Dysregulation of protein degradation pathways may mediate the liver injury and phospholipidosis associated with a cationic amphiphilic antibiotic drug

Highlights

• Identified susceptible and resistant mouse strains to liver injury induced by a CAD

• Liver injury characterized by single cell necrosis, and phospholipidosis

• Decreased gene expression associated with protein ubiquitination in sensitive mice

• Altered protein ubiquitination may cause oxidized protein accumulation in the liver.

Abstract

A large number of antibiotics are known to cause drug-induced liver injury in the clinic; however, interpreting clinical risk is not straightforward owing to a lack of predictivity of the toxicity by standard preclinical species and a poor understanding of the mechanisms of toxicity. An example is PF-04287881, a novel ketolide antibiotic that caused elevations in liver function tests in Phase I clinical studies. In this study, a mouse diversity panel (MDP), comprised of 34 genetically diverse, inbred mouse strains, was utilized to model the toxicity observed with PF-04287881 treatment and investigate potential mechanisms that may mediate the liver response. Significant elevations in serum alanine aminotransferase (ALT) levels in PF-04287881-treated animals relative to vehicle-treated controls were observed in the majority (88%) of strains tested following a seven day exposure. The average fold elevation in ALT varied by genetic background and correlated with microscopic findings of hepatocellular hypertrophy, hepatocellular single cell necrosis, and Kupffer cell vacuolation (confirmed as phospholipidosis) in the liver. Global liver mRNA expression was evaluated in a subset of four strains to identify transcript and pathway differences that distinguish susceptible mice from resistant mice in the context of PF-04287881 treatment. The protein ubiquitination pathway was highly enriched among genes associated with PF-04287881-induced hepatocellular necrosis. Expression changes associated with PF-04287881-induced phospholipidosis included genes involved in drug transport, phospholipid metabolism, and lysosomal function. The findings suggest that perturbations in genes involved in protein degradation leading to accumulation of oxidized proteins may mediate the liver injury induced by this 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.