IPEC-J2 MDR1, a Novel High-Resistance Cell Line with Functional Expression of Human P-glycoprotein (ABCB1) for Drug Screening Studies

Abstract

The P-glycoprotein (P-gp) efflux pump has been shown to affect drug distribution and absorption in various organs and to cause drug resistance in cancer therapy. The aim of this work was to develop a cell line to serve as a screening system for potential substrates of P-gp. This requires a cell line with high paracellular tightness, low expression of nonhuman ABC transporters, and high expression of functional human P-gp (ABCB1). The porcine intestinal epithelial cell line, IPEC-J2, was selected as a transfection host, due to its ability to form extremely high-resistance monolayers (>10,000 Ω·cm(2)) and its low endogenous expression of ABC-type efflux transporters. The IPEC-J2 cells were transfected with a plasmid that contained the sequence of the human MDR1 gene, which encodes P-gp, followed by a selection of successfully transfected cells with geneticin and puromycin. The resulting cell line, IPEC-J2 MDR1, retained its high transepithelilal resistance (>15,000 Ω·cm(2)), which translated into low permeability of the small hydrophilic tracer, mannitol (P < 10(-7) cm·s(-1)). The lipophilic compound, diazepam, displayed high permeability resulting in a dynamic range of 1500 (PDiazepam/Pmannitol) to separate high and low permeability compounds. Human P-gp was expressed predominantly in the apical membrane, as demonstrated by immunocytochemistry, Western blots, and a high efflux ratios (Pbasolateral-apical/Papical-basolateral) of known P-gp substrates. P-gp was demonstrated to be responsible for the efflux transport by substrate profiling, combined with application of P-gp and BCRP inhibitors. Furthermore, the compounds atenolol, citalopram, and mitoxantrone were identified as P-gp substrates. Functional P-gp expression was shown to be stable through at least 10 cell passages. In conclusion, the IPEC-J2 MDR1 cell line displays high paracellular tightness combined with high expression of human P-gp and low expression of porcine ABC transporters, and it may serve as a useful tool in drug development studies.

Revisiting atenolol as a low passive permeability marker

“…However, these inhibitors are not specific and also act on other transporters. In addition, polarized transport of atenolol was found in a Pgp-transfected IPEC-J2 cell lines and Caco-2 cell with an efflux ratio of 3.5 and 2.3, respectively, which were decreased by addition of Pgp inhibitors (zosuquidar and verapamil) [ 60 , 61 ]. In a collaborative study comparing Caco-2 cells from 10 laboratories, atenolol showed highly variable permeability and its efflux ratios ranged from 0.18 to 3.76, indicating the possibility of an involvement of transporter-mediated transport [ 38 ].…”

Efflux transport of nicotine, cotinine and trans‐3′‐hydroxycotinine glucuronides by human hepatic transporters

“…Nicotine is not actively effluxed in the human intestinal epithelial cell line Caco‐2, which indicates no contribution of intestinal transporters such as MRPs, BCRP or MDR1 . Lack of MDR1‐mediated transport of nicotine has also been found in a recombinant cell line expressing human MDR1 . In addition, nicotine and cotinine do not stimulate the ATPase activity of the human recombinant MDR1 .…”

“…Nicotine and cotinine are highly membrane‐permeable compounds that belong to Class 2 of the extended clearance classification system . Compounds in this class undergo extensive metabolism, while transporters have only negligible effect on their disposition.…”

“…Nicotine and cotinine are highly membrane-permeable compounds that belong to Class 2 of the extended clearance classification system. 29,30 Compounds in this class undergo extensive metabolism, while transporters have only negligible F I G U R E 2 Transport of nicotine-G (A and D), cotinine-G (B and E) and OH-cotinine-G (C and F) by MRP1-6, BCRP, MDR1 and control vesicles. Transport activity was studied at two substrate concentrations, 5 µmol/L (A-C) and 50 µmol/L (D-F).…”

“…31 Lack of MDR1-mediated transport of nicotine has also been found in a recombinant cell line expressing human MDR1. 30 In addition, nicotine and cotinine do not stimulate the ATPase activity of the human recombinant MDR1. 32 Thus, our findings that nicotine does not inhibit human MRP2-4, MDR1 or BCRP ( Figure 5) are consistent with previous reports that suggest no interaction between nicotine and these efflux transporters.…”