In vitro exploration of potential mechanisms of toxicity of the human hepatotoxic drug fenclozic acid

Abstract

The carboxylic acid NSAID fenclozic acid exhibited an excellent preclinical safety profile and promising clinical efficacy, yet was withdrawn from clinical development in 1971 due to hepatotoxicity observed in clinical trials. A variety of modern in vitro approaches have been used to explore potential underlying mechanisms. Covalent binding studies were undertaken with [(14)C]-fenclozic acid to investigate the possible role of reactive metabolites. Time-dependent covalent binding to protein was observed in NADPH-supplemented liver microsomes, although no metabolites were detected in these incubations or in reactive metabolite trapping experiments. In human hepatocytes, covalent binding was observed at lower levels than in microsomes and a minor uncharacterizable metabolite was also observed. In addition, covalent binding was observed in incubations undertaken with dog and rat hepatocytes, where a taurine conjugate of the drug was detected. Although an acyl glucuronide metabolite was detected when liver microsomes from human, rat and dog were supplemented with UDPGA, there was no detectable UDPGA-dependent covalent binding. No effects were observed when fenclozic acid was assessed for P450-dependent and P450-independent cytotoxicity to THLE cell lines, time-dependent inhibition of five major human cytochrome P450 enzymes, inhibition of the biliary efflux transporters BSEP and MRP2 or mitochondrial toxicity to THLE or HepG2 cells. These data suggest that Phase 1 bioactivation plays a role in the hepatotoxicity of fenclozic acid and highlight the unique insight into mechanisms of human drug toxicity that can be provided by investigations of biotransformation and covalent binding to proteins.

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

“…The radiochemical purities of [7-3 H] BSEP and Multidrug Resistance-Associated Protein 2 Transporter Activity. The effects of the ERA compounds on ATP-dependent uptake of probe substrates into inside-out membrane vesicles prepared from baculovirus-infected Spodoptera frugiperda Sf21 insect cells expressing human BSEP or multidrug resistance-associated protein 2 (MRP2) proteins were quantified using rapid filtration assays in 96-well plate format, as described by Rodrigues et al (2013) (v/v). For the studies in MRP2 vesicles, incubations were also undertaken using 5 mM AMP in place of ATP at each test compound concentration to quantify substrate accumulation independent of active transport.…”

Highlight report: acetaminophen hepatotoxicity

“…Currently, intensive research activities are ongoing to establish in vitro systems particularly for hepatotoxicity (Grinberg et al 2014;Ramaiahgari et al 2014;Rodrigues et al 2013;Schug et al 2013), nephrotoxicity (Sanchez-Niño et al 2014;Fujiki et al 2013;, neurotoxicity (Barbosa et al 2014;Sisnaiske et al 2014;Frimat et al 2010) and developmental toxicity (Krug et al 2013;Rempel et al 2015;Balmer et al 2014;Zimmer et al 2014). For validation of in vitro systems with hepatocytes APAP represents a popular reference compound (Jennings et al 2014;Hengstler et al 2014).…”

Highlight report: biomarkers of acetaminophen-induced liver injury

“…Moreover, large efforts are undertaken to establish in vitro systems for hepatotoxicity testing (Ramaiahgari et al 2014;Grinberg et al 2014;Tolosa et al 2013;Hewitt et al 2007;Godoy et al 2015;Schug et al 2013) and elucidating mechanisms of hepatotoxicity (Rodrigues et al 2013;Ghallab 2014a, b;Reif 2014a, b;Godoy 2011). Besides its clinical relevance, the translational biomarkers discussed in the review of Beger et al (2015) are also of high interest for scientists optimizing in vitro systems for hepatotoxicity testing.…”