Quantifying Transporter Activity: An Absolute Scaling Approach Using Digoxin

Area of Science:

• Pharmacokinetics and Drug Metabolism
• Cellular Transport Mechanisms
• Biophysical Chemistry

Background:

• Commonly used cell lines like LLC-PK1 and MDCK are vital for studying drug transport and P-glycoprotein (P-gp) activity.
• Significant variability in experimental outcomes, particularly P-gp activity, hinders drug development and transporter research.
• Conventional methods for assessing P-gp activity show interlaboratory variability, risking underestimation of transporter impact in models.

Purpose of the Study:

• To quantify P-gp protein expression levels across eight different cell lines used in various laboratories.
• To evaluate and compare conventional analysis methods with an in vitro modeling approach for estimating P-gp activity.
• To develop a physiologically based pharmacokinetic (PBPK) model for digoxin incorporating scaled P-gp activity.

Main Methods:

• Quantification of P-gp protein expression levels in eight distinct cell lines.
• Evaluation of digoxin transfer in a Transwell assay using conventional and modeling-based approaches for P-gp activity estimation.
• Development of a PBPK model for digoxin, scaling P-gp activity based on measured expression levels in key organs.

Main Results:

• Modeling-based P-gp activity estimates strongly correlated with absolute transporter abundance across cell lines.
• Conventional methods yielded parameters (Papp, Km,app, Jmax) with notable interlaboratory variability.
• The developed PBPK model accurately incorporated digoxin's P-gp-mediated transport, scaled by expression.

Conclusions:

• Quantifying transporter expression in vitro is crucial for accurate drug transport assessment.
• An expression-function relationship allows reliable scaling of P-gp activity across tissues.
• Integrating transporter expression data into modeling frameworks enables robust in vitro-to-in vivo extrapolation (IVIVE).