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A UPLC-MS Based Exploration of the Xenobiotic and Endogenous Metabolic Phenotypes of Pre-Clinical Models of Hepatotoxicity

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A UPLC-MS Based Exploration of the Xenobiotic and Endogenous Metabolic Phenotypes of Pre-Clinical Models of Hepatotoxicity A UPLC-MS Based Exploration of the Xenobiotic and Endogenous Metabolic Phenotypes of Pre-Clinical Models of Hepatotoxicity Isobelle Grant Computational and Systems Medicine Imperial College London Submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy of Imperial College London Declaration of Originality The author certifies that this thesis, and the experiments it refers to, are their own work and that all else is appropriately referenced or acknowledged. Copyright Declaration The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work. 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. 3 Funding The author was funded through a Medical Research Council (MRC) Doctoral Training Partnership studentship. Additional funding was provided by the MRC to support a three- month internship at Oncology Innovative Medicines, AstraZeneca, Cheshire. The Imperial College London Graduate School is acknowledged for funding a 4-week research placement at the National University of Singapore. 4 Supervisors and Collaborators Supervisors: Dr. Muireann Coen Computational and Systems Medicine, Primary supervisor Imperial College London Dr. Elizabeth Want Computational and Systems Medicine, Secondary supervisor Imperial College London Prof. Jeremy Nicholson Department of Surgery and Cancer, Secondary supervisor Imperial College London Additional guidance at Imperial was provided by: Prof. Ian Wilson Computational and Systems Medicine, LC-MS method development (5) Imperial College London and APAP study (6) Dr. Leanne Nye Imperial International Phenome Training Technical guidance for LC-MS Centre, Waters & Imperial College method development (5) External placement supervisors: Dr. Filippos Oncology Innovative Medicines, Industrial placement supervisor Michopoulos AstraZeneca, Cheshire IPC-MS/MS assistance (4/5) Prof. Eric Chan & Department of Pharmacy, Placement supervision, Dr. Lian Yee Yip National University of Singapore GC-MS analyses (4*) External collaborators: Prof. Sidney Nelson & Department of Medicinal Chemistry, TA/TAI synthesis & animal study Dr. Peter Rademacher University of Washington, U.S. design (3/4/5) Prof. Robert Roth & Department of Pharmacology and TA/TAI animal work and study colleagues Toxicology, Michigan State University, US design (3/4/5) Dr. Simone Stahl & DMPK, Drug Safety and Metabolism, APAP animal work and study colleagues AstraZeneca, Alderley Park, Cheshire design (6) “(3/4/5/6)” refers to the chapter the work is featured in. (*) data are referred to but not presented 5 Table of Contents Declaration of Originality .................................................................................................... 2 Copyright Declaration ......................................................................................................... 2 1 CHAPTER 1: INTRODUCTION .................................................................................... 21 1.1 Introduction ................................................................................................................. 22 1.1.1 Aims ................................................................................................................................ 23 1.2 Drug Induced Liver Injury ............................................................................................. 24 1.3 Mechanisms of DILI ...................................................................................................... 24 1.3.1 Drug metabolism and chemically reactive metabolites (CRMs) ..................................... 25 1.3.2 Intrinsic toxicity .............................................................................................................. 26 1.3.3 Idiosyncratic drug reactions ........................................................................................... 27 1.4 Biomarkers of DILI ........................................................................................................ 29 1.4.1 New approaches to biomarker discovery: ‘-omics’ ........................................................ 29 1.5 TieniLic Acid and TieniLic Acid Isomer ............................................................................ 31 1.5.1 Summary of the clinical features associated with TA toxicity ........................................ 31 1.5.2 TA/TAI metabolism and chemically reactive metabolite formation ............................... 32 1.5.3 Immune mechanism of TA toxicity ................................................................................. 34 1.5.4 Intrinsic/ direct toxicity of TA and TAI ............................................................................ 35 1.5.5 New approaches to study TA toxicity ............................................................................. 35 1.6 ParacetamoL ................................................................................................................. 37 1.6.1 APAP metabolism ........................................................................................................... 37 1.6.2 Mechanism of toxicity .................................................................................................... 39 2 CHAPTER 2: METHODOLOGY .................................................................................... 41 2.1 Introduction ................................................................................................................. 42 2.1.1 Aims ................................................................................................................................ 42 2.2 LiQuid Chromatography ................................................................................................ 44 2.2.1 RP chromatography for medium polar and non-polar metabolite analyses .................. 44 2.2.2 Ion-pairing and HILIC for polar metabolite analyses ...................................................... 44 2.3 Mass Spectrometry ...................................................................................................... 46 2.3.1 Electrospray ionisation ................................................................................................... 46 2.3.2 Quadrupole and Time-of-Flight Mass Analysers ............................................................. 47 6 2.4 Data Pre-Processing of Untargeted LC-MS Data ............................................................ 50 2.5 Chemometrics .............................................................................................................. 53 3 CHAPTER 3: TA AND TAI DRUG METABOLISM .......................................................... 55 3.1 Introduction ................................................................................................................. 56 3.1.1 Rationale and aims ........................................................................................................
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