Interindividual Variability in 2-Hydroxylation, 3-Sulfation, and 3-Glucuronidation of Ethynylestradiol in Human Liver

Abstract

Ethynylestradiol (EE 2 ) is commonly used as the major estrogenic component in many oral contraceptive formulations. Orme et al. 1) reported that EE 2 is metabolized by multiple pathways, and there is a large interindividual variability in the routes and rates of metabolism of EE 2 in humans. In addition, marked interindividual differences in the pharmacokinetic parameters of EE 2 have been reported.1) The reported systemic bioavailability of orally ingested EE 2 ranges from 20 to 65%. 1) Factors that could affect oral clearance of a drug include absorption from the gastrointestinal tract, protein binding, intrinsic hepatic clearance, hepatic blood flow, and renal clearance. Urinary excretion of unchanged EE 2 was very low, 2) suggesting that EE 2 is mainly eliminated by metabolism. The 2-hydroxylation of EE 2 is mainly catalyzed by CYP3A, CYP2C, and CYP2E enzymes, 3,4) and this route is one of the main metabolic pathways in humans.5,6) Additionally, sulfation and glucuronidation also play important roles in the metabolism of EE 2 . EE 2 -3-glucuronide and EE 2 -3-sulfate are eliminated into the gastrointestinal tract, hydrolyzed by gut bacteria to EE 2 , and subsequently reabsorbed. Rifampicin, an inducer of CYP3A, has been reported to significantly increase the oral clearance of EE 2 .7) This interaction is thought to reflect the significant contribution of CYP3A enzymes to the metabolism of EE 2 . It is well known that there are large interindividual differences in the expression levels and catalytic activities of CYP3A enzymes in human liver. 8)Patki et al. 9) indicated that the intrinsic clearance of triazolam, a CYP3A substrate, and CYP3A protein in human liver are reduced in the elderly.In vitro studies are useful for clarifying the role of respective enzymes on the systemic drug clearance. Plasma concentrations of EE 2 achieved during normal therapy are very low (Ͻ2 nM).7) However, most previous in vitro studies used substantially high EE 2 substrate concentrations (Ͼ10 mM). A recent study using human hepatocytes in vitro at a substrate concentration of 1 nM demonstrated that the major EE 2 metabolites are direct conjugates, with hydroxylation representing minor pathways, and suggested that the production of EE 2 -3-sulfate was increased by pretreatment of hepatocytes with rifampicin.10) The initial rate of conjugate formation was reported to be EE 2 -3-sulfateϾEE 2 -3-glucuronide in human hepatocytes. 10) In the current study, we investigated interindividual variability of the 2-hydroxylation, 3-glucuronidation, and 3-sulfation of EE 2 using human liver microsomes and cytosol in the presence of the respective cofactors. Additionally, interindividual variability of the relative contribution of CYP3A to the 2-hydroxylation of EE 2 in human liver microsomes was estimated from the degree of inhibition by ketoconazole, a potent inhibitor of CYP3A. All enzyme activities were measured at an EE 2 substrate concentration of 0.1 mM, which is under a nearly linear condition (Ͻ1/23 of K m values for the respectiv...

Interindividual Variability in 5-Fluorouracil Metabolism and Procainamide N-Acetylation in Human Liver Cytosol

“…Briefly, the incubation mixture consisted of 2-100 mM [6][7][8][9][10][11][12][13][14] C] labeled 5-fluorouracil, liver cytosol (0.5 mg protein/ml), 0.25 mM NADPH, and 35 mM potassium phosphate buffer (pH 7.4) in a final volume of 250 ml. The reaction was started by adding NADPH and the mixture was incubated at 37°C for 15 min.…”

“…6) Recently, we have reported interindividual variations in the 2-hydroxylation (CYP3A4), sulfation, and glucuronidation of ethynylestradiol in human microsomes and cytosol. 7) On the other hand, intrinsic metabolic clearance (CL int ) is calculated as a ratio of V max to K m in order to predict in vivo drug disposition. [8][9][10] Sugiyama and colleagues 8,9) proposed the prediction of in vivo metabolic clearance in experimental animals and humans from in vitro biochemical parameters such as hepatic metabolism and plasma protein binding, based on anatomically and physiologically realistic pharmacokinetic models.…”

Risk–Benefit Assessment of Ethinylestradiol Using a Physiologically Based Pharmacokinetic Modeling Approach

“…Assignment of the scaled‐up hepatic metabolic CL int of 66.77 μl/min/mg protein due to 2‐hydroxylation (which constitutes greater than 90% of the hydroxylated metabolites) of EE from the in vitro study carried out by Shiraga et al . in human liver microsomes (HLM), resulted in a contribution of ∼0.2 to the overall systemic clearance of EE (fm CYP ). Half of this (0.1) was assigned to CYP3A4, with the remainder apportioned to CYP2C9, 2C8, and 1A2, as indicated in the study with recombinant CYP enzymes .…”

“…The unbound metabolic intrinsic clearance (CL int ) estimated from in vitro studies in recombinant CYP3A4, 2C9, 2C8, and 1A2 enzymes expressed in baculovirus‐infected SF21 cells amounted to 11.38 μl/min/mg protein . However, this CL int estimate was much lower than that obtained when EE was directly incubated in human liver microsomes . Given that Shiraga et al .…”

Selective isolation of in vitro phase II conjugates using a lipophilic anionic exchange solid phase extraction method

“…Major metabolites of 17␣-ethynylestradiol reported in the literature are 2-hydroxylation, 3-sulfation, 3 and 17-glucuronidation products [19,20]. Considering that 17␣-ethynylestradiol leads to phase II metabolism and that authentic standards of the phase II conjugates are commercially available, it was selected as the first prototypical compound to test the selective isolation method using mixed phase anionic SPE cartridges.…”