High-Activity P-Glycoprotein, Multidrug Resistance Protein 2, and Breast Cancer Resistance Protein Membrane Vesicles Prepared from Transiently Transfected Human Embryonic Kidney 293-Epstein-Barr Virus Nuclear Antigen Cells

Abstract

ABSTRACT:Membrane-bound transporter proteins play an important role in the efflux of drugs from cells and can significantly influence the pharmacokinetics of drug molecules. This study describes the production of large amounts of high-activity transporter membrane vesicles from human embryonic kidney 293-Epstein-Barr virus nuclear antigen cells transiently transfected using a Gateway-adapted pCEP4 plasmid. Transfections were scaled up to 10-liter cell cultures, and vesicle preparations were optimized using ultracentrifugation with a sucrose cushion, which enabled us to produce hundreds of milligrams of membrane vesicles expressing human efflux transporter proteins Pglycoprotein (P-gp)/multidrug resistance 1 (ABCB1), multidrug resistance protein 2 (MRP2) (ABCC2), and breast cancer resistance protein (BCRP) (ABCG2). Assays were developed and optimized for analyzing the ATP-dependent functionality of the transporters using probe substrates and specific inhibitors. Excellent signal/noise ratios of ATP-stimulated uptake for P-gp, MRP2, and BCRP vesicles were obtained, indicating high expression of functioning transporters. The uptake kinetics of the transporters was investigated by determining K m and V max using the model substrates N-methylquinidine (P-gp), estradiol-17␤-glucuronide (MRP2), and estrone-3-sulfate (BCRP). The ATP-dependent transport was inhibited by the model inhibitors verapamil (P-gp), benzbromarone (MRP2), and sulfasalazine (BCRP). The vesicles are thus well suited to screen for possible substrates and inhibitors in high throughput systems or are used for detailed mechanistic investigations of transporter kinetics of specific substances.

Development of a Highly Sensitive Method Using Liquid Chromatography-Multiple Reaction Monitoring to Quantify Membrane P-Glycoprotein in Biological Matrices and Relationship to Transport Function

“…HEK293-Epstein-Barr virus nuclear antigen cells were cultured and transiently transfected with human P-gp as described earlier (Karlsson et al, 2010). Purified P-gp membrane vesicles from transfected HEK293-Epstein-Barr virus nuclear antigen cells were prepared as described previously (Karlsson et al, 2010).…”

“…The P-gp levels were measured in both HEK293 membrane vesicles expressing human P-gp (Karlsson et al, 2010) and Caco-2 cells of different time in culture. Furthermore, the P-gp protein expression level was correlated with mRNA expression level and functional analysis.…”

Key to Opening Kidney for In Vitro–In Vivo Extrapolation Entrance in Health and Disease: Part I: In Vitro Systems and Physiological Data

“…An important limitation for some of these cell lines is that functional expression of drug transporters, particularly OAT1 (SLC22A6) and OAT3 (SLC22A8), or other renal characteristics can be lost (46), although some of the recent advanced molecular biology tools may be useful to overcome such issues (47). Cell membrane vesicles can be isolated from tissue samples, cell lines and recombinantly expressed transporter systems (48,49), allowing multiple or individual transporters to be studied. The inside-out vesicular transport assay is particularly useful for studying transport mediated by efflux transporters, which can be challenging when in standard cellbased assays.…”

Variability in P-Glycoprotein Inhibitory Potency (IC50) Using Various in Vitro Experimental Systems: Implications for Universal Digoxin Drug-Drug Interaction Risk Assessment Decision Criteria

“…Membrane vesicles prepared from cells expressing P-gp were obtained from Solvo Biotechnology (Budapest, Hungary) following the method described by Keppler et al (1998) or were generated in-house at the participating laboratory (Table 4) (Karlsson et al, 2010).…”