Bull, 23, 1528C1531 (2000). direction. RTV had a significant inhibitory effect on the efflux transport of DRV in Caco-2 Budesonide 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 SOS2 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 is a transport substrate of P-glycoprotein. The purpose of this study was to Budesonide investigate the involvement of P-glycoprotein in transcellular transport of DRV across monolayers of the human intestinal epithelial Caco-2 cell line and the renal epithelial LLC-PK1 cell line stably transfected with the ABCB1 gene. MATERIALS AND METHODS Chemicals Saquinavir (SQV) was obtained form Nippon Roche Co. (Tokyo Japan). Nelfinavir (NFV) and ritonavir (RTV) were a gift from JT Co. (Tokyo Japan) and Abbott Laboratories Co. (Illinois, U.S.A.), respectively. Darunavir (DRV) was synthesized in a convergent manner as previously described by Ghosh for 15 min were used for the HPLC assay. The remaining cells were lysed with 1 ml of 1 1 N NaOH, and used for the protein assay using a Bio-Rad protein assay kit (Bio-Rad Laboratories; Richmond, CA, U.S.A.) with bovine -globulin as a standard. Calcein-AM Efflux Assay Efflux assays were performed as described previously.19) A kinetic fluorometric assay was used to study the interaction of DRV with P-glycoprotein. For the calcein-AM efflux assay, L-MDR1 and LLC-PK1 cells were seeded in 96-well tissue culture plates at a cell density of 1105 cells/well. Cells were cultured in 200 P-glycoprotein in L-MDR1 cells. Open in a separate window Fig. 3. Transepithelial Transport and Intracellular Accumulation of DRV in LLC-PK1 Cells and L-MDR1 Cells Transport of DRV (10 reported that P-glycoprotein expression levels in lymphocytes of patients coadministered with RTV and SQV were negatively correlated with the cellular accumulation of these PIs.21) It was also suggested that P-glycoprotein expressed in the blood-brain barrier and blood-placenta barrier participate in the restricted distribution of indinavir, SQV, NFV or amprenavir into the brain and placenta, respectively.28,29) Therefore, P-glycoprotein is thought to play a key role in the pharmacokinetics and therapeutic efficacy of most PIs. It was reported that DRV had a weak inhibitory effect (IC50 100 reported that indinavir, SQV and RTV were transported by MRP2, but not by MRP1 and MRP3.34) Moreover, Williams reported that SQV could be a substrate for P-glycoprotein, MRP1 and MRP2 with the transportability of P-glycoprotein MRP2 MRP1.35) Therefore, our results suggest Budesonide that MRP2 could mediate, at least in part, the apical efflux of DRV in Caco-2 cells. Novobiocin, Budesonide a typical BCRP inhibitor,24) stimulated the basal-to-apical transport of DRV in Caco-2 cells. This finding suggests that BCRP is not involved in the transcellular transport of DRV, because BCRP is localized in the apical membranes of Caco-2 cells where it mediates efflux of substrates. However, we cannot exclude the possibility that unidentified novobiocin-sensitive transporter(s) expressed in the basolateral membranes of Caco-2 cells may also mediate the efflux transport of DRV. The OATP non-specific inhibitors, rifamycin SV and BSP,32,33) showed weak but significant stimulatory effects on the apical-to-basal transport. Interestingly, this represents the opposite net direction of transport for DRV, suggesting a partial involvement of OATP-A and/or OATP-B in transcellular transport of this drug in Caco-2 cells. In the inhibition studies with glycylleucine and glycylsarcosine, we found no contribution of PEPT1 to apical DRV transport in Caco-2 cells. These findings suggest that P-glycoprotein mediates predominantly efflux transport of DRV in the apical membranes of Caco-2 cells, but other apical membrane-localized efflux transporters MRP2 and/or BCRP, and also OATP might be involved, at least in part, in transcellular transport of DRV. The present work using L-MDR1 cells provides the first direct evidence that DRV is definitely a transport.

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