P-glycoprotein mediates efflux transport of darunavir in human intestinal Caco-2 and ABCB1 gene-transfected renal LLC-PK1 cell lines

Abstract

Darunavir (DRV) is a nonpeptidic protease inhibitor (PI) approved for the treatment of human immunodeficiency virus (HIV) infection. DRV displays potent activity against HIV strains resistant to other available PIs. Coadministration with ritonavir (RTV) improves the oral bioavailability of DRV. Inhibition of cytochrome P450 by RTV has been proposed as a mechanism for enhanced DRV bioavailability. However, interaction of these drugs with intestinal transporters has not been elucidated. This study was performed to explore the involvement of P-glycoprotein in transcellular DRV transport in monolayers of human intestinal Caco-2 and in ABCB1 multidrug resistance 1, (MDR1) gene-transfected renal LLC-PK1 (L-MDR1) cell lines. Transepithelial transport of DRV in Caco-2 cell monolayers was 2-fold greater in the basal-to-apical direction compared to that in the opposite direction. RTV had a significant inhibitory effect on the efflux transport of DRV in Caco-2 cells. The apical-to-basal DRV transport was enhanced by P-glycoprotein inhibitors, cyclosporin A and verapamil, as well as multidrug resistance-related protein (MRP/ABCC) inhibitors, probenecid and MK571. Using the L-MDR1 cell line, basal-to-apical DRV transport was much greater than in the opposite direction. Furthermore, cyclosporin A markedly inhibited the basal-to-apical DRV transport. RTV significantly increased the apical-to-basal transport of DRV in L-MDR1 cells, but reduced transport in the opposite direction. DRV inhibited P-glycoprotein-mediated efflux of calcein-acetoxymethyl ester in L-MDR1 cells with the inhibitory potency of 121 microM. These findings suggest that DRV is a substrate of P-glycoprotein and MRP, most likely MRP2. RTV appeared to inhibit P-glycoprotein, thereby enhancing the absorptive transport of DRV.

Fig. 1.

Effect of RTV on the Transepithelial Transport of DRV in Monolayers of Caco-2 Cells Transport of DRV (10 µM) in the apical-to-basal direction (○, ●) and in the basal-to-apical direction (△, ▲) in the absence (control, open symbol) or presence (closed symbol) of RTV (20 µM). Each point represents the mean ± S.D. of three independent measurements. **p<0.01, significantly different from apical-to-basal transport without RTV. ^†† p<0.01, significantly different from basal-to-apical transport without RTV.

Fig. 2.

Effect of Various Inhibitors on Transepithelial Transport of DRV in Monolayers of Caco-2 Cells 6 h after Incubation with DRV Apical-to-basal transport (A), basal-to-apical transport (B) and the transport ratio of basal-to-apical transport divided by apical-to-basal transport (C). Each bar represents the mean±S.D. of three independent measurements. *p<0.05, **p<0.01; significantly different from control (DRV alone).

Fig. 3.

Transepithelial Transport and Intracellular Accumulation of DRV in LLC-PK1 Cells and L-MDR1 Cells Transport of DRV (10 µM) in the apical-to-basal direction (○, ●) and in the basal-to-apical direction (△, ▲) in the absence (control, open symbol) or presence (closed symbol) of cyclosporin A (10 µM) in LLC-PK1 (A) or L-MDR1 (B) cells. Each point represents the mean±S.D. of three independent measurements. **p<0.01, *p<0.05, significantly different from apical-to-basal transport without cyclosporin A. ^†† p<0.01, significantly different from basal-to-apical transport without cyclosporin A. Intracellular accumulation of DRV in the absence (control, open column) and presence of cyclosporin A (closed column) 6 h after addition of DRV (10 µM) to the apical side (C) and basal side (D) of the monolayer. Each point represents the mean±S.D. of three independent measurements. **p<0.01, significantly different from DRV accumulation in LLC-PK1 cells without cyclosporine *p<0.05, significantly different from DRV accumulation in L-MDR1 cells without cyclosporin A.

Fig. 4.

Effect of PIs on the Transepithelial Transport of DRV in L-MDR1 Cells 6 h after Incubation with DRV Transport of DRV (10 µM) in the apical-to-basal direction (A) or basal-to-apical direction (B). The transport ratio (basal-to-apical/apical-to-basal) is given in (C). Each point represents the mean±S.D. of three independent measurements. **p<0.01, *p<0.05, significantly different from control (DRV alone).

Fig. 5.

The Effect of DRV on Intracellular Uptake of Calcein-AM in LLC-PK1 and L-MDR1 Cells Monolayers LLC-PK1 cells without DRV (○), L-MDR1 cells without DRV (×), L-MDR1 cells with DRV at concentrations of 10 µM (▲), 25 µM (■), 50 µM (◆) and 100 µM (●). Each point represents the mean±S.D. of five independent measurements.