Bridging In Vitro and Clinical Data: Experimental Insights into the IC50 Variability of Dolutegravir as an Organic Cation Transporter 2 Inhibitor

Dolutegravir is a clinically confirmed inhibitor of organic cation transporter 2 (OCT2)/SLC22A2; however, its in vitro IC50 varies widely across studies. Using OCT2-expressing human embryonic kidney 293 cells, we investigated experimental conditions affecting IC50 determination. Based on clinical drug-drug interaction (DDI) data, an inhibition constant (Ki) of 0.0890 μM was estimated through pharmacokinetic (PK) modeling using both plasma concentration and urinary excretion profiles of metformin. In vitro, dolutegravir was found to be a noncompetitive inhibitor of OCT2. IC50 values increased with longer uptake times. A 2.9-fold difference was already observed between 1-minute and 5-minute uptake, and further extension to 30 minutes resulted in a 27-fold increase, attributable to the inhibition of both uptake and efflux processes. Preincubation of the cells with dolutegravir for 30 minutes additionally enhanced its inhibitory effect, resulting in a 5.8-fold decrease in IC50. Similar uptake time dependency was observed with other inhibitors (cimetidine and pyrimethamine). However, the extent of variability differed among metformin, N1-methylnicotinamide, atenolol, frovatriptan, and sumatriptan, likely reflecting differences in cellular kinetics. The lowest in vitro IC50 of dolutegravir for metformin uptake (0.126 μM), obtained under a 1-minute uptake and 30-minute preincubation condition, closely matched the estimated in vivo Ki. DDI predictions using in vitro IC50 values suggested repeated dolutegravir administration could raise the area under the curve of OCT substrates, such as atenolol, nadolol, and sulpiride beyond regulatory thresholds. In conclusion, we identified optimized in vitro conditions that yield IC50 values consistent with in vivo Ki estimates, offering valuable guidance for predicting OCT2-mediated DDIs. SIGNIFICANCE STATEMENT: This study identified experimental conditions affecting the IC50 variability of dolutegravir as an organic cation transporter 2 inhibitor. These findings offer valuable insights for improving in vitro to in vivo drug-drug interaction predictions and optimizing inhibition experiments for transporter-mediated drug-drug interactions.