Derivation of CYP3A4 and CYP2B6 degradation rate constants in primary human hepatocytes: A siRNA-silencing-based approach

Abstract

The first-order degradation rate constant (k_deg) of cytochrome P450 (CYP) enzymes is a known source of uncertainty in the prediction of time-dependent drug-drug interactions (DDIs) in physiologically-based pharmacokinetic (PBPK) modelling. This study aimed to measure CYP k_deg using siRNA to suppress CYP expression in primary human hepatocytes followed by incubation over a time-course and tracking of protein expression and activity to observe degradation. The magnitude of gene knockdown was determined by qPCR and activity was measured by probe substrate metabolite formation and CYP2B6-Glo™ assay. Protein disappearance was determined by Western blotting. During a time-course of 96 and 60 h of incubation, over 60% and 76% mRNA knockdown was observed for CYP3A4 and CYP2B6, respectively. The k_deg of CYP3A4 and CYP2B6 protein was 0.0138 h^-1 (±0.0023) and 0.0375 h^-1 (±0.025), respectively. The k_deg derived from probe substrate metabolism activity was 0.0171 h^-1 (±0.0025) for CYP3A4 and 0.0258 h^-1 (±0.0093) for CYP2B6. The CYP3A4 k_deg values derived from protein disappearance and metabolic activity were in relatively good agreement with each other and similar to published values. This novel approach can now be used for other less well-characterised CYPs.

Keywords: Cytochrome P450; Degradation rate constant; Drug–drug interactions; Half-life; Human hepatocytes; Mechanism-based inhibition; Physiologically-based pharmacokinetic modelling; Small-interfering RNA; Time-dependent inhibition.