A Correlation Between the In Vitro Drug Toxicity of Drugs to Cell Lines That Express Human P450s and Their Propensity to Cause Liver Injury in Humans

Abstract

Drug toxicity to T-antigen-immortalized human liver epithelial (THLE) cells stably transfected with plasmid vectors that encoded human cytochrome P450s 1A2, 2C9, 2C19, 2D6, or 3A4, or an empty plasmid vector (THLE-Null), was investigated. An automated screening platform, which included 1% dimethyl sulfoxide (DMSO) vehicle, 2.7% bovine serum in the culture medium, and assessed 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium reduction, was used to evaluate the cytotoxicity of 103 drugs after 24h. Twenty-two drugs caused cytotoxicity to THLE-Null cells, with EC₅₀ ≤ 200 μM; 21 of these drugs (95%) have been reported to cause human liver injury. Eleven drugs exhibited lower EC₅₀ values in cells transfected with CYP3A4 (THLE-3A4 cells) than in THLE-Null cells; 10 of these drugs (91%) caused human liver injury. An additional 8 drugs, all of which caused human liver injury, exhibited potentiated THLE-3A4 cell toxicity when evaluated using a manual protocol that included 0.2% or 1% DMSO, but not bovine serum. Fourteen of the drugs that exhibited potentiated THLE-3A4 cell toxicity are known to be metabolized by P450s to reactive intermediates. These drugs included troglitazone, which was shown to undergo metabolic bioactivation and covalent binding to proteins in THLE-3A4 cells. A single drug (rimonabant) exhibited marked THLE cell toxicity but did not cause human liver injury; this drug had very low reported plasma exposure. These results indicate that evaluation of toxicity to THLE-Null and THLE-3A4 cell lines during drug discovery may aid selection of drugs with reduced propensity to cause drug-induced liver injury and that consideration of human exposure is required to enhance data interpretation.

Multiple Compound-Related Adverse Properties Contribute to Liver Injury Caused by Endothelin Receptor Antagonists

“…The value of these cell lines for investigation of P450-independent and P450 metabolite-mediated cell toxicity has been demonstrated previously (Foster et al, 2013;Gustafsson et al, 2014). A marked and concentration-dependent reduction in MTS activity, which is indicative of reduced cell viability (Gustafsson et al, 2014), was observed when THLE-Null cells were exposed to sitaxentan for 24 hours ( Fig. 5A; Supplemental Table 1; Table 1).…”

“…The influence of P450 expression on cell toxicity caused by the ERAs was assessed using a panel of human liver-derived THLE cell lines, which expressed either no detectable P450 activities (THLE-Null cells) or the major human hepatic P450 enzymes CYP1A2, CYP2C9, CYP2C19, CYP3A4, or CYP2D6. The value of these cell lines for investigation of P450-independent and P450 metabolite-mediated cell toxicity has been demonstrated previously (Foster et al, 2013;Gustafsson et al, 2014). A marked and concentration-dependent reduction in MTS activity, which is indicative of reduced cell viability (Gustafsson et al, 2014), was observed when THLE-Null cells were exposed to sitaxentan for 24 hours ( Fig.…”

“…This drug also exhibited markedly potentiated toxicity to THLE-3A4 cells. Potentiated toxicity to THLE-3A4 cells has been observed with numerous other drugs that are bioactivated to reactive intermediates via CYP3A4 (Thompson et al, 2012;Foster et al, 2013;Gustafsson et al, 2014). Sitaxentan is metabolized by CYP3A4 (Erve et al, 2013) and exhibited a high in vitro f CVB to human hepatocytes; therefore, a likely explanation for the potentiated toxicity to THLE-3A4 cells is the formation of a cytotoxic reactive intermediate in these cells.…”

“…The threshold of concern for THLENull cell toxicity (,200 mM) was exhibited by 21 of 82 drugs that caused human DILI (26%) and by 1 of 21 drugs (5%) that did not cause DILI (Gustafsson et al, 2014). The threshold for potentiated THLE-3A4 cell toxicity (THLE-Null/THLE-3A4 EC 50 ratio $1.4) was exhibited by 10 of 82 drugs that caused DILI (12%) and by 1 of 21 drugs (5%) that did not (Gustafsson et al, 2014).…”

“…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.…”