In Vitro Reaction Phenotyping of Cytochrome P450 Enzymes

Cytochrome P450s (CYPs) reaction phenotyping is a technique used to identify the major enzyme(s) responsible for the metabolism of a new molecular entity in order to assess clinical drug–drug interaction potential and estimate its relative contribution to the overall metabolic clearance in human. CYP phenotyping is commonly carried out using recombinant CYPs, and/or human liver microsomes (HLM) in combination with isoform-specific chemical inhibitors. For drug candidates with both CYP- and non-CYP-mediated metabolism, human hepatocytes can be a preferred in vitro system for enzyme reaction phenotyping. The protocol described here mainly focuses on the eight common CYP isoforms (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A5) using both recombinant enzymes and HLM as the in vitro system, respectively. These rapid approaches provide qualitative information about the CYP involvement in metabolic clearance of a drug candidate, which can be followed by a more in-depth characterization of metabolic reactions using alternative in vitro systems as needed when the compound is advanced to the next stage of development.