Exploring concepts of in vitro time-dependent CYP inhibition assays

Expert Opin Drug Metab Toxicol. 2014 Feb;10(2):157-74. doi: 10.1517/17425255.2014.856882. Epub 2013 Nov 21.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Review

MeSH terms

  • Animals
  • Cytochrome P-450 Enzyme Inhibitors*
  • Cytochrome P-450 Enzyme System / metabolism
  • Drug Interactions / physiology
  • Enzyme Inhibitors / metabolism
  • Enzyme Inhibitors / pharmacology
  • Hepatocytes / drug effects
  • Hepatocytes / enzymology
  • High-Throughput Screening Assays / methods*
  • High-Throughput Screening Assays / trends
  • Humans
  • Microsomes, Liver / drug effects
  • Microsomes, Liver / enzymology
  • Time Factors


  • Cytochrome P-450 Enzyme Inhibitors
  • Enzyme Inhibitors
  • Cytochrome P-450 Enzyme System