Dysregulation of protein degradation pathways may mediate the liver injury and phospholipidosis associated with a cationic amphiphilic antibiotic drug
Introduction
Drug-induced liver injury (DILI) is the major adverse drug event that leads to regulatory actions on drugs (Abboud and Kaplowitz, 2007), and antibiotics are among the most commonly implicated culprits (Hussaini and Farrington, 2007, Robles et al., 2010). While both the widespread prescription of antibiotics and the large doses required for therapeutic effect contribute to the prevalence of adverse reactions, exposure alone does not fully explain the hepatotoxicity associated with these compounds. The mechanisms responsible for these adverse reactions are not well understood owing in part to a lack of experimental models to study the toxicity. As a result, liver injury remains one of the major liabilities in the development of new antibiotic therapies.
PF-04287881 is a ketolide antibiotic that was in development at Pfizer, Inc. for the treatment of acute bacterial respiratory infections. Preclinical toxicology studies to evaluate the safety of this compound were performed in both Sprague–Dawley rats and beagle dogs, and the liver was identified as one of the target organs of toxicity based on increased serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and microscopic findings in the liver consistent with phospholipidosis. The microscopic findings were not surprising given the propensity of other cationic amphiphilic antibiotics (e.g. azithromycin and erythromycin) to cause phospholipidosis in animal and in vitro models, despite positive clinical utility (Baronas et al., 2007, Matsumori et al., 2006). Overall, PF-04287881 was well tolerated in both animal species tested at doses where systemic exposure was approximately 30 times the anticipated clinical efficacious exposure. However, mild elevations in liver function tests (LFTs) were observed in study participants enrolled in the Phase I clinical study and the development of this compound was discontinued. Because there was inter-individual variation in the liver response profile across study subjects, it was hypothesized that genetic variation may, in part, underlie sensitivity to the adverse liver effects of PF-04287881.
A mouse diversity panel (MDP) has been previously shown to provide a population-based approach to modeling and predicting inter-individual variability in adverse drug reactions. An MDP is a commercially available panel of inbred strains that are genetically different from one another and selected to maximize genetic variation across strains (Bogue and Grubb, 2004, McClurg et al., 2007). This resource can be utilized to model human genetic diversity, and was recently shown to both predict and provide mechanistic insight into the kidney injury induced by DB289, a promising drug therapy for sleeping sickness that was suspended after kidney toxicity was observed in clinical studies (Harrill et al., 2012). The genetic diversity of an MDP has also been shown to enable the identification of genomic, transcriptomic, and metabolomic biomarkers associated with acetaminophen toxicity that inform human sensitivity to acetaminophen-induced liver injury (Court et al., 2013, Harrill et al., 2009a, Harrill et al., 2009b, Liu et al., 2010).
To better understand inter-individual sensitivity to PF-04287881, an MDP was utilized to identify mouse strains that showed sensitivity or resistance to PF-04287881-induced liver injury. Select strains demonstrating differential pathologic findings in the liver following treatment were then utilized to identify transcripts and pathways that distinguish susceptible and resistant strains in the context of PF-04287881 treatment. It was hypothesized that these transcriptional changes are important for mediating PF-04287881-induced liver injury, and would inform mechanisms associated with the hepatotoxicity induced by antibiotic drugs.
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Animals
Female mice aged 6–8 weeks were obtained from The Jackson Laboratory (129S1/SvlmJ, A/J, AKR/J, BALB/cJ, BTBR T + tf/J, BUB/BnJ, C3H/HeJ, C57BL/6J, C57BLKS/J, C57BR/cdJ, C58/J, CBA/J, CE/J, DBA/2J, FVB/NJ, I/LnJ, KK/HIJ, LG/J, LP/J, MA/MyJ, MRL/MpJ, NOD/ShiLtJ, NON/ShiLtJ, NOR/LtJ, NZW/LacJ, P/J, PL/J, PWK/PhJ, RIIIS/J, SEA/GnJ, SJL/J, SM/J, SWR/J, WSB/EiJ) or Charles River (CD-1) and housed in the Laboratory Animal Resources and Technical Support Facility at The Hamner Institutes. Mice were
Treatment of an MDP with PF-04287881
The potential for PF-04287881 to cause liver injury in inbred mouse strains of an MDP was confirmed by the observation of significant elevations in group mean serum ALT levels in PF-04287881-treated animals relative to vehicle-treated controls (Fig. 1A). All but the MA/MyJ, SM/J, SJL/J, and A/J strains (30/34, 88%) demonstrated significant, treatment-induced increases in ALT. The average fold elevation varied across strains (range: 1.6–14.2), indicating that genetic variation affected
Discussion
In this study, the liver response of 34 genetically diverse inbred mouse strains was evaluated following PF-04287881 treatment with the goal of identifying susceptible strains in which to investigate possible mechanisms leading to the liver injury. PF-04287881-induced liver injury, as evidenced by increases in serum ALT, AST, and ALP levels, and microscopic observations of hepatocellular single cell necrosis and Kupffer cell vacuolation consistent with phospholipidosis, was observed with
Conflict of interest
The authors declare that there are no conflicts of interest.
Acknowledgments
The authors extend their gratitude to Dr. Paul Watkins for the helpful discussions during this work, Brian Rago for the analysis of PF-04287881 drug concentration, and Richard Giovanelli for the clinical chemistry analysis.
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These two authors contributed equally to this work.