Dose Metrics in Repeatedly Dosed In Vitro Toxicity Assays

摘要

Aim: The emphasis in toxicological risk assessment is moving away from animal testing towards integrating in vitro cell-based assays with computational modeling to extrapolate in vitro derived effective concentrations to human-relevant doses. To improve in vitro assays to predict repeat-dose systemic toxicity, testing strategies are developed where batteries of complex in vitro assays with highly differentiated cells of human origin and representing multiple organs are repeatedly dosed for extended periods of time. One such strategy was developed during the EU FP7 Predict-IV project, where RPTEC/TERT1 kidney cells, primary rat and human hepatocytes, HepaRG liver cells and 2D and 3D primary brain cultures were dosed daily or every other day for 14 days to a selection of drugs varying in their mechanism of pharmacological action. Molecular perturbations induced by the drugs were assessed by integrating a suite of omics technologies. Dose response analyses and physiologically based pharmacokinetic models were developed to relate daily oral exposure to in vitro derived points of departures. To address the increasingly acknowledged problem that traditionally used in vitro nominal concentrations are not necessarily proportional to the concentration at the target site, which is directly related to the initial molecular changes caused by the drugs, one aim of the project was to assess whether and how knowledge of the kinetics of drugs in in vitro assays helps explain the variations in observed effect between drugs, cell types and assay setup. Methods: The concentration of a selection of drugs in cells, labware, cell attachment matrices, and exposure medium was measured over time. Results: Results indicate that lipophilic drugs like chlorpromazine bind significantly to serum constituents in the exposure medium and plastic labware. A few drugs, including less lipophilic drugs like ibuprofen, bind to cell-attachment matrices. Chemicals that reach high concentrations is cells, including cyclosporin A and amiodarone, significantly accumulate over time after repeated dosing, partly explaining their increased toxicity after repeated dosing, compared to a single dose. Conclusions: The results of these studies clearly suggest that integrating knowledge of the differences in concentration over time of drugs in cells in vitro between dosing regimens, as well as knowledge of transporter and biotransformation enzyme function, allows for the development of a mechanistic understanding of the observed in vitro toxicity.