Analytical Strategies to Study the Interaction Between Microbiota and Drug Metabolism Using Targeted and Untargeted LC-MS-based Metabolomics Marine Letertre (Presenter) Imperial College London Presenter Bio: I am currently a second year PhD student at Imperial College London, studying the interactions between gut microbiota and drug metabolism. I am mainly using targeted and untargeted LCMS as well as NMR metabolic profiling and amplicon sequencing. I have graduated in 2015 from the University of Nantes (France), with a Master’s Degree (Hons), specialized in the use of Natural Active Ingredients for a pharmaceutical application. Although I am a chemist by training, I have always worked at the interface of Chemistry and Biology, allowing me to take part on diverse projects such as understanding the toxicity of Bisphenol A on human testicular explants by MALDI-Imaging Mass Spectrometry (Rennes, France) or developing a novel class of irreversible kinase inhibitors using organic chemistry technics as well as enzyme assays and molecular modelling (Auckland, New Zealand).

Authors: Marine Letertre (1), Nyasha C. Munjoma (2), Lesley Hoyles (1), Aadra Bhatt (3), Anne L. McCartney (4), Muireann Coen (5), Matthew Redinbo (3), Jeremy K. Nicholson (1), Jonathan R. Swann (1), & Ian D. Wilson (1)
(1) Imperial College London, London, UK. (2) Waters Corporation, Wilmslow, UK. (3) University of North Carolina, Chapel Hill, USA. (4) The University of Reading, Reading, UK. (5) AstraZeneca, Cambridge, UK.

Metabonomics, and particularly pharmacometabonomics, is a useful tool for patient stratification and to establish individualized drug therapy and limited ADRs to improve patient journey. We have used the pharmacometabonomics principle on animal experiments to study the interaction between the microbiome and several drugs. The general workflow applied both untargeted LC-MS for endogenous metabolic phenotyping and a targeted approach to quantify the drug of interest and its metabolites, in complement of NMR and amplicon sequencing. It enabled the impact of drugs on the gut microbiota community structure to be investigated as well as the influence of the microbiome on the metabolism of these drugs and their metabolites.

Introduction

Understanding drug-microbiome-host interactions should now be considered a priority to determine the pharmacological profile of a drug as well as its toxicological profile. On one side, the gut microbiota can, directly and indirectly, affect the pharmacological profile of orally administered drugs (or drugs/metabolites excreted into the gut via e.g. the bile), and on the other, the drug itself can perturb the microbiome structure and its functionality with potential ramifications for host health. A multifaceted approach is required to study the wide range of gut microbial-drug interactions. This project focus on methotrexate and the potential to therapeutically target the gut microbiota.

Methods

In each sub-project, animal experiments were performed and pharmacometabonomics principle was used through the general workflow as pre- and postdose samples were collected in each study. Faecal, urine and if available plasma and tissues samples from animal studies were analysed by a dual LC-MS untargeted metabolic profiling and targeted approach to quantify the drug of interest and its metabolites. In parallel, metataxonomics analysis was performed on faecal samples to determine microbes profile. As well as these in vivo approaches, we are also seeking to develop a rapid in vitro method to determine the interaction of a given drug with the gut microbiota.

Results

The simultaneous use of targeted and untargeted LCMS metabolic profiling allowed to (1) monitor the detoxification of methotrexate by the bacterial enzyme FGCP and (2), to observed dose-related effects of the drug on the urine and faecal metabolic profile and microbial community. We also showed that targeting the microbiome with a selective inhibitor of the bacterial β-glucuronidase does not alter the metabolic profile of the animals which received the inhibitor compared to the untreated ones. This result suggests that this inhibitor could dramatically reduce adverse drug reactions due to irinotecan in mice. Finally, preliminary results of an in-vitro model based on static batch cultures and semi-targeted LCMS showed drug and donor-specific effects on compound metabolism.

Conclusions & Discussion

By using targeted and untargeted LCMS-based metabolomics, we have been able to have a deeper understanding of the interaction between methotrexate and a bacterial inhibitor. We were also able to test a new in-vitro model aiming to quickly determine the interaction of the gut microbiota with a given drug. Based on these promising early data, further development will be undertaken to confirm these observations and extend the range of molecules studied. These approaches are complementary and form part of a strategy designed to provide tools to further illuminate the complex interactions between drugs, gut microbiota and the host.