Advertisement
Research ArticleArticle
Open Access

Inhibition of the Aromatase Enzyme by Exemestane Cysteine Conjugates

Irina Teslenko, Christy J.W. Watson, Gang Chen and Philip Lazarus
Molecular Pharmacology November 2022, 102 (5) 216-222; DOI: https://doi.org/10.1124/molpharm.122.000545

Abstract

Exemestane(EXE) is an aromatase inhibitor used to treat hormone-dependent breast cancer. EXE is extensively metabolized, with unchanged EXE and its active metabolite 17β-dihydroexemestane (17β-DHE) accounting for 17% and 12%, respectively, of total plasma EXE in vivo. The major circulating EXE metabolites are the cysteine conjugates of EXE and 17β-DHE, and the 17β-DHE glucuronide, which together account for 70% of the total plasma EXE in vivo. The goal of the present study was to examine the inhibition potential of major metabolites of EXE through inhibition assays using aromatase-overexpressing cells and pooled ovarian tissues. Estrone formation was used as a measure of aromatase activity and was detected and quantified using ultraperformance liquid chromatography–mass spectrometry. 6-Methylcysteinylandrosta-1,4-diene-3,17-dione (EXE-cys), 17β-dihydroexemestane (17β-DHE), and 17β-DHE-cysteine all exhibited inhibition of estrone formation at both 1 µM and 10 µM concentrations, with 17β-DHE and EXE-cys showing significant inhibition of estrone formation (63% each) at 10 µM. In contrast, 17β-hydroxy-EXE-17-O-β-D-glucuronide displayed minimal inhibition (5%–8%) at both concentrations. In ovarian tissue, EXE-cys and 17β-DHE showed similar patterns of inhibition, with 49% and 47% inhibition, respectively, at 10 µM. The IC50 value for EXE-cys (16 ± 10 µM) was similar to 17β-DHE (9.2 ± 2.7 µM) and higher than EXE (1.3 ± 0.28 µM), and all three compounds showed time-dependent inhibition with IC50 shifts of 13 ± 10, 5.0 ± 2.5 and 36 ± 12-fold, respectively. Given its high circulating levels in patients taking EXE, these results suggest that EXE-cys may contribute to the pharmacologic effect of EXE in vivo.

SIGNIFICANCE STATEMENT The current study is the first to examine the major phase II metabolites of exemestane (EXE): [6-methylcysteinylandrosta-1,4-diene-3,17-dione (EXE-cys), 17β-DHE-cysteine, and 17β-hydroxy-EXE-17-O-β-D-glucuronide] for inhibition potential against the target enzyme aromatase (CYP19A1). EXE-cys was found to significantly inhibit aromatase in a time-dependent manner. Given its high circulating levels in patients taking EXE, this phase II metabolite may play an important role in reducing circulating estrogen levels in vivo.

Introduction

Breast cancer continues to be the most commonly diagnosed cancer in women, with an estimated 290,000 new cases and 43,000 breast cancer–related deaths expected in the United States in 2022 (Siegel et al., 2022). More than two-thirds of all diagnosed breast cancers are estrogen receptor–positive (ER+), characterized by the presence of estrogen receptors that ultimately bind to estrogen to stimulate breast gland cell proliferation (https://seer.cancer.gov/archive/csr/1975_2013/; Osborne and Schiff, 2011). A major strategy to managing tumor growth and proliferation in ER+ breast cancer patients is through antiestrogenic therapy, by blocking estrogen biosynthesis in the final step of the conversion of androgens to estrogens, by inhibiting the key enzyme aromatase with aromatase inhibitors (Untch and Thomssen, 2010; Early Breast Cancer Trialists’ Collaborative Group(EBCTCG), 2015; Robertson et al., 2016). This strategy is particularly effective in postmenopausal women due to circulating estrogen levels being lower than in premenopausal women (Ramchand et al., 2019) with the addition of aromatase inhibitors suppressing circulating estrogens to almost undetectable levels (Campos, 2004; Kittaneh and Glück, 2011).

Exemestane (EXE) belongs to a class of steroidal aromatase inhibitors that are structurally related to the natural substrate of aromatase, androstenedione (Lombardi, 2002; Yadav et al., 2015). Since EXE has an androstenedione-like structure, it interacts with the substrate binding site of aromatase, forming a reactive intermediate that covalently binds to the enzyme and causes time-dependent (irreversible) inhibition (TDI) (Lombardi, 2002). EXE has shown superior clinical efficacy with fewer life-threatening side effects, such as endometrial cancers and thromboembolic complications, as compared with the commonly used selective estrogen modulator, tamoxifen(Coombes et al., 2007; Goss et al., 2011). In addition, results from the international Mammary Prevention 3 trial show that EXE reduced the risk of breast cancer by 65% in postmenopausal women who were at high risk for developing breast cancer based on their Gail risk score (Goss et al., 2011). Recently, updated clinical practice guidelines suggest that EXE be prescribed as an extended therapy (up to 10 years) for postmenopausal women who have been diagnosed with lymph node–positive ER+ breast cancer (Burstein et al., 2019; Wazir et al., 2019).

