Validated HepaRG™ mechanistic methods as a tool for dynamic and kinetic regulatory applications

The toxicity profile of an exogenous chemical (xenobiotic) to which the body is exposed depends not only on the toxicity of the parent compound but also (1) on the interplay between dynamic and kinetic processes, (2) on any toxicologically relevant metabolites that may be formed during metabolism and (3) on the xenobiotic's ability to modulate biotransformation enzymes that affect its rate of metabolism. Information on metabolism is useful in toxicity testing strategies, for example, to support in vitro to in vivo extrapolation. At the regulatory level, an ADME toolbox is needed providing both kinetic and dynamic information. A xenobiotic’s response is determined within certain limits by the concentration of the xenobiotic at the target and its mechanism of action. However, the concentration at the active site under in vivo conditions cannot be easily measured, and surrogates such as plasma concentration are used instead. The emergence of validated in vitro approaches to study some primary kinetic and dynamic processes in simpler experimental set-ups is key to better mechanistic understanding and provide ways to estimate the concentration at the active site, which then can be used in a more reliable extrapolation and prediction of in vivo behavior and action of xenobiotics. In this context biotransformation enzyme induction or inhibition are sensitive biomarkers for phenotypic metabolic competence of in vitro test systems and are key events associated with e.g. thyroid disruption, and can be considered as biomarkers for toxicologically relevant nuclear receptor-mediated pathways. The seminar will summarise the potential of test systems like human HepaRG™ cells to obtain in vitro mechanistic method study data gaining insight into how xenobiotic receptors regulate many endogenous functions and signalling pathways, and how xenobiotic exposure thus may dysregulate an array of fundamental cell functions. With the current OECD programmes on the adverse outcome pathway (AOP) concept, these receptors are being and will be assigned as molecular initiating events or key events in numerous discrete toxicity pathways. During the seminar, also the results of a multi-laboratory validation study of two in vitro methods based on the use of cryopreserved primary human hepatocytes (PHH) and human HepaRG™ cells that assess the potential of xenobiotics to induce cytochrome P450 (CYP) enzyme activity, in particular, CYP1A2, CYP2B6, and CYP3A4 will be presented.

Speaker: Dr. Sandra Coecke  

Dr. Sandra Coecke, senior scientist at European Commission Joint Research Centre.  Dr. Coecke has a Science, Technology, Engineering and Mathematics-based education (Engineer Biotechnology, Free University Brussels) complemented with a Ph.D. in the faculty of Medicine and Pharmacy entitled „Hormonal regulation of flavin-containing monooxygenase and glutathione S-transferases, an in vitro approach“. She has been managing and leading scientific teams since the 90’ies first in the pharmaceutical industry and later joining the European Commission Joint Research Centre’s ECVAM. Dr. Coecke has more than 30 years' experience in vitro toxicology, including research and activities related to the development and validation of new in vitro cell and tissue-based methods. In 1994 she was already awarded the International Prize from Foundation for the Substitution of Animal Experimentation in Luxembourg based on her work in the field of metabolism and novel in vitro cell culture systems in general. This year she has been announced as the Björn Ekwall Memorial Award (BEMA) winner 2020 for the development and validation of new in vitro cell- and tissue-based methods for in vitro toxicity testing for the replacement of animal experiments. A significant part of her work has been dedicated to leading the development and implementation of internationally accepted quality standards, such as Good Laboratory Practices (GLP), for in vitro methods. She has been instrumental in the development of the European Commission Joint Research Centre’s guidance document on Good Cell Culture Practice (GCCP) and the OECD’s guidance document on Good In Vitro Method Practices (GIVIMP). Dr. Coecke has established and currently manages the European Union Network of Laboratories for the Validation of Alternative Methods (EU-NETVAL), which includes 35 high-quality laboratories across Europe, and she is the coordinator of a large EU-NETVAL validation study with 17 mechanistic methods for the detection of chemicals that may disrupt normal thyroid function. She continues to be an ambassador for stimulating the use in vitro and in silico toxicokinetic and metabolism methods in regulatory decision-making and led the EURL ECVAM validation study assessing HepaRG™ and human hepatocyte-based in vitro methods for human cytochrome P450 enzyme induction. She published over 100 in vitro toxicological peer-reviewed papers and several book chapters and has been invited speaker and lecturer in many international conferences.