Importance of Probe Substrate Selection in Assessing Interactions of Tyrosine Kinase Inhibitors with Polyspecific Organic Cation Transporters.

The organic cation transporters 1-3 (hOCT1-3) and multidrug and toxin extrusion proteins 1 and 2-K (hMATE1/2-K) are important polyspecific transporters involved in the disposition of many clinically used therapeutics. Evaluation of transporter-mediated drug-drug interactions (DDIs) is a critical step during drug development. Tyrosine kinase inhibitors (TKIs) are a rapidly growing class of targeted anticancer agents designed to target specific tyrosine kinases that are fused, mutated, or overexpressed in cancer. Previously, using known inhibitors, we and others have shown that the inhibition of hOCT2 and hMATEs is substrate-dependent, with hOCT2 exhibiting greater substrate-dependence than hMATEs. The goal of this study is to characterize the interactions of TKIs with polyspecific organic cation transporters and determine if TKI inhibition of hOCT1-3 and hMATE1/2-K is substrate- and isoform-dependent. Using HEK293 cell lines stably transfected with hOCT1-3 and hMATE1/2-K, we determined the inhibitory potency of selected TKIs (erlotinib, lorlatinib, crizotinib, and brigatinib) using three different substrates (meta-iodobenzylguanidine (mIBG), metformin, and 1-methyl-4-phenylpyridinium (MPP+)). Among the tested TKIs, brigatinib potently inhibited mIBG, metformin and MPP+ uptake by hOCT3 and hMATE1, and metformin uptake by hOCT1, with their half maximal inhibitory concentration (IC ) being in the low micromolar range. Crizotinib also potently inhibited metformin and MPP+ uptake by hOCT3 and hMATE1 and metformin uptake by hOCT1, hOCT2 and hMATE2-K. Except for hMATE1, substantial substrate-dependent inhibition of transporters was observed for both crizotinib and brigatinib, with metformin being the most sensitive to inhibition among the three tested substrates. Brigatinib was approximately 23-, 18-, 4-, and 11-fold more potent towards hOCT1, hOCT2, hOCT3 and hMATE2-K, respectively, when metformin is used as the substrate instead of MPP+, while crizotinib was approximately 17-, 16-, 5-, and 12- fold more potent, respectively, when metformin is used. Interestingly and consistent with our previous observation, hMATE1 inhibition by both TKIs is influenced minimally by the choice of substrate, with similar IC values of less than 1.5-fold differences across substrates. Together, our results demonstrated that TKI inhibition of hOCTs and hMATE2-K is substrate-dependent, which underscore the importance of selecting clinically relevant probe substrates when assessing the DDI potential for these transporters using in vitro systems.