Metabolism-Dependent Inhibition of CYP3A4 by Lapatinib: Evidence for Formation of a Metabolic Intermediate Complex with a Nitroso/Oxime Metabolite Formed via a Nitrone Intermediate

Abstract

Metabolism-dependent inhibition (MDI) of cytochrome P450 (P450) enzymes has the potential to cause clinically relevant drug-drug interactions. In the case of several alkylamine drugs, MDI of P450 involves formation of a metabolite that binds quasi-irreversibly to the ferrous heme iron to form a metabolic intermediate (MI) complex. The specific metabolites coordinately bound to ferrous iron and the pathways leading to MI complex formation are the subject of debate. We describe an approach combining heme iron oxidation with potassium ferricyanide and metabolite profiling to probe the mechanism of MI complex-based CYP3A4 inactivation by the secondary alkylamine drug lapatinib. Ten metabolites formed from lapatinib by CYP3A4-mediated heteroatom dealkylation, Chydroxylation, N-oxygenation with or without further oxidation, or a combination thereof, were detected by accurate mass spectrometry. The abundance of one metabolite, the N-dealkylated nitroso/ oxime lapatinib metabolite (M9), correlated directly with the prevalence or the disruption of the MI complex with CYP3A4. Nitroso/ oxime metabolite formation from secondary alkylamines has been proposed to occur through two possible pathways: (1) sequential N-dealkylation, N-hydroxylation, and dehydrogenation (primary hydroxylamine pathway) or (2) N-hydroxylation with dehydrogenation to yield a nitrone followed by N-dealkylation (secondary hydroxylamine pathway). All intermediates for the secondary hydroxylamine pathway were detected but the primary N-hydroxylamine intermediate of the primary hydroxylamine pathway was not. Our findings support the mechanism of lapatinib CYP3A4 inactivation as MI complex formation with the nitroso metabolite formed through the secondary hydroxylamine and nitrone pathway, rather than by N-dealkylation to the primary amine followed by N-hydroxylation and dehydrogenation as is usually assumed.

Cytochrome P450 3A4 and CYP3A5-Catalyzed Bioactivation of Lapatinib

“…An increase in lapatinib serum concentrations and t 1/2 over time was reported with multiple dosing, suggesting time-dependent autoinhibition of lapatinib clearance (Burris et al, 2005). Previous in silico docking and metabolic profiling studies suggested that lapatinib N-dealkylation and N-hydroxylation are predominately catalyzed by CYP3A4, whereas CYP3A5 may preferentially catalyze the O-dealkylation pathway (Takakusa et al, 2011;Chan et al, 2012;Barbara et al, 2013). The results of the present study are consistent with these findings.…”

“…Similar to previous reports, N-dealkylated and N-hydroxylated metabolites were higher by CYP3A4 compared with CYP3A5. The marked decline in the levels of N-OH-LAP during longer incubation times with CYP3A4 may be because N-OH-LAP can be further oxidized to a nitroso intermediate (Takakusa et al, 2011;Barbara et al, 2013). On the other hand, formation of LAP-OH was higher in incubations with CYP3A5 compared with CYP3A4 during longer incubations.…”

“…One pathway is via N-hydroxylation and N-dealkylation of the secondary amine to form a reactive nitroso intermediate (Takakusa et al, 2011). Previous reports indicate that the nitroso intermediate forms a metabolic intermediate complex with the P450 heme iron resulting in quasiirreversible inactivation of CYP3A4 (Takakusa et al, 2011;Barbara et al, 2013). The second bioactivation pathway is via lapatinib O-dealkylation and further oxidation to form of a reactive quinoneimine (Teng et al, 2010).…”

“…Lapatinib undergoes extensive metabolism by cytochromes P450 (P450) CYP3A4 and CYP3A5 and, to a lesser extent, 2C8, through three major routes: O-dealkylation, N-dealkylation, and N-hydroxylation ( Fig. 1) (Takakusa et al, 2011;Castellino et al, 2012) plus some C-hydroxylation (Barbara et al, 2013). The O-dealkylated metabolite can be further oxidized to a reactive quinoneimine (Teng et al, 2010), which may covalently modify cellular proteins, potentially leading to cell stress and/or immune activation (Park et al, 2005).…”

Stereoselective Inhibition of CYP2C19 and CYP3A4 by Fluoxetine and Its Metabolite: Implications for Risk Assessment of Multiple Time-Dependent Inhibitor Systems

“…Hence, it is unclear whether the concentration of the parent TDI can be used as a surrogate for the concentration of the metabolite that ultimately complexes with the P450 heme. In addition, MIC formation from alkylamine TDIs may arise from two competing pathways: initial N-dealkylation or N-hydroxylation (Hanson et al, 2010;Barbara et al, 2013). Thus, it is not clear whether inclusion of The unbound fractions (f u ) for the enantiomers of fluoxetine and norfluoxetine in pooled HLMs at 0.1 mg/ml and 1.0 mg/ml microsomal protein and in plasma TABLE 3 Stereospecific risk assessment of the inhibition of CYP2C19 and CYP3A4 after fluoxetine administration…”