The effect of shikonin on the metabolism of lapatinib in vitro, and in vivo.

ABSTRACT

PURPOSE: The purpose of this study was to develop an assay for simultaneous determination of lapatinib and its metabolites (N-dealkylated lapatinib and O-dealkylated lapatinib) by ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), and to determine the interaction between shikonin and lapatinib in vitro, in vivo, in silico and its mechanism of action. METHODS: A new UPLC-MS/MS method for the determination of the concentrations of lapatinib and its metabolites was developed. In vivo, Sprague-Dawley (SD) rats were given lapatinib with or without shikonin. In vitro, to study the interaction mechanism, rat liver microsomes (RLMs), human liver microsomes (HLMs) and recombinant human CYP3A4.1 were used for determining enzyme kinetics. Lastly, we used in silico molecular docking to investigate the molecular mechanism of inhibition. RESULTS: The selectivity, precision, accuracy, stability, matrix effect and recovery of UPLC-MS/MS all met the requirements of quantitative analysis of biological samples. Administration of lapatinib combined with shikonin resulted in significantly increased pharmacokinetic parameters (AUC(0-t) and Cmax) of lapatinib, indicating that shikonin increased the exposure of lapatinib in rats. Moreover, in vitro kinetic measurements indicated that shikonin was a time-independent inhibitor, which inhibited the metabolism of lapatinib through a competitive mechanism in RLMs, while noncompetitive inhibition type in both HLMs and CYP3A4.1. Molecular docking analysis further verified the non-competitive inhibition of shikonin on lapatinib in CYP3A4.1. CONCLUSION: We developed an UPLC-MS/MS assay for simultaneous determination of lapatinib and its metabolites. It could be successfully applied to the study of pharmacokinetic interaction of shikonin on the inhibition of lapatinib metabolism in vivo and in vitro. In the end, further studies are needed to determine if such interactions are indeed valid in humans and if the interaction is clinically relevant.