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Evaluation of time-dependent inhibition (TDI) properties in drug candidates is generally required for any compound entering development. Methods to evaluate TDI, particularly in abbreviated formats, differ widely among laboratories and there appears to be lack of consensus how to address certain assay shortcomings.As a first objective of this work, we provide commentary on experimental and theoretical considerations in the conduct of abbreviated TDI testing. Methods considered are the single K(obs), the progress curve, the '2 + 2' method, the measurement of partition ratios and the IC₅₀ shift assay. The merits of multiple experimental variations in the IC₅₀ shift assay, including in depth discussion on the use of a dilution step are explored. Growing evidence suggests that the use of hepatocytes provides certain advantages over liver microsomes. Therefore, a second major objective of this work is to consider merits of the use of hepatocytes in TDI testing.An in-depth technical understanding of methods to evaluate TDI is critical to enable a selection of an assay aimed at efficiency while minimizing erroneous classification of TDI properties.

Time-dependent inhibition (TDI) of CYP refers to a change in potency during an in vitro incubation or dosing period in vivo. Potential mechanisms include the formation of inhibitory metabolites and mechanism-based inhibition (MBI). In vitro experiments are configured to assess TDI and MBI is inferred, at least initially. MBI is more profound after multiple-dosing and the recovery period is independent of continued drug exposure. Advances in in vitro-in vivo extrapolations for competitive inhibition and the potential relationship between MBI and reactive metabolite-mediated toxicity, have redirected emphasis to CYP TDI. In contrast, with reversible inhibition, strategies for projecting the risks from TDI are less developed and the traditional I/K(i) model often yields a dramatic underprediction. This review explores the contribution of TDI to drug-drug interactions and idiosyncratic drug toxicity.