Cytochrome P450 reaction phenotyping: State of the art

Cytochrome P450 reaction phenotyping refers to the in vitro experimental approach that estimates the quantitative contributions of individual P450 enzymes to the metabolism of a drug. Methods for this are well-established and have existed for over three decades and include the use of selective inhibitors, individually expressed P450 enzymes, and human-derived in vitro systems such as liver microsomes and hepatocytes. The results from P450 reaction phenotyping experiments are used to inform patient safety, clinical trial designs, and physiologically-based pharmacokinetic models, and this information is an expectation from government regulatory authorities when developing a new drug candidate. Despite widespread use, P450 reaction phenotyping methods possess shortcomings. These include sub-optimal selectivity of P450 inhibitors, scaling factors that can differ among substrates, challenges measuring low turnover substrates, and considerations of non-P450 routes of drug clearance (e.g. active transport, other drug metabolizing enzyme families). A recently described "sequential qualitative-then-quantitative" approach to P450 reaction phenotyping is described along with a more comprehensive experimental design that considers incomplete selectivity of P450 inhibitors. This approach addresses some of the aforementioned shortcomings, however it is still important to consider the contribution of P450 enzymes to the overall dispositional profile that is obtained from in vivo studies, such as radiolabel human absorption/distribution/metabolism/excretion (ADME) studies.