Derivation of a System-Independent Ki for P-glycoprotein Mediated Digoxin Transport from System-Dependent IC50 Data.

It has been previously demonstrated that IC50 values for inhibition of digoxin transport across confluent polarized cell monolayers are system-dependent. Digoxin IC50 data from five laboratories participating in the P-glycoprotein (P-gp) IC50 Initiative, using Caco-2, MDCKII-hMDR1 or LLC-PK1-hMDR1 cells, were fitted by the structural mass action kinetic model for P-gp-mediated transport across confluent cell monolayers. We determined their efflux-active P-gp concentration [T(0)], inhibitor elementary dissociation rate constant from P-gp (krQ), digoxin basolateral uptake clearance (kB), and inhibitor binding affinity to the digoxin basolateral uptake transporter (KQB). We also fitted the IC50 data for inhibition of digoxin transport through monolayers of primary human proximal tubule cells (HPTCs). All cell systems kinetically required a basolateral uptake transporter for digoxin, which also bound to all inhibitors. The inhibitor krQ was cell system-independent, thereby allowing calculation of a system-independent Ki. The variability in efflux-active P-gp concentrations and basolateral uptake clearances in the five laboratories was about an order of magnitude. These laboratory-to-laboratory variabilities can explain more than 60% of the IC50 variability found in the principal component analysis plot in a previous study, supporting the hypothesis that the observed IC50 variability is primarily due to differences in expression levels of efflux-active P-gp and the basolateral digoxin uptake transporter. HPTCs had 10- to 100-fold lower efflux-active P-gp concentrations than the overexpressing cell lines, whereas their digoxin basolateral uptake clearances were similar. HPTC basolateral uptake of digoxin was inhibited 50% by 10 μM ouabain, suggesting involvement of OATP4C1.