PT  - JOURNAL ARTICLE
AU  - Johan E. Karlsson
AU  - Catherine Heddle
AU  - Aleksei Rozkov
AU  - Joke Rotticci-Mulder
AU  - Ola Tuvesson
AU  - Constanze Hilgendorf
AU  - Tommy B. Andersson
TI  - 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
AID  - 10.1124/dmd.109.028886
DP  - 2010 Apr 01
TA  - Drug Metabolism and Disposition
PG  - 705--714
VI  - 38
IP  - 4
4099  - http://dmd.aspetjournals.org/content/38/4/705.short
4100  - http://dmd.aspetjournals.org/content/38/4/705.full
SO  - Drug Metab Dispos2010 Apr 01; 38
AB  - 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 P-glycoprotein (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 Km and Vmax using the model substrates N-methylquinidine (P-gp), estradiol-17β-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. Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics