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

Abstract

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. MichaelisMenten (Km) constants of [ 3 H] Gly-Sar uptake across the AP and BL directions in MDCKII-MDR1 cell line were found to be 457 ± 37 μM and 464 ± 85 μM respectively. Vmax 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 ± 0.034 pmol/min/mg 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.

Peptide prodrugs: Improved oral absorption of lopinavir, a HIV protease inhibitor

“…The apical cell layer expresses functional efflux (P-gp or MRP2) as well as influx (peptide) transporters; which makes it convenient for us to compare the permeability values of both the parent drug and the prodrugs in the same cell line. This study was performed at pH 4 because highest permeabilities of Gly-Sar were found to be at this pH in the MDCKII-MDR1 cell line (Agarwal et al, 2007b). Permeabilities of VVL and GVL were found to be 6.37 and 2.58*10 −6 cm/sec respectively relative to 1.32*10 −6 cm/sec for LVR in MDCKII-MDR1 cells.…”

Dipeptide prodrug approach to evade efflux pumps and CYP3A4 metabolism of lopinavir

“…Previously, we have reported that peptide transporters and efflux pumps (MDR1 and MRP2) are expressed on the apical surface of these cell lines (Agarwal et al, 2007a; Agarwal et al, 2007b). Moreover, we have also demonstrated that MDR1 and MRP2 efflux pumps play a significant role in diminishing A-B transport of LPV (Agarwal et al, 2008; Agarwal et al, 2007b; Patel et al, 2014b).…”

“…Samples were stored at −80 °C until further analysis with LCMS/MS. Transport studies across Caco2 cells were conducted at pH 5 and MDCK cells at pH 7.4 (Agarwal et al, 2008; Agarwal et al, 2007a). …”

Stereoisomeric Prodrugs to Improve Corneal Absorption of Prednisolone: Synthesis and In Vitro Evaluation

“…For investigating substrate affinity towards peptide transporters, cellular uptake of [3H]-Gly-Sar was conducted in the presence and absence of prodrugs in MDCK-MDR1 cells. This cell line was selected as it highly expresses peptide transporters (44). Results obtained from this study are depicted in Fig.…”