P220 Establishing a platform of uptake transporters in HEK-293 cells for the analysis of possible drug-drug-interactions

Jia Jia , PRIMACYT Cell Culture Technology GmbH, Schwerin, Germany
Anett Ullrich , PRIMACYT Cell Culture Technology GmbH, Schwerin, Germany
Claudia Garve , PRIMACYT Cell Culture Technology GmbH, Schwerin, Germany
Dieter Runge , PRIMACYT Cell Culture Technology GmbH, Schwerin, Germany
Markus Keiser , Department of Pharmacology and Clinical Pharmacology, Center of Drug Absorption and Transport (C_DAT), University Medicine of Greifswald, Greifswald, Germany
Membrane transporters are major variables for disposition, efficacy and safety of many drugs. Organic anion transporting polypeptides (OATPs, gene family: SLCO), Na+-taurocholate co-transporting polypeptide (NTCP, gene family SLC10A1), Organic cation transporters (OCT, gene family SLC22) and Organic anion transporters (OAT, gene family gene family SLC22) belong to the uptake transporters and mediate the uptake of a broad range of substrates including several widely prescribed drugs into cells. We have established a cell platform using stably transfected cells expressing pharmacologic relevant uptake transporters to analyze drug affinities. Here, the transporter activities are analyzed with fluorescent substances in assays that can be performed in standard laboratories.

Transporter activities of OATP1A2, -1B1, -1B3, -2B1 and NTCP as well as OCT1, -2, -3 and OAT2, -3 were analyzed with five fluorescent substances (Fluorescein methotrexate (FMTX), Fluorescein, Rhodamine 123, Dibromofluorescein (DBF), Cholyl-Lysyl-Fluorescein (CLF)).

FMTX was specifically transported by OATP1B1, -1B3 and OAT2, -3. Fluorescein is a substrate for OATP1B1 and OAT2, -3, while Rhodamine 123 is one for HEK-OATP1A2 and OCT1, -2, -3. CLF was characterized as substrate for a wide range of transporters: NTCP, OATP1B1 and -1B3. The compound DBF is transported by HEK-OATP2B1. Rifampicin functions as inhibitor for all investigated OATPs. The transport activity of all 3 OCTs could be inhibited by Quinidine. Cholate efficiently inhibited the uptake of CLF mediated by NTCP. Diclofenac functions as inhibitor for OAT2 and OAT3.

In summary, the transport function of stably transfected HEK-293 cells expressing the above mentioned transporters could be analysed with fluorescent substances. This platform can be used for identification of specific transporters involved in drug uptake and drug-drug-interactions.