LAUSR

ABSTRACT The generation of reliable kinetic parameters to describe P‐glycoprotein (P‐gp) activity is essential for predicting the impact of efflux transport on gastrointestinal drug absorption. The compound‐specific selection of in vitro assay designs and ensuing data analysis methods is explored in this manuscript. We measured transcellular permeability and cellular uptake of five P‐gp substrates in Caco‐2 and LLC‐PK1 MDR1 cells. Kinetic parameters of P‐gp‐mediated efflux transport (Km, Vmax) were derived from conventional and mechanistic compartmental models. The estimated apparent Km values based on medium concentrations in the conventional permeability model indicated significant differences between the cell lines. The respective intrinsic Km values based on unbound intracellular concentrations in the mechanistic compartmental models were significantly lower and comparable between cell lines and assay formats. Non‐specific binding or lysosomal trapping were shown to cause discrepancies in the kinetic parameters obtained from different assay formats. A guidance for the selection of in vitro assays and kinetic assessment methods is proposed in line with the Biopharmaceutics Drug Disposition Classification System (BDDCS). The recommendations are expected to aid the acquisition of robust and reproducible kinetic parameters of P‐gp‐mediated efflux transport.