan P (2019) Predicting drug-induced liver injury with bayesian machine learning. Chem Res Toxicol. https://doi.org/10.1021/acs.chemrestox.9b00264
Wolenski FS, Zhu AZX, Johnson M et al (2017) Fasiglifam (TAK-875) alters bile acid homeostasis in rats and dogs: a potential cause of drug induced liver injury. Toxicol Sci 157(1):50–61. https://doi.org/10.1093/toxsci/kfx018
Yu DD, Lin W, Forman BM, Chen T (2014) Identification of trisubstituted-pyrazol carboxamide analogs as novel and potent antagonists of farnesoid X receptor. Bioorg Med Chem 22(11):2919–2938. https://doi.org/10.1016/j.bmc.2014.04.014
Yucha RW, He K, Shi Q et al (2017) In vitro drug-induced liver injury prediction: criteria optimization of efflux transporter IC50 and physicochemical properties. Toxicol Sci 157(2):487–499. https://doi.org/10.1093/toxsci/kfx060
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LMN: conceived of/designed the study, performed research, analyzed data and wrote the paper. CK, JM, WRP: conceived of/designed the study and wrote the paper. AF: analyzed data. CL, LL, TK: performed research. JB: analyzed data.
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Norona, L.M., Fullerton, A., Lawson, C. et al. In vitro assessment of farnesoid X receptor antagonism to predict drug-induced liver injury risk. Arch Toxicol 94, 3185–3200 (2020). https://doi.org/10.1007/s00204-020-02804-4
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DOI: https://doi.org/10.1007/s00204-020-02804-4