Biokinetics in repeated-dosing in vitro drug toxicity studies
Graphical abstract
Introduction
Many derivatives of a lead compound exhibiting a desired pharmacological effect are synthesised early on in the development of a new pharmaceutical entity in order to identify the ones with the most optimal pharmacological response. In order to assess the safety of these lead compounds before the ‘first dose in man’, toxicity testing of a large number of chemicals on animals is necessary and a costly endeavour (Sasseville et al., 2004). This testing represents one of the major bottlenecks in drug development as toxicity testing in preclinical studies is time consuming and requires large numbers of animals and considerable amounts of test compound. In addition, the high costs of toxicity testing are exacerbated by the high drug attrition rate, where 23% of registered drugs are retracted due to adverse reactions not predicted in animal models (Kola, 2008). These adverse reactions are often idiosyncratic and occur after repeated dosing. Major reasons for the suboptimal correlation between animal and human toxicity are the inter- and intra-species differences in pharmacokinetics (Park et al., 2011).
In light of both ethical and financial costs associated with drug safety testing on animals, human cell-based in vitro assays are increasingly used to screen drug candidates for human-relevant pharmacokinetic properties and molecular mechanisms of toxicity prior to pre-clinical testing in animals. However, the move from using in vitro assays for hazard identification, i.e. the mere potential of a chemical to cause an effect, to hazard characterization in drug development, i.e. dose–response assessment, is still in its early stage of development. It is generally accepted that no single stand-alone in vitro test sufficiently replaces an animal-based toxicity test and thus an integrated strategy is required. Such strategy calls for a battery of in vitro assays employing long-lived, highly functional organotypic cell cultures and a mechanistic understanding of the molecular events leading to adverse health effects (Adler et al., 2011). For such in vitro test battery to be used in a risk assessment procedure, a point of departure needs to be derived from the set of dose–response relationships obtained from these assays and translated into a toxicologically equivalent dose in humans. Indeed, the pharmacokinetics (i.e. the absorption, distribution, metabolism and excretion from a body) of a drug determines the concentration over time of the drug (or its toxicologically relevant metabolite) at the target site, which strongly dictates the drug's toxicity. These processes need to be integrated into a meaningful in vitro-based drug safety testing strategy (Adler et al., 2011).
To improve the predictivity of in vitro systems for unwanted effects of drugs after repeated dosing, the aim of the EU 7th Framework Project, Predict-IV, was to develop such a testing strategy integrating in vitro systems with knowledge of cell biology, mechanistic toxicology and in silico (pharmacokinetic) modelling. The project focussed its efforts on developing testing strategies by using in vitro assays with cells of human origin (whenever possible) and representing target organs most frequently affected by poorly predicted drug toxicity, namely the liver, kidney and central nervous system. It opted for using primary human hepatocytes, primary rat hepatocytes and the human hepatoma cell line HepaRG as its main liver models (Mueller et al., 2015), 2D mouse and 3D rat primary brain cell models with an in vitro blood brain barrier (BBB) model to predict neurotoxicity (Culot et al., 2008, Culot et al., 2013, Schultz et al., 2015), and the human renal proximal tubule cell line RPTEC/TERT1 (Wieser et al., 2008, Aschauer et al., 2015b) as its model of choice for predicting nephrotoxicity. Culture conditions were adapted to maintain highly differentiated organotypic cells in culture for 14 days, during which the cells were exposed daily to a selection of 27 drugs varying in their mechanism of pharmacological action and known to cause hepatotoxicity, nephrotoxicity and/or neurotoxicity after repeated use. For a holistic, mechanistic approach, in depth characterization of molecular perturbations induced by the drugs was performed by integrating a suite of ‘omics’ technologies (e.g. Wilmes et al., 2013). Moreover, exposure conditions and changes within the assays over the 14-day exposure period were monitored and modelled (e.g. Pomponio et al., 2015a, Pomponio et al., 2015b, Truisi et al., 2015). Dose response analyses and physiologically based pharmacokinetic (PBPK) models were developed to relate daily oral exposure to in vitro derived points of departures (Hamon et al., 2015).
Section snippets
Role of in vitro biokinetics in quantitative in vitro–in vivo extrapolation (QIVIVE) studies
Predict-IV uniquely devoted a separate work package (WP3 ‘Non animal-based models for in vitro kinetics and human kinetic prediction’) to propose and apply a step-wise strategy to measure and model cell exposure levels over time of a selected number of drugs in the developed in vitro assays. The aim was to assess whether and how knowledge of the kinetics of drugs in in vitro assays helps to explain the variations in observed effects between drugs, cell types and assay setup, and – in so doing –
Chemical stability
Chemical stability in solution determines the concentration in cells and subsequently its potential to perturb molecular pathways in vitro. The concentration in stock solutions and exposure medium of drugs listed in Table 1 were measured over time. Whereas most drugs were chemically stable in exposure medium as well as in the vehicles used to prepare stock solutions, i.e. distilled water, methanol and DMSO, adefovir dipivoxil hydrolysed significantly in exposure medium of RTPEC/TERT1 cells at 37
Expression and activity of drug transporters and biotransformation enzymes
To identify specific mechanisms of toxicity and new toxicity pathways, it is important to distinguish between an increase in cytotoxic potency of a drug in vitro that is due to higher intracellular concentrations of the drug (i.e. due to differences in kinetics) and an intrinsic sensitivity of a cell type to a drug (i.e. the drug's ability to activate selective toxicity pathways). If a significant fraction of a drug is taken up by cells, repeated dosing of the cells, as done in the Predict-IV
Conclusions: a strategy for future in vitro testing
The aim of the EU FP7 Predict-IV project was to improve the predictivity of in vitro assays for unwanted effects of drugs after repeated dosing. The project assessed the added benefit of integrating long-lived in vitro cell systems, representing the liver, the kidney and the central nervous system, with ‘omics’ technologies and in silico modelling, including systems biology and pharmacokinetic assessments. WP3 studied the distribution over time (i.e. kinetics) of a selection of drugs, varying
Acknowledgements
The authors declare no conflicts of interest. This work was supported by the EU FP 7 Predict-IV programme (grant 202222). The authors are grateful to the many PhD students and scientists performing the work cited in this overview paper. The authors would also like to acknowledge the Doerenkamp Zbinden Foundation for financially supporting N.I. Kramer and B.J. Blaauboer at Utrecht University.
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