Underlying Mechanisms of Pharmacology and Toxicity of a Novel PPAR Agonist Revealed Using Rodent and Canine Hepatocytes

Abstract

Marked species-specific responses to agonists of the peroxisome proliferator-activated alpha receptor (PPAR alpha) have been observed in rats and dogs, two species typically used to assess the potential human risk of pharmaceuticals in development. In this study, we used primary cultured rat and dog hepatocytes to investigate the underlying mechanisms of a novel PPAR alpha and -gamma coagonist, LY465608, relative to fenofibrate, a prototypical PPAR alpha agonist. As expected, rat hepatocytes incubated with these two agonists demonstrated an increase in peroxisome number as evaluated by electron microscopy, whereas the peroxisome number remained unchanged in dog hepatocytes. Biochemical analysis showed that rat hepatocytes responded to PPAR agonists with an induction of both peroxisomal and mitochondrial beta-oxidation (PBox and MBox) activities. Dog hepatocytes treated with both PPAR agonists, however, did not show increased PBox activity but did demonstrate increased MBox activity. Analysis of peroxisomal beta-oxidation gene expression markers by quantitative real-time PCR confirmed that PPAR agonists induced the peroxisomal enzymes, acyl-coenzyme A (CoA) oxidase (Acox), enoyl-CoA hydratase/L-3-hydroxyacyl-CoA dehydrogenase (Ehhadh), and 3-ketoacyl-CoA thiolase (Acaa1) at the transcriptional level in rat hepatocytes, but not dog hepatocytes. Expression of mRNA for the mitochondrial beta-oxidation gene hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase (Hadhb), however, increased in both rat and dog hepatocytes, consistent with biochemical measurements of peroxisomal and mitochondrial beta-oxidation. Repeat-dose nonclinical safety studies of LY465608 revealed abnormities in mitochondrial morphology and evidence of single-cell necrosis following 30 days of dosing exclusively in dogs, but not in rats. Microarray analysis indicated that dog hepatocytes, but not rat hepatocytes, treated with LY465608 had an expression profile consistent with abnormalities in the regulation of cell renewal and death, oxidative stress, and mitochondrial bioenergetics, which may explain the canine-specific toxicity observed in vivo with this compound. This increased sensitivity to mitochondrial toxicity of canine hepatocytes relative to rat hepatocytes identified using gene expression was confirmed using the fluorescent indicator tetramethylrhodamine ethyl ester (TMRE) and flow cytometry. At doses of 0.1 microM LY465608, canine hepatocytes showed a greater shift in fluorescence indicative of mitochondrial damage than observed with rat hepatocytes treated at 10 microM. In summary, using rat and dog primary hepatocytes, we replicated the pharmacologic and toxicologic effects of LY465608 observed in vivo during preclinical development and propose an underlying mechanism for these species-specific effects.