Acute liver effects, disposition and metabolic fate of [14C]-fenclozic acid following oral administration to normal and bile-cannulated male C57BL/6J mice

Abstract

The distribution, metabolism, excretion and hepatic effects of the human hepatotoxin fenclozic acid were investigated following single oral doses of 10 mg/kg to normal and bile duct-cannulated male C57BL/6J mice. Whole body autoradiography showed distribution into all tissues except the brain, with radioactivity still detectable in blood, kidney and liver at 72 h post-dose. Mice dosed with [14C]-fenclozic acid showed acute centrilobular hepatocellular necrosis, but no other regions of the liver were affected. The majority of the [14C]-fenclozic acid-related material recovered was found in the urine/aqueous cage wash, (49%) whilst a smaller portion (13%) was eliminated via the faeces. Metabolic profiles for urine, bile and faecal extracts, obtained using liquid chromatography and a combination of mass spectrometric and radioactivity detection, revealed extensive metabolism of fenclozic acid in mice that involved biotransformations via both oxidation and conjugation. These profiling studies also revealed the presence of glutathione-derived metabolites providing evidence for the production of reactive species by mice administered fenclozic acid. Covalent binding to proteins from liver, kidney and plasma was also demonstrated, although this binding was relatively low (less than 50 pmol eq./mg protein).Electronic supplementary materialThe online version of this article (doi:10.1007/s00204-016-1894-5) contains supplementary material, which is available to authorized users.

The metabolic fate of fenclozic acid in chimeric mice with a humanized liver

“…As indicated in the experimental section, following oral administration of fenclozic acid to PXB-mice at 10 mg/kg, to both intact and bile duct cannulated animals, metabolite profiles were determined by UHPLC–MS for plasma, bile, and urine, as well as in extracts of liver, kidney, and faeces [as described for previous studies in the C57BL/6 mouse (Pickup et al 2017 )]. The metabolites detected in the various samples are listed in Table 1 (further mass spectral data and mass spectra supporting the assignment of structures are provided in Online Resource 1, Table S1 and Figs.…”

“…The metabolites detected in the various samples are listed in Table 1 (further mass spectral data and mass spectra supporting the assignment of structures are provided in Online Resource 1, Table S1 and Figs. S8–S30) using the numbering system employed in Martin et al ( 2014 ) and Pickup et al ( 2017 ), with additions to take into account the novel metabolites (M25–M37) detected in the present study. To facilitate inter-species, inter-strain, and inter-model comparison, this table also lists the metabolites observed in recent rodent studies (Martin et al 2014 ; Pickup et al 2017 ).…”

“…The mass error for each proposed metabolite and fragment ion structure was < 5 ppm (range, e.g., 0.03–1.27 mDa for metabolites seen in plasma) compared to the theoretical exact mass. For those metabolites where no MSMS data could be acquired, identification was based on the accurate mass of the protonated and/or sodiated molecules and reference to previously published work from our group (Martin et al 2014 and; Pickup et al 2017 ).…”

“…Thus, fenclozic acid presents an interesting problem for the safety assessment of new drug candidates as, in preclinical species, it was not a hepatotoxin but when administered to humans showed itself to result in dose-dependent DILI. As a result, we have explored a range of in vitro systems (Rodrigues et al 2013 ) and performed a number of in vivo studies in rodents (Martin et al 2014 ; Pickup et al 2014 , 2017 ; Akingbasote et al 2017 ) in efforts to better understand the mechanism(s) underlying the toxicity of fenclozic acid. It was also of interest to see if the methods currently available for evaluating the potential of a new chemical entity (NCE) to cause human toxicity were more able to predict the toxicity of fenclozic acid than those used at the time which the drug was in preclinical development.…”

“…As well as in microsomal incubations, fenclozic acid was assessed in vitro for both P450-dependent and -independent cytotoxicity to THLE cell lines (SV40 T-antigen-immortalized human liver epithelial cells), time-dependent inhibition of important human cytochrome P450 enzymes, inhibition of BSEP (bile salt efflux pump) and MRP2 (human multidrug resistance-associated protein 2), and mitochondrial toxicity to THLE or HepG2 cells, but was without effect (Rodrigues et al 2013 ). Support for the hypothesis that P450-based bioactivation and covalent binding to proteins might have a role in the DILI observed with fenclozic acid in humans was provided by a number of in vivo metabolism studies in rodents which, unlike the in vitro investigation, revealed the presence of glutathione-derived metabolites in rat- and mouse-derived samples (Martin et al 2014 ; Pickup et al 2017 ). In addition, a whole-body autoradiography study using [ 14 C]-labelled fenclozic acid in the mouse showed retention of radioactivity in blood, kidney, and liver at 72 h post-dose together with the demonstration of covalent binding to proteins.…”