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To determine the effect of the protease inhibitors ritonavir, nelfinavir and indinavir on the P-glycoprotein (P-gp)-mediated transport of saquinavir in Caco-2 cell monolayers. To study the modulation of P-gp function in human lymphocytes by saquinavir, ritonavir, nelfinavir and indinavir.We examined the effect of the protease inhibitors on P-gp function in human lymphocytes by using Rhodamine123 (Rh123; a fluorescent substrate of P-gp) by flow cytometry. Efflux of Rh123 correlates with P-gp function and inhibition of P-gp results in dye retention. Verapamil, a P-gp modulator and inhibitor of active transport at 4°C was used as a positive control. The transport of [14C]saquinavir (1μM) across Caco-2 cell monolayers was investigated, alone and in the presence of verapamil and ketoconazole (500μM) and the protease inhibitors at 100μM. Caco-2 cells are an in vitro model of the intestinal epithelium that is widely used for the study of P-gp function. The transport of saquinavir was determined in both the apical to basolateral (AP-BL) and basolateral to apical (BL-AP) directions.Saquinavir and ritonavir (10μM) markedly inhibited Rh123 efflux with an increase in fluorescence intensity similar to that obtained with verapamil. A small but statistically significant increase in fluorescence intensity was observed with nelfinavir; however indinavir did not modulate Rh123 efflux. In Caco-2 cells the apparent permeability coefficient for BL-AP efflux of saquinavir exceeded that for AP-BL efflux by a factor of 26: this is indicative of an active efflux pump. Known P-gp modulators caused a decrease in BL-AP efflux and an increase in AP-BL transport. The protease inhibitors displayed some P-gp modulation with ritonavir having the most potent effect.We have demonstrated that saquinavir is a substrate for P-gp and that ritonavir, nelfinavir and indinavir modulate P-gp function in both human lymphocytes and Caco-2 cells.