Down‐Regulation of OATP1B1‐Mediated Transport by Mammalian Target of Rapamycin (mTOR) Inhibitors Everolimus and Sirolimus: Potential Mechanism and Implication in OATP‐Mediated Drug‐Drug Interactions

Purpose Organic anion transporting polypeptides (OATP) 1B1 is an important determinant of transporter‐mediated drug‐drug interactions (DDIs). The OATP substrates statins are often co‐administered with immunosuppressants including mammalian target of rapamycin (mTOR) inhibitors everolimus (EV) and sirolimus (SIR) to reduce cardiovascular risks in transplant recipients. OATP1B1 has putative phosphorylation sites by the mTOR kinase. The aim of this study is to determine the effects of mTOR inhibitors on OATP1B1‐mediated transport and to elucidate the potential underlying mechanism and implication in OATP‐mediated DDIs. Methods Phosphorylation of OATP1B1 was determined by P32‐orthophosphate labeling. Effects of EV and SIR on accumulation of [ 3 H]E 2 17G, [ 3 H]estrone sulfate and [ 3 H]rosuvastatin were determined in HEK293‐OATP1B1 cells. The effects of EV and SIR on wild‐type (WT) OATP1B1‐ and mTOR kinase‐dead mutant (MT) OATP1B1‐mediated transport were compared. The IC 50 of EV and SIR against OATP1B1 was determined with or without pre‐incubation with EV or SIR. The maximum plasma concentration (C max )/IC 50 and plasma concentration‐time curve (AUC) ratio [AUCR=1+(f u × I in,max /IC 50 )] were calculated based on the FDA draft guidance for OATP‐inhibition tree. The FDA Adverse Events Reporting System (FAERS) was used for pharmacoepidemiological studies to assess whether concurrent usage of EV or SIR with metabolically stable statins, pitavastatin, rosuvastatin and pravastatin, leads to a higher risk for myopathy than statins alone. Results OATP1B1 is a phosphorylated protein. EV (0.2–5 μM) and SIR (0.2–5 μM) pretreatment rapidly down‐regulates OATP1B1‐mediated transport, as early as 10 min after pretreatment and in a concentration‐dependent manner. [ 3 H]E 2 17G accumulation‐mediated by WT‐ and MT‐OATP1B1 was significantly decreased to 0.29 ± 0.02 and 0.48 ± 0.03 fold of control, respectively, by EV pretreatment (1 μM, 1 h), and to 0.35 ± 0.01 and 0.55 ± 0.02 fold of control, respectively, by SIR pretreatment (1 μM, 1 h). For both EV and SIR, the fold change in [ 3 H]E 2 17G accumulation‐mediated by WT‐OATP1B1 was significantly different from that by the MT‐OATP1B1. Pretreatment with EV and SIR decreased the IC 50 against OATP1B1, and increased the C max /IC 50 ‐ and AUCR‐values. Without and with pre‐incubation, the AUCR of EV ranged 1.20–1.38 and 1.43–1.98, respectively, and the AUCR of SIR ranged 1.04–1.08 and 1.07–1.26, respectively. Statins alone have a myopathy risk of 9.2% in patients. Concurrent usage of statins with EV, but not SIR, leads to an increased myopathy risk of 16.42% (relative risk=1.94, p=0.05). Conclusions We report novel findings that OATP1B1 is a phosphorylated protein and that mTOR phosphorylation sites are involved in the down‐regulation of OATP1B1‐mediated transport by EV and SIR. Both in vitro and pharmacoepidemiological studies suggest that EV, but not SIR, has the potential to cause OATP‐mediated clinically significant DDIs against statins. The predicted DDI potential of EV is consistent with a previous publication reporting a ~3 fold increase in the AUC of rosuvastatin when co‐administered with EV. Multidisciplinary approaches were utilized in current studies to assessing OATP‐mediated DDI potential of EV and SIR, which encompass in vitro functional and mechanism study and myopathy DDI risk analysis using the FAERS. The current study highlights the importance of post‐translational regulation of OATP1B1 function by perpetrator drugs. Support or Funding Information Supported by NIH R01 GM094268 and GM094268‐06S1 (WY), DK102694, GM10448301 and R01LM011945 (LL)