A UPLC-MS based exploration of the xenobiotic and endogenous metabolic phenotypes of pre-clinical models of hepatotoxicity

Abstract

To reduce late stage attrition during drug development, and improve the diagnosis of drug induced liver injury (DILI), a greater mechanistic understanding of DILI and improved predictive biomarkers are required. In this thesis, the xenobiotic and endogenous metabolic phenotypes of model hepatotoxins are studied in the rat using an ultra-performance liquid chromatography- mass spectrometry (UPLC-MS) based metabonomics approach. The idiosyncratic hepatotoxin Tienilic Acid (TA), was compared to its structural analogue, Tienilic Acid Isomer (TAI), which is an intrinsic hepatotoxin. TAI dosing resulted in elevated ALT activity and liver necrosis, whereas TA showed no signs of toxicity. The untargeted UPLC-MS approach revealed both previously reported and novel TA drug metabolites, including likely acyl-glucuronides and amino acid conjugates. In contrast, the TAI metabolites detected were predominantly glutathione (GSH) related; reflective of higher reactive metabolite formation. Untargeted UPLC-MS and targeted ion-pair-LC-MS revealed numerous endogenous metabolic alterations, including an elevation in hepatic and plasma ophthalmic acid, common to TA and TAI treated animals. In addition, a unique elevation in pyroglutamate was detected in response to TAI. Interestingly, both ophthalmic acid and pyroglutamic acid have previously been associated with hepatic GSH depletion and oxidative stress. Hepatic oxidative stress is a well-established mechanism in intrinsic toxicity, but has a less established role in idiosyncratic toxicity. To enable further assessment of these compounds, and other glutathione related metabolites, as potential biomarkers of hepatic oxidative stress, a quantitative UPLC-MS/MS assay was developed. Interestingly, despite TA and TAI both depleting hepatic GSH and elevating ophthalmic acid, they were found to impact other circulating metabolites in different ways. To further explore the dynamics of these metabolites, the assay was applied to plasma from paracetamol (APAP) dosed rats; a model GSH depleting hepatotoxin. Quantitative data such as these may contribute to the further development and validation of mathematical models to predict hepatic glutathione status from multiple circulating plasma biomarkers. This thesis demonstrates the utility of a UPLC-MS based approach for hypothesis generation and biomarker development.Open Acces