Inhibition of the Aromatase Enzyme by Exemestane Cysteine Conjugates

Exemestane(EXE) is an aromatase inhibitor used to treat hormone-dependent breast cancer. EXE is extensively metabolized, with unchanged EXE and its active metabolite 17β-dihydroexemestane (17β-DHE) accounting for 17% and 12%, respectively, of total plasma EXE in vivo. The major circulating EXE metabolites are the cysteine conjugates of EXE and 17β-DHE, and the 17β-DHE glucuronide, which together account for 70% of the total plasma EXE in vivo. The goal of the present study was to examine the inhibition potential of major metabolites of EXE through inhibition assays using aromatase-overexpressing cells and pooled ovarian tissues. Estrone formation was used as a measure of aromatase activity and was detected and quantified using ultraperformance liquid chromatography–mass spectrometry. 6-Methylcysteinylandrosta-1,4-diene-3,17-dione (EXE-cys), 17β-dihydroexemestane (17β-DHE), and 17β-DHE-cysteine all exhibited inhibition of estrone formation at both 1 µM and 10 µM concentrations, with 17β-DHE and EXE-cys showing significant inhibition of estrone formation (63% each) at 10 µM. In contrast, 17β-hydroxy-EXE-17-O-β-D-glucuronide displayed minimal inhibition (5%–8%) at both concentrations. In ovarian tissue, EXE-cys and 17β-DHE showed similar patterns of inhibition, with 49% and 47% inhibition, respectively, at 10 µM. The IC(50) value for EXE-cys (16 ± 10 µM) was similar to 17β-DHE (9.2 ± 2.7 µM) and higher than EXE (1.3 ± 0.28 µM), and all three compounds showed time-dependent inhibition with IC(50) shifts of 13 ± 10, 5.0 ± 2.5 and 36 ± 12-fold, respectively. Given its high circulating levels in patients taking EXE, these results suggest that EXE-cys may contribute to the pharmacologic effect of EXE in vivo. SIGNIFICANCE STATEMENT: The current study is the first to examine the major phase II metabolites of exemestane (EXE): [6-methylcysteinylandrosta-1,4-diene-3,17-dione (EXE-cys), 17β-DHE-cysteine, and 17β-hydroxy-EXE-17-O-β-D-glucuronide] for inhibition potential against the target enzyme aromatase (CYP19A1). EXE-cys was found to significantly inhibit aromatase in a time-dependent manner. Given its high circulating levels in patients taking EXE, this phase II metabolite may play an important role in reducing circulating estrogen levels in vivo.