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

Abstract

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 17b-dihydro-EXE (17b-DHE) and subsequent glucuronidation to 17b-hydroxy-EXE-17-O-b-D-glucuronide (17b-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 17b-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-17b-hydroxy-3-one (6-17b-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 17b-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 6-17b-DHE-cys made up 77% of total EXE metabolites in urine (vs. 1.7%, 0.14%, and 21% for EXE, 17b-DHE, and 17b-DHE-Gluc, respectively) and 35% in plasma (vs. 17%, 12%, and 36% for EXE, 17b-DHE, and 17b-DHE-Gluc, respectively).

Characterization of Cytosolic Glutathione S-Transferases Involved in the Metabolism of the Aromatase Inhibitor, Exemestane

“…Glutathionylation is an important step in the xenobiotic detoxification pathway of many compounds and might play an important role in the rate of excretion and elimination of EXE, thereby contributing to the variation observed in EXE treatment efficacy and side effects (Jancova et al, 2010). As shown in previous studies, cysteine conjugates of EXE comprise 77% of total urinary metabolites, while in plasma cysteine conjugate levels are similar to another major metabolite,17β-DHE-Gluc (Luo et al, 2018). These data suggest that the formation of cysteine conjugates is a major route of metabolism of EXE.…”

“…Subsequently, the glutamyl moiety is removed by γ-glutamyltransferases (GGT) and the glycyl moiety is removed by dipeptidases, and cysteine conjugates are excreted in the urine (Hanna and Anders, 2019). This three-step pathway has been verified for EXE through in vitro biosynthesis, with the resulting enzymatically synthesized cysteine conjugate of EXE matching both chemically synthesized EXE-cys, as well as through ex vivo analysis of urinary EXE-cys detected in subjects taking EXE (Luo et al, 2018).…”

High‐throughput liquid chromatography tandem mass spectrometry assay as initial testing procedure for analysis of total urinary fraction

“…This transition (348 > 321), albeit minor, was found to be exceptionally specific with nearly zero background and helped identify presumptive adverse analytical findings (AAFs) that would otherwise remain unnoticed (Figure 4). Another example is cysteinyl exemestane, 36 which unlike exemestane or 17β‐hydroxyexemestane does not suffer from endogenous interferences. Given the polarity of the cysteinyl conjugate, its REC might be low, but this metabolite was clearly detectable in the excretion urine distributed among antidoping laboratories by WADA as part of external quality assessment scheme in 2017 (Figure 5).…”

Steroidal alkaloids efficient aromatase inhibitors with potential for the treatment of postmenopausal breast cancer

“…Aromatase inhibitors (AIs) can be divided into two groups. One is Non‐steroidal, and other is steroidal inhibitors (Banting et al, ; Luo et al, ). The non‐steroidal are the derivatives of AG and also include mostly imidazole or triazole compounds such as anastrazole and letrozole while steroidal aromatase inhibitors are the derivatives of natural substrate androstenedione for the aromatase enzyme such as exemestane and formestane (Lonning & Lien, ).…”