Abstract
Exemestane (EXE) is a hormonal therapy used to treat estrogen receptor positive (ER+) breast cancer by inhibiting the final step of estrogen biosynthesis catalyzed by the enzyme aromatase. Cysteine conjugates of EXE and its active metabolite 17β-dihydro-EXE (17β-DHE) are the major metabolites found in both the urine and plasma of patients taking EXE. The initial step in cysteine conjugate formation is glutathione conjugation catalyzed by the glutathione-S-transferase (GST) family of enzymes. The goal of the present study was to identify cytosolic hepatic GSTs active in the GST-mediated metabolism of EXE and 17β-DHE. Twelve recombinant cytosolic hepatic GSTs were screened for their activity against EXE and 17β-DHE, with glutathionylated EXE and 17β-DHE conjugates detected by ultra-performance liquid chromatography-tandem mass spectrometry. GSTA1, GSTM3, and GSTM1 were active against EXE while only GSTA1 exhibited activity against 17β-DHE. GSTM1 exhibited the highest affinity against EXE with a KM value that was 3.8- and 7.1-fold lower than that observed for GSTA1 and GSTM3, respectively. Of the three GSTs, GSTM3 exhibited the highest intrinsic clearance against EXE (ClINT=0.14 nl·min-1·mg-1). The KM values observed for human liver cytosol against EXE (46 µM) and 17β-DHE (77 µM) were similar to that observed for recombinant GSTA1 (53 µM and 30 µM, respectively). Western blot analysis revealed that GSTA1 and GSTM1 comprised 4.3% and 0.57%, respectively, of total protein in human liver cytosol; GSTM3 was not detected. These data suggest that GSTA1 is the major hepatic cytosolic enzyme involved in the clearance of EXE and its major active metabolite, 17β-DHE.
Significance Statement Most previous studies related to the metabolism of the aromatase inhibitor, exemestane (EXE), have focused mainly on phase I metabolic pathways as well as the glucuronidation phase II metabolic pathway. However, recent studies have indicated that glutathionylation is the major metabolic pathway for EXE. The present study is the first to characterize hepatic GST activity against EXE and 17β-DHE and to identify GSTA1 and GSTM1 as the major cytosolic GSTs involved in the hepatic metabolism of EXE.
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