The Impact of Endogenous Breast Cancer Resistance Protein on Human P-Glycoprotein-Mediated Transport Assays Using LLC-PK1 Cells Transfected With Human P-Glycoprotein |
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Authors: | Rei Miyamoto Takashi Nozawa Koichi Shiozuka Kenji Tabata |
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Institution: | 1. Drug Metabolism and Pharmacokinetics Research Division, Astellas Research Technologies Corporation, Ltd., Ibaraki, Japan;2. Analysis and Pharmacokinetics Research Labs, Astellas Pharma Inc., Ibaraki, Japan |
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Abstract: | Lilly Laboratories cell porcine kidney 1 (LLC-PK1) cells transfected with human P-glycoprotein (LLC-PK1-P-gp) are widely used in transport assays to identify drug candidates that function as substrates of this efflux transporter. Endogenous transporters expressed in LLC-PK1 cells may complicate the interpretation of findings from P-gp-mediated transport assays. We investigated the impact of porcine breast cancer resistance protein (Bcrp) in P-gp-mediated transport assays in LLC-PK1 cells. Porcine Bcrp mRNA was detected in both LLC-PK1 wildtype (WT) and LLC-PK1-P-gp cells by quantitative RT-PCR. To investigate the activity and impact of porcine Bcrp, we conducted transport assays using 6 typical BCRP substrates in LLC-PK1 cells. Efflux ratios (ER) of the 6 BCRP substrates in LLC-PK1 WT cells were >2, and were reduced in the presence of the BCRP inhibitor Ko143. The efflux activities of the 6 BCRP substrates were confirmed using MDCKII cells transfected with human BCRP. Net ERs of prazosin and fluvastatin, dual substrates of P-gp and BCRP, determined by dividing ERs in LLC-PK1-P-gp cells by those in LLC-PK1 WT cells, were <2, but increased to >2 in the presence of Ko143. These results indicated that endogenous Bcrp in LLC-PK1 cells was involved in the transport of BCRP substrates and may interfere with the identification of P-gp substrates. |
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Keywords: | ABC transporters P-glycoprotein breast cancer resistance protein drug transport efflux pump permeability coefficient permeability polymerase chain reaction BCRP/Bcrp breast cancer resistant protein ER efflux ratio FDA food and drug administration LC-MS/MS liquid chromatography-tandem mass spectrometry LLC-PK1-P-gp LLC-PK1 transfected with human P-gp MDCKII-BCRP MDCKII transfected with human BCRP Mrp2 multidrug resistance-associated protein 2 NER net efflux ratio apparent permeability coefficient P-gp P-glycoprotein qRT-PCR quantitative RT-PCR WT wildtype |
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