title: Transporter studies with the 3-O-sulfate conjugate of 17alpha-ethinylestradiol: assessment of human kidney drug transporters.
abstract: 17alpha-Ethinylestradiol (EE2), a synthetic and potent estrogen receptor agonist, is extensively metabolized in both intestine and liver and is largely excreted in bile and urine as the 3-O-sulfate (EE2-Sul) and 3-O-glucuronide. In the present study, EE2-Sul was evaluated as a substrate of various transporters known to be expressed in the kidney. Uptake studies were performed with human epithelial cells [human embryonic kidney (HEK)-293] that contained individually expressed organic cation transporter 2 (OCT2), organic anion transporter (OAT) forms 3 and 4, and multidrug and toxin extrusion 1 (MATE1). The transporter phenotyping studies were extended to include insect cell (Sf9) membrane vesicles that expressed multidrug resistance-associated protein 4 (MRP4) and Madin-Darby canine kidney cells that expressed OAT1. Based on the results obtained, we concluded that EE2-Sul serves as a substrate of OAT3 and OAT4, but not OCT2, OAT1, MATE1, and MRP4. First, EE2-Sul uptake was highly increased in OAT3/HEK-293 cells (versus mock/HEK-293 cells) and was inhibited by OAT3 inhibitors such as bromosulfophthalein (BSP), cimetidine, and probenecid. OAT3-mediated uptake also conformed to single-K(m) (Michaelis constant) kinetics (K(m) = 21.1 microM). Second, EE2-Sul uptake was also significantly higher in OAT4/HEK-293 cells and was inhibited by BSP, methotrexate, and probenecid. In contrast to OAT3, OAT4-dependent uptake was characterized by a two-K(m) model (K(m1) = 1.6 microM; K(m2) = 195 microM). Based on the results of this study, we hypothesize that EE2-Sul is taken up into renal proximal tubule cells by OAT3, and OAT4 plays a role in its secretion into the renal brush border lumen.
authors.affiliation: Department of Metabolism and Pharmacokinetics, Pharmaceutical Candidate Optimization, Bristol-Meyers Squibb, Princeton, New Jersey 08543-4000, USA. yong-hae.han@bms.com
authors.forename: Yong-Hae
authors.initials: YH
authors.lastname: Han
authors.forename: Dennis
authors.initials: D
authors.lastname: Busler
authors.forename: Yang
authors.initials: Y
authors.lastname: Hong
authors.forename: Yuan
authors.initials: Y
authors.lastname: Tian
authors.forename: Cliff
authors.initials: C
authors.lastname: Chen
authors.forename: A David
authors.initials: AD
authors.lastname: Rodrigues
mesh_terms: D000818:Animals; D001692:Biological Transport / Q000187:drug effects* / Q000235:genetics; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics; D002461:Cell Line, Transformed; D004285:Dogs; D004997:Ethinyl Estradiol / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D007313:Insecta; D007668:Kidney / Q000378:metabolism*; D007700:Kinetics; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000096:biosynthesis / Q000235:genetics; D027362:Organic Anion Transport Protein 1 / Q000096:biosynthesis / Q000235:genetics; D029362:Organic Anion Transporters, Sodium-Independent / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D027701:Organic Cation Transport Proteins / Q000096:biosynthesis / Q000235:genetics; D000075102:Organic Cation Transporter 2; D013379:Substrate Specificity; D014162:Transfection / Q000379:methods
chemical_list: D002352:Carrier Proteins; D004997:Ethinyl Estradiol; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D027362:Organic Anion Transport Protein 1; D029362:Organic Anion Transporters, Sodium-Independent; D027701:Organic Cation Transport Proteins; D000075102:Organic Cation Transporter 2; C491013:SLC22A2 protein, human; C498886:SLC22A9 protein, human; C521869:SLC47A1 protein, human; C114338:organic anion transport protein 3; C025810:ethinylestradiol-3-sulfate