Value of quantifying ABC transporters by mass spectrometry and impact on in vitro-to-in vivo prediction of transporter-mediated drug-drug interactions of rivaroxaban.

ABC transporters, such as P-gp and BCRP, are involved in rivaroxaban pharmacokinetics and can lead to drug-drug interactions (DDIs). Investigations of the victim role for rivaroxaban and transporter-mediated DDI are commonly performed using in vitro models. However, interpretation of rivaroxaban efflux transport and DDI studies in cell models may be influenced by P-gp and BCRP transporter abundance. This study aimed to develop an LC-MS/MS quantification method for assessing the relationship between transporter expression and functionality in Caco-2, Caco-2, MDCK-MDR1, MDCK-BCRP cell models. First, the relative and absolute quantities of the transporters were determined by LC-MS/MS. P-gp and BCRP expression was then confirmed by western blotting and immunofluorescence staining. Finally, P-gp and BCRP functional activities and half-inhibitory concentrations (IC50s) of two specific inhibitors (verapamil and ko143) were determined by bidirectional transport experiments. P-gp and BCRP protein expression was detected at the cell membrane and was greater in the respective transfected models. Efflux ratios were correlated with P-gp and BCRP quantities. The lowest IC50s were obtained in the MDCK-MDR1 and MDCK-BCRP models for verapamil and ko143, respectively. In conclusion, this study demonstrated that LC-MS/MS can accurately quantify P-gp and BCRP efflux transporters and thereby improve the interpretation of transport data and in vitro-in vivo correlations.