Quantifying Transporter Activity: An Absolute Scaling Approach Using Digoxin

Lilly Laboratories porcine kidney 1 (LLC-PK₁) and Madin-Darby canine kidney (MDCK) cell lines are commonly used in studying both passive and active drug transport, particularly through permeation assays, and, when transfected with human MDR1, to assess P-glycoprotein (P-gp)-mediated drug clearance. However, the substantial variability in experimental outcomes, including P-gp activity, presents a significant challenge to drug development and transporter research. In this study, P-gp protein expression levels were quantified in 8 cell lines used across different laboratories to evaluate digoxin transfer in a Transwell assay. P-gp activity was estimated using both conventional analysis methods and an in vitro modeling approach. Parameters derived from conventional methods, such as apparent permeability (P_app) and Michaelis-Menten parameters (K_m,app and J_max), exhibited notable interlaboratory variability and could increase the risk of underestimating the impact of transporters and their saturation when used in a physiologically based pharmacokinetic (PBPK) model. By contrast, modeling-based estimates of P-gp activity demonstrated a strong correlation with absolute transporter abundance in the cell lines. This expression-function relationship supports the accurate scaling of the P-gp activity across tissues based on expression levels. A PBPK model for digoxin was developed incorporating P-gp activity in key organs, scaled according to measured expression. These findings underscore the importance of quantifying transporter expression in in vitro systems and integrating this information into a modeling framework to enable reliable in vitro-to-in vivo extrapolation (IVIVE).