Exemestane (EXE) is a hormonal
therapy used to treat
estrogen receptor-positive
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 (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, and glutathionylated EXE and 17β-DHE conjugates were detected by ultra-performance liquid chromatography tandem mass spectrometry. GST α (GSTA)
isoform 1, GST μ (GSTM)
isoform 3 and
isoform 1 were active against EXE, whereas only GSTA1 exhibited activity against 17β-DHE. GSTM1 exhibited the highest affinity against EXE with a Michaelis-Menten constant (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 (intrinsic clearance = 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 those observed for recombinant GSTA1 (53 and 30 μM, respectively). Western blot analysis revealed that GSTA1 and GSTM1 composed 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 and 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
glutathione S-transferase (GST) activity against EXE and 17β-dihydro-EXE and to identify GST α 1 and GST μ 1 as the major cytosolic
GSTs involved in the hepatic metabolism of EXE.