P347 Rapid Screening of Human OATP1B1 and OATP1B3 Mediated Drug Interactions in Stably Transfected Human Embryonic Kidney HEK-293 Cell Lines Using Flow Cytometry and Fluorescence Microplate Methods

Steve Wright , Department of Biology, Carson-Newman College, Jefferson City, TN
Ying Wang , Absorption Systems LP, Exton, PA
Jibin Li , Absorption Systems LP, Exton, PA
Albert Owen , Absorption Systems LP, Exton, PA
Ismael Hidalgo , Absorption Systems LP, Exton, PA

Human organic anion transporting polypeptides (OATP) 1B1 and 1B3 are predominantly expressed at the sinusoidal membrane of hepatocytes and play an important role in the hepatic uptake of numerous drugs including statins. Inhibition of OATP1B1/1B3-mediated drug transport may reduce statin liver uptake and/or efficacy, and elevate systemic exposure. Therefore, it is imperative to have reliable assay systems for identifying potential OATP1B1- and OATP1B3-mediated adverse drug reactions. The objective of this study was to establish rapid in vitro inhibition assays for screening OATP1B1- and OATP1B3-mediated drug interactions in stably transfected HEK293 cells using either flow cytometry or a fluorescence microplate reader.  


For flow cytometry, HEK293 cells, stably transfected with OATP1B1 or OATP1B3 or the control vector, were cultured as monolayers, trypsinized, and then assayed in suspension; for the fluorescence microplate reader, cells were cultured in 96-well plates. Fluorescein-methotrexate (FMTX) was used as a probe substrate. Cellular uptake of FMTX in transfected and control cells was determined by flow cytometry or the fluorescence microplate reader.


The suitability of the assays was determined by Z’ factor, a simple statistical parameter for evaluating high throughput screening assays. The calculated Z’ factors for the OATP1B1 and OATP1B3 assays were 0.83 and 0.89, respectively, by flow cytometry, and 0.76 and 0.75, respectively, by the fluorescence microplate reader method. Those Z’ factor values indicate that both methods are suitable for measuring inhibition of OATP1B1- and OATP1B3-mediated transport. The application of these two methods for screening drug interactions with OATP1B1 and OATP1B3 transporters was illustrated by investigating some well-known OATP substrates and/or inhibitors including statins, E3S, and digoxin. FMTX uptake assays using the fluorescence microplate reader conducted in the presence of those compounds accurately reflected their inhibitory potency against OATP1B1 and OATP1B3. Compounds known to interact with OATPs inhibited OATP-mediated FMTX cellular uptake relative to control cells and displayed concentration-dependent inhibition. IC50 values were determined for those individual compounds. E3S and pitavastatin had lower IC50 values in OATP1B1 cells than OAPT1B3s, indicating higher inhibition potency towards OATP1B1. For example, the IC50 value of E3S was 0.08 µM in OATP1B1 but 25.95 µM in OATP1B3 cells. Such results are consistent with previous findings that E3S and pitavastatin are more OATP1B1 specific substrates. 


The in vitro assays using either flow cytometry or fluorescence microplate methods demonstrated utility for identifying potential drug-drug interactions associated with human OATP1B1 and OATP1B3 transporters.