Purpose: To investigate the role of carrier mechanisms in: [1] the polarized transport of the bis(pivaloyloxymethyl)- [bis(POM)-] ester prodrug of the antiviral agent 9-(2-phosphonylmethoxyethyl)adenine [PMEA] and [2] the directional secretion of its metabolites.
Methods: Caco-2 monolayers were used to study the modulation effect of carriers on the transport of bis(POM)-PMEA and the efflux of intracellularly formed metabolites mono(POM)-PMEA and PMEA from the cells. The interaction of bis(POM)-PMEA and its metabolites with the efflux mechanisms present in Caco-2 monolayers was investigated by testing the effect of various concentrations of verapamil (30, 100, 300, microns) or indomethacin (10-500 microns) on transport and efflux.
Results: Polarity in transport of bis(POM)-PMEA (50 micron) across Caco-2 monolayers was noted: transport of total PMEA [=bis(POM)-PMEA, mono(POM)-PMEA and PMEA] was significantly higher in basolateral (BL) to apical (AP) direction (14.5 +/- 0.4%) than transport in the opposite (AP to BL) direction (1.7 +/- 0.2%). This difference was reduced in a concentration dependent way when verapamil (0-100 microns) was included in both AP and BL incubation media. After loading the cells with bis(POM)-PMEA (100 micron) for 1 hr, studies on efflux of PMEA and mono(POM)-PMEA from the Caco-2 monolayers over a 3 hr period, revealed that both metabolites were preferentially secreted towards the AP compartment. Efflux of PMEA toward AP and BL compartments amounted to 14.6 +/- 1.1% and 5.3 +/- 0.4, respectively, of the initial intracellular amount of total PMEA, while efflux of mono(POM)-PMEA towards AP and BL compartments was limited to 2.3 +/- 0.1% and 0.5 +/- 0.1%, respectively. When 10 micron indomethacin was included in the AP incubation medium, efflux of PMEA was decreased to 7.8 +/- 0.3% and 3.3 +/- 0.3% towards the AP and BL compartments, respectively. The decrease in efflux by indomethacin was concentration-dependent up to 100 micron. Transepithelial transport of total PMEA was also reduced in the presence of 30 micron indomethacin, as reflected in smaller concentrations of PMEA and mono(POM)-PMEA in the acceptor compartment, irrespective of the transport direction.
Conclusions: The data obtained in this study suggest that bis(POM)-PMEA is substrate for a P -glycoprotein-like carrier mechanism in Caco-2 monolayers, while its metabolites mono(POM)-PMEA and PMEA are transported by a non-P-glycoprotein efflux protein.