Abstract
17α-Ethinylestradiol (EE2), a component of oral contraceptives, is known to undergo considerable first-pass 3-O-sulfation in the intestine and liver. Once formed, the 3-O-sulfate conjugate (EE2-Sul) is detected in circulation at appreciable levels (versus parent EE2) and is present in bile. Therefore, hepatic uptake of EE2-Sul was assessed with suspensions of cryopreserved human primary hepatocytes. In this instance, there was evidence for active (temperature-dependent) uptake, which was described by a two-Km (Michaelis constant) model (Km1 = 220 nM; Km2 = 15.5 μM). Uptake was inhibited (∼90%) by bromosulfophthalein but not by tetraethylammonium or p-aminohippurate. In agreement, EE2-Sul was shown to be a substrate of recombinant organic anion transporter peptides (OATP1B1 and OATP2B1), and Na+/taurocholate-cotransporting polypeptide (NTCP), expressed individually in human embryonic kidney (HEK) 293 cells. Transport by OATP1B1 was described by two Km values (87 nM and 141 μM), whereas OATP2B1- and NTCP-mediated uptake into HEK-293 cells conformed to single Km kinetics (10.7 and 2.6 μM, respectively). EE2-Sul was also assessed as an efflux transporter substrate using membrane vesicles expressing bile salt export pump, breast cancer resistance protein (BCRP), and individual forms of multidrug resistance-associated protein (MRP1, MRP2, and MRP3). Transport studies were also conducted with a cell line expression P-glycoprotein. Only vesicles that contained BCRP exhibited ATP-dependent uptake of EE2-Sul (Km1 = 2.9 and Km2 = 307 μM). Collectively, the data show that hepatic uptake of EE2-Sul can be mediated by three transporters (OATP1B1, OATP2B1, and NTCP), whereas biliary excretion of EE2-Sul into bile likely involves BCRP.
Footnotes
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.109.031518.
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ABBREVIATIONS:
- EE2
- 17α-ethinylestradiol
- UGT
- UDP-glucuronosyltransferase
- EE2-Sul
- 17α-ethinylestradiol-3-O-sulfate
- SULT
- sulfotransferase
- OAT
- organic anion transporter
- OCT
- organic cation transporter
- OATP
- organic anion-transporting polypeptide
- MRP
- multidrug resistance-associated protein
- BCRP
- breast cancer resistance protein
- P-gp
- P-glycoprotein
- BSEP
- bile salt export pump
- EE2-Glu
- 17α-ethinylestradiol 3-O-glucuronide
- HEK
- human embryonic kidney
- NTCP
- sodium taurocholate-cotransporting polypeptide
- MDCK
- Madin-Darby canine kidney
- MPP
- 1-methyl-4-phenylpyridinium
- CCK-8
- cholecystokinin octapeptide
- PAPS
- 3′-phosphoadenosine-5′-phosphosulfate
- MDR1
- multidrug-resistant protein 1
- HPLC
- high-performance liquid chromatography
- FRT
- Flp recombination target
- PCR
- polymerase chain reaction
- RQ
- relative quantification
- BSP
- bromosulfophthalein
- PAH
- p-aminohippurate
- HBSS
- Hank's balanced salt solution
- Pc
- permeability coefficient
- GF-120918
- N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide
- DHEAS
- dehydroepiandrosterone-3-sulfate
- OST
- organic-solute transporter.
- Received December 2, 2009.
- Accepted April 1, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
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