The roles of multiple transporters in the intestinal abosrption of saquinavir, a peptidomimetic HIV protease inhibitor

The contributions of human multidrug resistance-associated protein 1 (hMRP1), multidrug resistance-associated protein 2 (hMRP2) and P-glycoprotein (hPgp) to saquinavir transport were investigated using MDCKII cells overexpressing hMRP 1 (MDCKII-MRP1), hMRP2 (MDCKII-MRP2) or hPgp (MDCKII-PGP) in comparison with MDCKII/wild type (MDCKII/wt) cells. Saquinavir transport and cytotoxicity were measured. The effects of GF120918 (specific Pgp inhibitor) and MK-571 (MRP family inhibitor) were determined.; To develop a standard protocol for MDCKII cell studies, the effects of seeding density and substratum on saquinavir transport through, and interactions with MDCKII-PGP cell monolayers were investigated. Saquinavir presence significantly disrupted MDCKII-PGP monolayer integrity. To minimize monolayer disruption, all cell lines were seeded with 5 × 104 cells/cm 2 on collagen-coated polycarbonate membranes. Collagen-coating increased Pgp expression and saquinavir efflux ratios (ratios of BL to AP/AP to BL permeability) correlated with Pgp expression in this rank order: MDCKII-PGP (on collagen-coated polycarbonate) > MDCKII-PGP (on polycarbonate) > MDCKII/wt (on collagen-coated polycarbonate).; For MDCKII-PGP cells, GF120918 reduced saquinavir efflux ratios but did not fully inhibit saquinavir efflux. Saquinavir efflux was fully inhibited, in a concentration dependent manner, by MK-571 in the presence GF120918. The MDCKII-MRP1 (LD₅₀ = 10.5 μM), MDCKII-MRP2 (LD₅₀ = 27.1 μM) and MDCKII-PGP (LD₅₀ = 33.7 μM) cell lines exhibited statistically greater viability than the MDCKII/wt cells (LD₅₀ = 7.8 μM). Relative to the MDCKII/wt, the MDCKII-MRP1 cells had a significantly reduced saquinavir efflux ratio due to basolaterally directed transport by hMRP1 competing with apically directed endogenous cMRP2. The MDCKII-MRP2 cells had a significantly increased saquinavir efflux ratio due to apically directed transport by hMRP2.; To influence specific basolateral or apical transport events in association with the known localization of hMRP1 and hMRP2, MK-571 was selectively applied with saquinavir to apical or basolateral surfaces of MDCKII-MRP1, MDCKII-MRP2 or MDCKII/wt monolayers. Only basolateral application of MK-571 to MDCKII/wt and MDCKII-MRP1 monolayers significantly reduced saquinavir efflux ratios, causing increased AP to BL transport.; Collectively, these results indicate saquinavir transport by at least hMRP1, human and canine MRP2, and human and canine Pgp. These results further suggest saquinavir transport by an unidentified MK-571 sensitive BL to AP transport mechanism with basolateral localization.