Development and validation of an in vitro, seven-in-one human cytochrome P450 assay for evaluation of both direct and time-dependent inhibition.

Introduction: Direct and time-dependent inhibition (TDI) of cytochrome P450 enzymes (CYP) raises drug safety concerns and has major implications in drug development. This study describes the development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) based screening tool to simultaneously assess both the direct and the time-dependent inhibitory potential of xenobiotics on the seven major CYPs using a two-step approach.

Methods: The in vitro cocktail of FDA recognized model substrates was incubated with human liver microsomes (HLM) and consisted of caffeine (CYP1A2), bupropion (CYP2B6), rosiglitazone (CYP2C8), tolbutamide (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6) and midazolam (CYP3A4). Direct and time-dependent inhibitory profiles of direct and time-dependent reference inhibitors for each CYP were studied. For validation, the results were compared to those obtained with the traditional single substrate approach. Statistical uncertainty was quantified using the bootstrap method.

Results: The direct inhibition assay showed an acceptable fold bias of 1.35 (geometric mean fold absolute deviation, range 1.01-2.61) in the IC50 values for the cocktail assay compared to the single substrate results with no trend for under- or overestimation. Using a single point inactivation assay to assess TDI, we were able to identify all seven tested time-dependent reference inhibitors, without any false negatives.

Discussion: The presented design enhances throughput by assessing the seven major CYPs simultaneously and allows for detection of and discrimination between direct and time-dependent CYP inhibition via IC50 and single point inactivation experiments. For the latter, a threshold of 10% TDI is proposed for carrying out more detailed inactivation kinetic experiments.