Transporter Studies with the 3-O-Sulfate Conjugate of 17α-Ethinylestradiol: Assessment of Human Liver Drug Transporters

Abstract

ABSTRACT:17␣-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-K m (Michaelis constant) model (K m1 ‫؍‬ 220 nM; K m2 ‫؍‬ 15.5 M). Uptake was inhibited (ϳ90%) 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 K m values (87 nM and 141 M), whereas OATP2B1-and NTCP-mediated uptake into HEK-293 cells conformed to single K m kinetics (10.7 and 2.6 M, 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 Pglycoprotein. Only vesicles that contained BCRP exhibited ATPdependent uptake of EE2-Sul (K m1 ‫؍‬ 2.9 and K m2 ‫؍‬ 307 M). 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.

Transporter Studies with the 3-O-Sulfate Conjugate of 17α-Ethinylestradiol: Assessment of Human Kidney Drug Transporters

“…EE2-Sul can cycle back into the gut (via bile secretion) and be hydrolyzed to parent EE2, or it can be taken up by OATP2B1 therein. At the present time, the mechanism by which EE2-Sul undergoes efflux across the basolateral membrane (from the enterocyte into the portal blood) is unknown, but OST␣ and OST␤ may be the candidate transporters (Han et al, 2010). The fraction of EE2-Sul that survives liver first pass can circulate, can be taken up by OAT3 in the kidney and secreted into urine via both OAT4 and BCRP.…”

“…It is concluded that circulating EE2-Sul is taken up by OAT3 and effluxed by OAT4 at the brush-border membrane of human renal proximal tubule cells. The latter may function coordinately with BCRP, because EE2-Sul is also a BCRP substrate (Han et al, 2010). Therefore, at least three transporters may play a role in the renal elimination of EE2-Sul.…”

“…In an accompanying manuscript (Han et al, 2010), EE2-Sul was shown to be a substrate of numerous liver-expressed transporters (OATP1B1, OATP2B1, NTCP, and BCRP). However, information related to the transporters involved in the renal excretion of EE2-Sul is lacking.…”

Efflux Transport Is an Important Determinant of Ethinylestradiol Glucuronide and Ethinylestradiol Sulfate Pharmacokinetics

“…In contrast to EEG, EES systemic exposure was decreased (up to an order of magnitude) in TR Ϫ rats. EES is a BCRP substrate (Han et al, 2010b), so these alterations are primarily attributed to the greatly decreased Bcrp expression and function in TR Ϫ rats (Yue et al, 2011). In the studies presented here, EES biliary excretion in TR Ϫ rat liver perfusions was negligible.…”

“…Likewise in mice, the reduction was noted only after oral administration. Although Han et al (2010b) and Chu et al (2004) concluded that EES is not a substrate of MRP2, the Chu et al (2004) study demonstrated a very low level of MRP2 transport activity, although it did not meet the prespecified substrate criteria. The Mrp2-knockout mouse data presented here and the fact that the decrease in EES exposure was greater in TR Ϫ rats than either Mrp2-or Bcrp-knockout mice alone suggest that EES may be a poor Mrp2 substrate and that this transporter by itself has a very modest influence on EES pharmacokinetics.…”