Identification and Quantification of Novel Major Metabolites of the Steroidal Aromatase Inhibitor, Exemestane

Exemestane (EXE) is an aromatase inhibitor used for the prevention and treatment of estrogen receptor–positive breast cancer. Although the known major metabolic pathway for EXE is reduction to form the active 17β-dihydro-EXE (17β-DHE) and subsequent glucuronidation to 17β-hydroxy-EXE-17-O-β-D-glucuronide (17β-DHE-Gluc), previous studies have suggested that other major metabolites exist for exemestane. In the present study, a liquid chromatography–mass spectrometry (LC-MS) approach was used to acquire accurate mass data in MS(E) mode, in which precursor ion and fragment ion data were obtained simultaneously to screen novel phase II EXE metabolites in urine specimens from women taking EXE. Two major metabolites predicted to be cysteine conjugates of EXE and 17β-DHE by elemental composition were identified. The structures of the two metabolites were confirmed to be 6-methylcysteinylandrosta-1,4-diene-3,17-dione (6-EXE-cys) and 6-methylcysteinylandrosta-1,4-diene-17β-hydroxy-3-one (6-17β-DHE-cys) after comparison with their chemically synthesized counterparts. Both underwent biosynthesis in vitro in three stepwise enzymatic reactions, with the first involving glutathione conjugation. The cysteine conjugates of EXE and 17β-DHE were subsequently quantified by liquid chromatography–mass spectrometry in the urine and matched plasma samples of 132 subjects taking EXE. The combined 6-EXE-cys plus 6-17β-DHE-cys made up 77% of total EXE metabolites in urine (vs. 1.7%, 0.14%, and 21% for EXE, 17β-DHE, and 17β-DHE-Gluc, respectively) and 35% in plasma (vs. 17%, 12%, and 36% for EXE, 17β-DHE, and 17β-DHE-Gluc, respectively). Therefore, cysteine conjugates of EXE and 17β-DHE appear to be major metabolites of EXE in both urine and plasma.