Derivation of a System-Independent K_i 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 (k_rQ), digoxin basolateral uptake clearance (k_B), and inhibitor binding affinity to the digoxin basolateral uptake transporter (K_QB). 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 k_rQ 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 microM ouabain, suggesting involvement of OATP4C1.