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-2ATCC, Caco-2ECACC, 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.

中文翻译：

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质谱定量ABC转运蛋白的价值及其对利伐沙班转运蛋白介导的药物相互作用的体内到体外预测的影响。

ABC转运蛋白（例如P-gp和BCRP）参与利伐沙班的药代动力学，并可能导致药物相互作用。通常使用体外模型对利伐沙班和转运蛋白介导的DDI的受害者作用进行调查。但是，在细胞模型中利伐沙班外排转运和DDI研究的解释可能受P-gp和BCRP转运蛋白丰度的影响。这项研究旨在开发一种LC-MS / MS定量方法，用于评估Caco-2ATCC，Caco-2ECACC，MDCK-MDR1，MDCK-BCRP细胞模型中转运蛋白表达与功能之间的关系。首先，通过LC-MS / MS确定转运蛋白的相对和绝对量。然后通过蛋白质印迹和免疫荧光染色确认P-gp和BCRP表达。最后，通过双向转运实验确定了两种特异性抑制剂（维拉帕米和ko143）的P-gp和BCRP功能活性以及半抑制浓度（IC50）。在细胞膜上检测到P-gp和BCRP蛋白表达，并且在各自转染的模型中更高。外排率与P-gp和BCRP量相关。在维拉帕米和ko143的MDCK-MDR1和MDCK-BCRP模型中，IC50最低。总之，这项研究表明，LC-MS / MS可以准确定量P-gp和BCRP外排转运蛋白，从而改善转运数据的解释和体内外相关性。在细胞膜上检测到P-gp和BCRP蛋白表达，并且在各自转染的模型中更高。外排率与P-gp和BCRP量相关。在维拉帕米和ko143的MDCK-MDR1和MDCK-BCRP模型中，IC50最低。总之，这项研究表明，LC-MS / MS可以准确定量P-gp和BCRP外排转运蛋白，从而改善转运数据的解释和体内外相关性。在细胞膜上检测到P-gp和BCRP蛋白表达，并且在各自转染的模型中更高。外排率与P-gp和BCRP量相关。在维拉帕米和ko143的MDCK-MDR1和MDCK-BCRP模型中，IC50最低。总之，这项研究表明，LC-MS / MS可以准确定量P-gp和BCRP外排转运蛋白，从而改善转运数据的解释和体内外相关性。