A novel method for predicting human cardiotoxicity without animal testing

The development of reliable non-animal testing strategies is the holy grail in current human safety testing of chemicals and drugs. However, adverse effective concentration obtained from in vitro models represent concentrations in the tissue and are not equivalent to the exposure doses. Hence concentration-response curves are inadequate for human risk and safety assessment because risk assessment requires in vivo dose-response curves from which points of departure. To bridge this gap, a so-called physiologically based kinetic (PBK) modeling based reverse dosimetry approach have been developed, which allows the translation of in vitro data to the in vivo situation. Cardiotoxicity has been considered as an important endpoint in pharmaceutical safety testing given that it has been a leading cause of drug attrition during the development. Cardiotoxicity is also a relevant endpoint in chemical/food safety risk assessment given that many natural alkaloids from botanicals and botanical preparation show the potential cardiotoxicity. The current project aims to provide a proof-of-principle that electrophysiological cardiotoxicity of chemicals or drugs for human can be predicted using a quantitative in vitro in vivo extrapolation (QIVIVE) approach, which could contribute the utility of alternatives of animal testing in risk assessment and safety evaluation of chemicals and drugs. Methadone and ibogaine, two anti-addiction drugs with known in vivo cardiotoxicity, were selected as model compounds.Chapter 1 as the introduction chapter starts with the background information on alternative test strategies/ NAM and the aim of the present PhD project. It also provides a definition on cardiotoxicity, followed by the summaries of toxicokinetic and toxicodynamic profiles of the two model compounds used for the studies and their metabolites. Subsequently the main approaches applied in the present project, including two in vitro cardiotoxicity assays, PBK modelling and Monte Carlo simulations are introduced. Chapter 2 evaluates a mouse and a human stem cell-based in vitro model, namely the mESC-CM beating arrest assay and the hiPSC-CM MEA assay, for cardiotoxicity screening of chemicals. Eleven cardiotoxic chemicals with different modes of action were used as reference compounds. The results obtained from the two models were compared to each other and to in vivo cardiotoxicity data, to provide insight into their applicability domains and to enable selection of a suitable toxicity assay for QIVIVE in subsequent chapters. In Chapter 3 the cardiotoxicity of methadone and its metabolites EDDP and EMDP were quantified using the hiPSC-CM MEA assay. A PBK model of racemic methadone was developed to enable the translation of the in vitro concentration-response curve obtained to an in vivo dose-response curve for methadone-induced QTc prolongation. The outcomes were compared with available human in vivo QTc prolongation data to evaluate the model performance. As follow-up of the work described in Chapter 3, Chapter 4 investigated the potential of the developed QIVIVE approach for the prediction of human inter-individual variability in in vivo cardiotoxicity of methadone. To this end in vitro cardiotoxicity and metabolic data were integrated with PBK models and Monte Carlo simulations to predict the effect of inter-individual and inter-ethnic kinetic variations on the cardiotoxicity of the two methadone enantiomers in the Caucasian and the Chinese population. CSAFs were defined and used to derive dose-response curves for the sensitive individuals. The kinetic variations obtained using individual human liver microsomes or recombinant cytochrome P450 enzymes (rCYPs) were compared. Chapter 5 investigates whether PBK modeling-based reverse dosimetry of in vitro data was able to adequately predict the human cardiotoxicity of the herbal alkaloid ibogaine and its metabolite noribogaine. The TEQ approach was incorporated in the PBK model enabling the evaluation of the role of noribogaine in ibogaine-induced in vivo cardiotoxicity. Chapter 6 summarizes the results obtained in the thesis, provides an overall discussion and presents the future perspectives that follow from the results obtained