Assessment of the role of nucleoside transporters, P-glycoprotein, breast cancer resistance protein, and multidrug resistance-associated protein 2 in the placental transport of entecavir using in vitro, ex vivo, and in situ methods.

The nucleoside analog entecavir (ETV) is a first-line pharmacotherapy for chronic hepatitis B in adult and pediatric patients. However, due to insufficient data on placental transfer and its effects on pregnancy, ETV administration is not recommended for women after conception. To expand knowledge of safety, we focused on evaluating the contribution of nucleoside transporters (NBMPR sensitive ENTs and Na+ dependent CNTs) and efflux transporters, P-glycoprotein (ABCB1), breast cancer resistance protein (ABCG2), and multidrug resistance-associated transporter 2 (ABCC2), to the placental kinetics of ETV. We observed that NBMPR and nucleosides (adenosine and/or uridine) inhibited [3H]ETV uptake into BeWo cells, microvillous membrane vesicles, and fresh villous fragments prepared from the human term placenta, while Na+ depletion had no effect. Using a dual perfusion study in an open-circuit setup, we showed that maternal-to-fetal and fetal-to-maternal clearances of [3H]ETV in the rat term placenta were decreased by NBMPR and uridine. Net efflux ratios calculated for bidirectional transport studies performed in MDCKII cells expressing human ABCB1, ABCG2, or ABCC2 were close to the value of one. Consistently, no significant decrease in fetal perfusate was observed in the closed-circuit setup of dual perfusion studies, suggesting that active efflux does not significantly reduce maternal-to-fetal transport. In conclusion, ENTs (most likely ENT1), but not CNTs, ABCB1, ABCG2, and ABCC2, contribute significantly to the placental kinetics of ETV. Future studies should investigate the placental/fetal toxicity of ETV, the impact of drug-drug interactions on ENT1, and interindividual variability in ENT1 expression on the placental uptake and fetal exposure to ETV.

Frog intestinal sac as an in vitro method for the assessment of intestinal permeability in humans: Application to carrier transported drugs.

The aim of this study was to investigate the presence of pharmaceutically relevant drug transporters in frog intestine which has been proposed as model for intestinal permeability screening assays of passively absorbed drugs in humans [Trapani, G., Franco, M., Trapani, A., Lopedota, A., Latrofa, A., Gallucci, E., Micelli, S., Liso, G., 2004. Frog intestinal sac: a new in vitro method for the assessment of intestinal permeability. J. Pharm. Sci. 93, 2909-2919]. The expression of transporters in frog intestine was supported by the following observations: (i) the involvement of purine nucleobase transport system was deduced by inhibition of acyclovir transport in the presence of adenine; (ii) baclofen or l-dopa transport was inhibited by the digitalis steroid ouabain and it may be related to the Na(+) electrochemical potential difference, presumably involving amino acid transporters; (iii) the presence of proton-dependent peptide transporters was argued evaluating the effect of the pH change (from pH 5.9 to pH 7.4) on the transport of glutathione; (iv) the possible expression in the frog intestine of an efflux system distinct from P-glycoprotein (Pgp) in the benzylpenicillin transport was deduced using a glucose enriched frog Ringer with or without the known Pgp inhibitor verapamil; (v) the contribution of Pgp-mediated efflux system in determining the frog intestinal absorption of drugs was supported by the specific inhibition of cimetidine or nadolol transport in the presence of verapamil. These results indicate that pharmaceutically relevant drug transporters should be also expressed in frog intestine. In this work, an attempt was also made to compare the measured P(app) values in the frog intestinal model for the aforementioned series of actively/effluxed transported drugs in humans to the corresponding literature values for the fraction absorbed. The P(app) values used in these comparisons were obtained at high concentrations of drugs at which probably saturation of the carrier occurs. Interestingly, it was found that drugs that are completely absorbed had P(app) values >3 x 10(-6)cm/s, while drugs absorbed <90% had P(app) values lower than 1 x 10(-6)cm/s. In these cases, indeed, a borderline region characterized by the apparent permeability coefficient P(app) value between 1 x 10(-6) and 3 x 10(-6)cm/s should be considered for which the prediction of the absorbed fraction after oral administration in humans become more uncertain by the frog intestinal sac system.