P321 ESTABLISHMENT OF STABLY TRANSFECTED CELL LINES EXPRESSING TRANSPORTERS KEY TO DRUG DISPOSITION

Gang Luo , Drug Metabolism and Disposition, Covance Laboratories Inc., Madison, WI
Kristen Cardinal , Drug Metabolism and Disposition, Covance Laboratories Inc., Madison, WI
Manuela Deavila , Drug Metabolism and Disposition, Covance Laboratories Inc., Madison, WI
Michele Hamrick , Drug Metabolism and Disposition, Covance Laboratories Inc., Madison, WI
Kristine Horabik , Drug Metabolism and Disposition, Covance Laboratories Inc., Madison, WI
Jeffrey Zuvon , Drug Metabolism and Disposition, Covance Laboratories Inc., Madison, WI
Liberty Liuperez , Drug Metabolism and Disposition, Covance Laboratories Inc., Madison, WI
Richard Ridgewell , Drug Metabolism and Disposition, Covance Laboratories Inc., Madison, WI
Over the last decade, pharmaceutical and academic research has shown an increasing relevance of transporters in the disposition of drugs. With this information, the International Transporter Consortium highlighted several transporters of emerging importance in drug development. These include the uptake transporters organic anion transporting peptide (OATP) 1B1 and 1B3, organic anion transporter (OAT) 1 and 3, and organic cation transporter (OCT) 2. Also included are the efflux transporters P‑glycoprotein (P-gp/MDR1) and breast cancer resistance protein (BCRP). In addition, OCT1 and multidrug resistance protein (MRP) 2 also play significant roles in the disposition of certain drugs. To assess drug-transporter interactions, we established stably transfected cell lines for these human drug transporters. The cDNA encoding each of these transporters was inserted into a pCMV6-entry vector containing a neomycin resistance gene as the selection marker. Chinese hamster ovary (CHO) and Madin-Darby canine kidney (MDCK) II cells were both transfected with either a sham vector pCMV6 or pCMV6 containing a single uptake or efflux transporter cDNA, respectively.  Stably transfected cells were established through selection with geneticin (G418). For each transporter, approximately 40 colonies were screened for functional activity using a radiolabeled substrate. All uptake experiments were conducted by determining radioactivity uptake.  OATP1B1 showed 7.2-fold uptake of 3H‑estradiol-17b-D-glucuronide, OATP1B3 8.5‑fold uptake of 3H-cholecystokinin, OAT1 26.0-fold uptake of 14C‑p‑aminohippurate, OAT3 5.5-fold uptake of 3H‑estrone-3-sulfate, while OCT1 and OCT3 showed 19.7- and 19.6-fold uptake, respectively, of 14C‑tetraethylammonium compared to the control cells.  In preliminary screening of P-gp, the apparent permeability of 3H‑digoxin from the basolateral to apical direction using 24-transwell plates was determined.  Also in preliminary screening, the diminished accumulation of radioactivity in the BCRP and MRP2 colonies compared to control cells was measured following dosing of 3H‑mitoxantrone (for BCRP) and 14C‑1-chloro-2,4-dinitrobenzene (for MRP2).  For secondary screening and further qualification of the efflux transporters, efflux ratios were calculated from the apparent permeability of both the basolateral to apical and apical to basolateral directions using 24-transwell plates.  P-gp, BCRP, and MRP2 showed more than two-fold higher efflux ratios with their respective radiolabeled substrates compared to the control cells.  Known inhibitors, namely, cyclosporine A for OATP1B1 and OATP1B3, probenecid for OAT1 and OAT3, quinidine for OCT1 and OCT2, zosuquidar for P-gp, Ko-143 for BCRP, and MK-571 for MRP2, showed significant inhibition of the functional activity of each of these cell lines.