Mechanism-based inactivation of CYP2D6 by imperatorin and drug-drug interaction in vitro and in vivo

Imperatorin (IMP) is a naturally occurring furanocoumarin with a wide range of pharmacological activities, including anti-cancer, anti-inflammatory, and anti-oxidant properties. This study preliminarily investigated the major cytochrome P450 (CYP) enzymes involved in the metabolism of IMP to heraclenin, identifying CYP3A4 as its primary metabolic enzyme. Furthermore, the impact of nine CYP3A4 variants on IMP metabolism was investigated, with CYP3A4.24 demonstrating the lowest relative clearance rate (6.26%). Additionally, the impact of napabucasin on IMP metabolism was investigated, revealing various degrees of inhibition in rat liver microsomes (RLM), human liver microsomes (HLM), and recombinant human CYP3A4. However, no significant alterations in pharmacokinetic parameters were observed during co-administration in rats. Mechanism-based inactivation studies revealed that IMP inactivated CYP2D6 in a time-, concentration-, and NADPH-dependent manner, with kinetic parameters of k inact = 0.024 min⁻¹ and K I = 2.814 µM. The competitive CYP2D6 inhibitor quinidine was found to attenuate this inactivation, while GSH or catalase/SOD did not demonstrate a similar effect. This observation suggested that the active intermediate acted within the enzyme’s active site. Subsequent to oxidation with potassium ferricyanide, there was partial recovery of CYP2D6 activity, thereby confirming the quasi-irreversible inactivator property of IMP. These findings suggested that IMP may exhibit clinically significant interactions with drugs metabolized by CYP2D6.