Sodium-taurocholate cotransporting polypeptide (NTCP) is the major bile acid uptake system in human hepatocytes. Previous studies showed NTCP transported rosuvastatin and its variants demonstrated significant different transport capacities. In addition, about 35% of rosuvastatin hepatic uptake is sodium dependent, thus implying that NTCP may play a critical role in rosuvastatin disposition in vivo. As a result, inhibition of NTCP-mediated drug transport may reduce liver uptake and/or efficacy of rosuvastatin or other drugs. The objective of this study was to establish a rapid in vitro inhibition assay using flow cytometry for screening NTCP-mediated drug interactions in stably transfected HEK293.
HEK293 cells, stably transfected with NTCP or the control vector, were cultured as monolayers, trypsinized, and then assayed in suspension. Chenodeoxychilyl-(Nε-NBD)-lysine (CDCA-NBD) was used as a probe substrate. Cellular uptake of CDCa-NBD in transfected and control cells was determined by flow cytometry.
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 NTCP was 0.93, indicating the assay is suitable for measuring inhibition of NTCP-mediated transport. The application of this assay for screening drug interactions with NTCP was illustrated by investigating some well-known NTCP substrates and/or inhibitors including bosentan, BSP, CsA, CDCA, glibenclamide, rifampicin, rifamycin SV, taurocholic acid, and three statins, atorvastatin, rosuvastatin and pravastatin. CDCA-NBD uptake assays conducted in the presence of well-known compounds accurately reflected their inhibitory potency against NTCP. Compounds known to interact with NTCP inhibited NTCP-mediated CDCA-NBD cellular uptake relative to control cells. Among the three statins, atovastatin at 400 μM showed 75%, inhibition followed by rosuvastatin with 20% inhibition while pravastatin had no effect on CDCA-NBD uptake. Based on the initial screen, concentration-dependent inhibition was investigated to determine the IC50 values for selected compounds. The IC50 values were 49.4 μM for CDCA, 131.5 μM for rifampicin, 258.0 μM for taurocholic acid, 639.3 μM for E3S and 763.7 μM for bosentan.
The in vitro assays using flow cytometry demonstrated utility for identifying potential drug-drug interactions associated with human NTCP.