Identification of Time-Dependent CYP Inhibitors Using Human Liver Microsomes (HLM)

Cytochrome P450s (CYPs) are responsible for the metabolism of a majority of marketed drugs and, as a consequence, alteration in CYP activity can result in clinically relevant drug-drug interactions (DDIs). Drugs that are time dependent inhibitors (TDIs) of CYPs have been reported to cause severe DDIs, leading to prescription adjustments and, in certain cases, have been withdrawn from the market (Zhou et al., Ther Drug Monit 29:687–710, 2007). Oftentimes, TDI is the result of mechanism-based inactivation (MBI), where CYPs catalyze the formation of reactive metabolites that irreversibly or quasi-irreversibly inhibit its own activity. In order to restore basal CYP activity lost as a result of MBI de novo enzyme synthesis is required, and therefore, MBI can have greater clinical consequences than reversible CYP inhibition. Methodologies capable of identifying MBIs in drug discovery are warranted to address this potential liability. The most commonly employed assay to identify MBIs is by measuring the IC₅₀ for CYP enzymes with and without pre-incubation of discovery compounds with human liver microsomes. An IC₅₀ shift assay for CYP3A will be described in greater detail given the enzyme’s prominent role in drug metabolism and association with severe clinical DDIs resulting from MBI; however, the overall assay design can easily be adopted for other CYPs. While the IC₅₀ shift assay can be used to build SAR to mitigate this liability in discovery, clinical risk assessment of an MBI requires the determination of the kinetic parameters K_I and k_inact .