Exploring concepts of in vitro time-dependent CYP inhibition assays

Abstract

Introduction: 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.

Areas covered: 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.

Expert opinion: 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.

Keywords::

• cytochrome P450
• time-dependent inactivation
• time-dependent inhibition

Acknowledgments

The authors thank G Zhang and I Smukste (Corning) and C Khojasteh and M Hop (Genentech) for providing a careful reading of the paper and their helpful comments. We thank A Mason for generating the data in Figure 2 and Table 2. We also thank J Sherwood for excellent editorial assistance.

Declaration of interest

This paper was sponsored by Corning Life Sciences, Genetech and AstraZeneca. D Stresser is an employee of Corning Gentest Contract Research Services, J Mao and J Kenny are employees of Genentech Inc., B Jones and K Grime are employees of AstraZeneca.

Notes

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