Purpose: Here we characterized the transport properties of morpholine-urea-phenylalanine-homophenylalanine-vinylsulfone-phenyl (K02), a newly developed peptidomimetic cysteine protease inhibitor, across monolayers of P-gp-expressed MDRI transfected MDCK cells (MDR1-MDCK) and Caco-2 cells.
Methods: MDR1-MDCK, MDCK and Caco-2 cells, grown to confluence on Transwell insert membranes, were used to investigate transcellular transport of [14C]-K02.
Results: The basolateral to apical (B-A) flux of 10 microM [14C]-K02 across MDR1-MDCK cells was markedly greater than its apical to basolateral (A-B) flux (ratio = 39). This specific B-A transport was temperature dependent and saturable, with an apparent Michaelis-Menten constant and maximum velocity of 69.1 +/- 19.5 microM and 148.9 +/- 16.3 pmol/min/cm2, respectively. This B-A flux was significantly inhibited by cyclosporine (IC50 = 17.1 +/- 0.7 microM), vinblastine (IC50 = 75.9 +/- 13.0 microM) and verapamil (IC50 = 236 +/- 63 microM). In Caco-2 cell monolayers, the B-A flux was reduced about 50% compared to that in MDR1-MDCK and the A-B flux was increased about 8-fold. The apparent Michaelis-Menten constant and maximum velocity values for the B-A transport were 71.8 +/- 45.9 microM and 35.3 +/- 9.0 pmol/min/ cm2. This B-A flux was also significantly inhibited by P-gp substrates/ inhibitors. Western blots showed that the P-gp expression in MDR1-MDCK cells was about 10-fold that in Caco-2 cells.
Conclusions: K02 is transported by P-gp in both MDR1-MDCK and Caco-2 cells, and the in vitro interactions between K02 and various P-gp substrates may provide strategies to overcome the bioavailability barrier by intestinal P-gp.