Selection of an Optimal In Vitro Model to Assess P-gp Inhibition: Comparison of Vesicular and Bidirectional Transcellular Transport Inhibition Assays

The multidrug resistance protein 1 (MDR1) P-glycoprotein (P-gp) is a clinically important transporter. In vitro P-gp inhibition assays have been routinely conducted to predict the potential for clinical drug-drug interactions (DDIs) mediated by P-gp. However, high interlaboratory and intersystem variability of P-gp IC₅₀ data limits accurate prediction of DDIs using static models and decision criteria recommended by regulatory agencies. In this study, we calibrated two in vitro P-gp inhibition models: vesicular uptake of N-methyl-quinidine (NMQ) in MDR1 vesicles and bidirectional transport (BDT) of digoxin in Lilly Laboratories Cell Porcine Kidney 1 cells overexpressing MDR1 (LLC-MDR1) using a total of 48 P-gp inhibitor and noninhibitor drugs and digoxin DDI data from 70 clinical studies. Refined thresholds were derived using receiver operating characteristic analysis, and their predictive performance was compared with the decision frameworks proposed by regulatory agencies and selected reference. Furthermore, the impact of various IC₅₀ calculation methods and nonspecific binding of drugs on DDI prediction was evaluated. Our studies suggest that the concentration of inhibitor based on highest approved dose dissolved in 250 ml divided by IC₅₀(I₂/IC₅₀) is sufficient to predict P-gp related intestinal DDIs. IC₅₀ obtained from vesicular inhibition assay with a refined threshold of I₂/IC₅₀ ≥ 25.9 provides comparable predictive power over those measured by net secretory flux and efflux ratio in LLC-MDR1 cells. We therefore recommend vesicular P-gp inhibition as our preferred method given its simplicity, lower variability, higher assay throughput, and more direct estimation of in vitro kinetic parameters, rather than BDT assay. SIGNIFICANCE STATEMENT: This study has conducted comprehensive calibration of two in vitro P-gp inhibition models: uptake in MDR1 vesicles and bidirectional transport in LLC-MDR1 cell monolayers to predict DDIs. This study suggests that IC₅₀s obtained from vesicular inhibition with a refined threshold of I₂/IC₅₀ ≥ 25.9 provide comparable predictive power over those in LLC-MDR1 cells. Therefore, vesicular P-gp inhibition is recommended as the preferred method given its simplicity, lower variability, higher assay throughput, and more direct estimation of in vitro kinetic parameters.