Characterization of the IPEC-J2 MDR1 (iP-gp) cell line as a tool for identification of P-gp substrates

Transepithelial fluxes of [3H]-labeled digoxin and rhodamine 123 were measured at varying donor concentrations, and kinetic parameters were estimated. Km and Vmax of P-gp mediated basolateral-to-apical (B-A) flux of rhodamine 123 were estimated to 332 ± 124 μM and 111 ± 16 pmol·cm− 2·min− 1 (n = 3, total N = 6), respectively. Vmax and Km of digoxin B-A flux could not be estimated due to the low aqueous solubility of digoxin. The half maximal inhibitory concentrations (IC50) of the selective P-gp inhibitor, zosuquidar (LY-335979), were estimated to 0.05 ± 0.01 μM (n = 3, total N = 6) and 0.04 ± 0.01 μM (n = 3, total N = 6) in transport experiments with digoxin and rhodamine 123 as substrates, respectively. Bidirectional fluxes of digoxin and rhodamine 123 were measured in transfected Madin Darby canine kidney cells (MDCK II MDR1) and compared with the fluxes obtained with the iP-gp cell monolayers. Efflux ratios were highest in the iP-gp cells, due to a tighter paracellular pathway. In conclusion, both digoxin and rhodamine 123 could be used to obtain IC50 values of inhibition, Ki values were only possible to obtain using rhodamine 123. The observed tightness, robustness towards solvents and the high efflux ratios confirmed that the iP-gp cell line may serve as a useful screening tool for investigations of substrate-P-gp interactions and modulation of P-gp function.