Characterization of 17-dihydroexemestane glucuronidation: potential role of the UGT2B17 deletion in exemestane pharmacogenetics

Abstract

Objective Exemestane is a third-generation aromatase inhibitor used in the treatment of breast cancer in postmenopausal women. Reduction to form 17-dihydroexemestane and subsequent glucuronidation to exemestane-17-O-glucuronide is a major pathway for exemestane metabolism. The goal of this study was to analyze 17-dihydroexemestane anti-aromatase activity, characterize the 17-dihydroexemestane glucuronidation pathway, and determine whether the functional polymorphisms in active UGTs could play a role in altered 17-dihydroexemestane glucuronidation. Methods Homogenates from a HEK293 aromatase-overexpressing cell line (HEK293-aro) were used to examine exemestane versus 17-dihydroexemestane anti-aromatase activities. UGT-overexpressing cell lines and a panel (n = 110) of human liver microsome (HLM) were screened for glucuronidation activity against 17-dihydroexemestane. UGT2B17 genotyping and liver mRNA expression were performed by real-time PCR. Results The inhibition of estrone formation from androst-4-ene-3,17-dione in HEK293-aro cell homogenates was similar for 17-dihydroexemestane (IC50 = 2.3 ± 0.83 μmol/l) and exemestane (IC50 = 1.4 ± 0.42 μmol/l). UGTs 2B17 and 1A4 were high-expression hepatic UGTs that exhibited activity against 17-dihydroexemestane, with UGT2B17 exhibiting a 17-fold higher Vmax/KM than UGT1A4. The rate of exemestane-17-O-glucuronide formation was shown to be significantly (P < 0.001) decreased (14-fold) in HLMs exhibiting the UGT2B17(*2/*2) deletion genotype versus wild-type UGT2B17(*1/*1) HLMs; a 36-fold lower Vmax/KM (P = 0.023) was observed in UGT2B17(*2/*2) versus UGT2B17(*1/*1) HLMs. A significant (P < 0.0001, R2 = 0.72) correlation was observed between HLM exemestane-17-O-glucuronide formation and liver UGT2B17 expression. Conclusion These data suggest that 17-dihydroexemestane is an active metabolite of exemestane and that the UGT2B17 deletion polymorphism could play an important role in determining levels of excretion of 17-dihydroexemestane and overall exemestane metabolism.

Sex Differences in UDP-Glucuronosyltransferase 2B17 Expression and Activity

“…UGT2B17 was shown previously to exhibit the highest glucuronidation activity of any hepatic UGT tested against 17-DHE, 3-HC, and SAHA (Balliet et al, 2009;Sun et al, 2010; G. Chen, N. Giambrone, R. M. Balliet, and P. Lazarus, submitted for publication), so those substrates were the focus of the glucuronidation studies by sex in this study. When we stratified by sex, significantly higher amounts of exemestane-17-O-glucuronide formation were observed in individuals with either one or two copies of UGT2B17 than in individuals with 0 copies of UGT2B17 for both women ( p ϭ 0.045 and 0.001, respectively) and men ( p Ͻ 0.001 for both) (Fig.…”

“…3-Hydroxycotinine (3-HC), 3-HC glucuronide, and deuterium-labeled internal standards cotinine-(methyl-D 3 )-3Ј-O-glucuronide were purchased from Toronto Research Chemicals Inc. (North York, ON, Canada). Structures of all compounds were provided in previous publications (Balliet et al, 2009;Sun et al, 2010; Chen, N. Giambrone, R. M. Balliet, and P. Lazarus, submitted for publication). UDP-glucuronic acid, alamethicin, and ␤-glucuronidase were purchased from Sigma-Aldrich (St. Louis, MO).…”

“…Microsomes were prepared and in vitro glucuronidation activity assays against three UGT2B17 substrates were performed as described previously (Balliet et al, 2009;Sun et al, 2010;G. Chen, N. Giambrone, R. M. Balliet, and P. Lazarus, submitted for publication).…”

“…17-DHE glucuronidation was analyzed as described previously (Sun et al, 2010) using an ACQUITY UPLC System (Waters, Milford, MA) with a 1.7-m ACQUITY UPLC BEH C18 analytical column (2.1 mm ϫ 50 mm; Waters Chromatography Ireland Ltd., Dublin, Ireland) in series with a 0.2-m assay frit filter (2.1 mm; Waters). The gradient elution conditions, using a flow rate of 0.3 ml/min, were as follows: starting with 19% acetonitrile and 81% buffer A (5 mM ammonium acetate, pH 5.0) for 1 min, a subsequent linear gradient to 75% acetonitrile and 25% buffer over 2 min, and then maintenance at 75% acetonitrile for 2 min.…”

UGT2B Gene Expression Analysis in Multiple Tobacco Carcinogen-Targeted Tissues

“…The UDP-glucuronosyltransferase (UGT) family of phase II metabolic enzymes play a central role in the excretion of numerous endogenous compounds, including bilirubin (Bosma et al, 1994) and steroid hormones (Belanger et al, 1998(Belanger et al, , 2003, as well as exogenous compounds, including various drugs (Nagar and Remmel, 2006) and chemotherapeutic agents (Kemp et al, 2002;Sun et al, 2006Sun et al, , 2007Sun et al, , 2010Balliet et al, 2009). The UGTs also play an important role in the metabolism and detoxification of multiple tobacco carcinogens, including tobacco-specific nitrosamines like 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) (Ren et al, 2000;Wiener et al, 2004a;Chen et al, 2008b), and polycyclic aromatic hydrocarbons like benzo(a)pyrene [B(a)P]-7,8-diol and dibenzo(a,l)pyrene-11,12-diol [DB(a,l)P] (Fang et al, 2002;Olson et al, 2011).…”