Abstract

PKI166, a specific inhibitor of the tyrosine kinase activity of two epidermal growth factor receptors, was under development for the treatment of cancer. In preclinical studies PKI166 was mainly cleared by metabolism, and its metabolites were eliminated by biliary excretion, emphasizing the role of liver transport processes for its disposition. Here the transport properties of [¹⁴C]PKI166 and its main metabolite [¹⁴C]ACU154, an O-glucuronide, were analyzed using 1) Madin-Darby canine kidney II (MDCKII) cells stably transfected with human multidrug resistance-associated protein 2 (MRP2) and/or human organic anion-transporting peptide 2 (OATP2) and 2) liver canalicular membrane vesicles (CMVs) prepared from Wistar and mrp2-deficient TR^- rats. Analysis of transport through MDCKII cells revealed that [¹⁴C]ACU154 was a substrate of MRP2 and OATP2. Rat mrp2 was shown to transport [¹⁴C]ACU154 with a K_m of approximately 1 μM. [¹⁴C]PKI166 efficiently crossed MDCKII cells, particularly toward the apical side, but expression of MRP2 and/or OATP2 did not increase the flux. The effect of PKI166 and ACU154 on transport of [³H]estradiol-17β-d-glucuronide (EG; via mrp2/MRP2 and OATP2) or [³H]taurocholic acid (TCA; via bile salt export pump (bsep) was analyzed. PKI166 inhibited the transport of [³H]EG by OATP2. ACU154 did strongly inhibit [³H]TCA uptake into CMVs from Wistar but not from TR^- rats, demonstrating a dependence of bsep inhibition on mrp2 activity. ATP-dependent uptake of [³H]EG into CMVs from Wistar rats was inhibited by ACU154 but up to 4-fold increased by PKI166. In conclusion, OATP2 and MRP2/mrp2 were identified as transporters involved in ACU154 transport into bile. Both PKI166 and its O-glucuronide ACU154 affected mrp2/MRP2-, OATP2-, and/or bsep-mediated transport processes.