Functional characterization of peptide transporters in MDCKII-MDR1 cell line as a model for oral absorption studies.

MDCKII-MDR1 cell line has been extensively selected as a model to study P-gp-mediated drug efflux. Recently, investigators have employed this cell line for studying influx of peptide prodrug derivatives of parent compounds, which are P-gp substrates. Therefore, the objective of this study is to functionally characterize the peptide mediated uptake and transport of [(3)H] Glycylsarcosine ([(3)H] Gly-Sar), a model peptide substrate across MDCKII-MDR1 cells. [(3)H] Gly-Sar uptake from apical (AP) and basolateral (BL) membranes was found to be time-dependent and saturable. Michaelis-Menten (K(m)) constants of [(3)H] Gly-Sar uptake across the AP and BL directions in MDCKII-MDR1 cell line were found to be 457+/-37 and 464+/-85microM, respectively. V(max) values in AP and BL directions for the peptide transporters in MDCKII-MDR1 cell line were calculated to be 0.035+/-0.001 and 0.35+/-0.034pmol/minmg protein, respectively. Uptake of [(3)H] Gly-Sar was significantly inhibited in the presence of aminocephalosporins and ACE-Inhibitors, known substrates for peptide transporters in both the AP and BL directions. Permeability of [(3)H] Gly-Sar in the BL direction was maximal at pH 4 as compared to pH 5, 6 and 7.4 whereas such permeability in the AP direction was optimal at pH 7.4. Transepithelial transport of [(3)H] Gly-Sar in the AP-BL direction was significantly lower than from BL-AP direction at all observed pHs. No statistical difference was observed in the transepithelial permeability of [(3)H] Gly-Sar across both AP and BL directions over 4-10 days of growth period. The present study indicates that peptide transporters are effectively involved in the bidirectional transport of Gly-Sar across MDCKII-MDR1 cell line; the BL peptide transporter can transport Gly-Sar at a greater rate as compared to the AP peptide transporter. Results from these studies suggest the application of MDCKII-MDR1 cell line as a rapid effective tool to study peptide mediated influx of compounds that may be substrates for both P-gp and peptide transporters.