Despite its effectiveness at reducing circulating estrogen levels (85%–95% at a relatively low dose of 25 mg), EXE has a low absolute bioavailability of 5% due to extensive first-pass metabolism (Singh et al., 2009; Kittaneh and Glück, 2011). In phase I of its metabolic pathway, EXE is reduced to the active metabolite 17β-dihydroexemestane (17β-DHE) by hepatic cytosolic aldo-keto reductases (AKR1C) 1–4, cytochrome P450s (1A2, 2C8, 2C9, 2C19, 2D6, 3A4, and 3A5), and carbonyl reductase (Kamdem et al., 2011; Platt et al., 2016; Peterson et al., 2017b). Further metabolism of exemestane and 17β-DHE proceeds through the phase II pathways of glutathionylation and glucuronidation. Glutathionylation begins with the conjugation of the tripeptide glutathione (GSH) to EXE or 17β-DHE, primarily via glutathione S-transferase (GST) A1 (Teslenko et al., 2021). Subsequent stepwise removal of the glutamine via γ-glutamyl transferase enzymes and the glycine via dipeptidases results in a stable cysteine conjugate (Hinchman and Ballatori, 1994; Hayes et al., 2005; Luo et al., 2018b; Teslenko et al., 2021). In the glucuronidation pathway, 17β-DHE is conjugated with a glucuronic acid moiety via the UDP-glucuronosyltransferase 2B17 enzyme to form 17β-hydroxy-EXE-17-O-β-D-glucuronide [17β-DHE-Gluc (Sun et al., 2010; Luo et al., 2018a)].

Cysteine conjugates of EXE and 17β-DHE comprise the major metabolites in the plasma (combined = 35% of total EXE) and urine (combined = 77% of total EXE) of postmenopausal women diagnosed with ER+ breast cancer and taking EXE as a monotherapy for at least 28 days (Luo et al., 2018b). In the same patient population, 17β-DHE-Gluc comprised 35% of the total EXE metabolites in plasma and 26% in urine (Luo et al., 2018b). This compared with only 17% and 12% in the plasma and 1.7% and 0.14% in the urine for EXE and 17β-DHE, respectively (Luo et al., 2018b). The pharmacologic effect of EXE and 17β-DHE have been extensively studied (Peterson et al., 2017a). However, the contribution of the phase II metabolites [6-methylcysteinylandrosta-1,4-diene-3,17-dione (EXE-cys), 17β-DHE-cysteine (17β-DHE-cys), and 17β-DHE-Gluc] that comprise 71% of the total EXE metabolites in plasma have not been elucidated. The focus of the current study is to comprehensively evaluate the effects of EXE metabolites on the inhibition of the aromatase enzyme.

Materials and Methods

Chemicals and Materials

Sigma-Aldrich (St Louis, MO) supplied 4-andostene-3,17-dione, estrone, and estrone-2,3,4-13C3. Corning (Bedford, MA) provided the NADPH regeneration system (1.3 mM NADP, 3.3 mM 6-phosphate, and 0.4 U/ml glucose 6-phosphate dehydrogenase) and DMSO. The 17β-DHE-Gluc and 17β-DHE were purchased from Toronto Research Chemicals (Toronto, ON), and EXE was purchased from Cayman Chemical Company (Ann Arbor, MI). Liquid chromatography–mass spectrometry (LC-MS)-grade formic acid and methanol as well as Dulbecco’s modified Eagle’s medium cell culture media and geneticin were obtained from Thermo-Fisher Scientific (Waltham, MA), and LC-MS–grade ammonium formate was purchased from Sigma-Aldrich. Acquity UPLC BEH C18 columns (1.7µm 2.1 × 50 mm) were purchased from Waters (Milford, MA). Pooled human liver cytosol was obtained from Xenotech (Kansas City, KS). FBS was provided by Avantor (Radnor Township, PA) and Pierce BCA protein assay kits were purchased from Thermo-Fisher Scientific. All other chemicals and reagents were purchased from Thermo Fisher Scientific and Sigma-Aldrich.

Inhibition Assays of Aromatase Activity

HEK293 cells with stable overexpression of the wild-type aromatase enzyme [CYP19A1 (Peterson et al., 2017a)] were used for activity screening assays. The cells were grown in Dulbecco’s modified Eagle’s medium containing 350 µg/mL of geneticin and 10% FBS and harvested, and an S9 fraction was prepared using previously described protocols (Peterson et al., 2017a). The total protein concentration was determined using the Pierce BCA assay. Major phase II metabolites (17β-DHE-Gluc, EXE-cys, and 17β-DHE-cys) were screened as potential inhibitors of estrone formation in the S9 fraction of the aromatase-overexpressing cells, using EXE and its phase I metabolite 17β-DHE as positive controls. 17β-DHE-Gluc, EXE, and 17β-DHE were purchased commercially (described above), and EXE-cys and 17β-DHE-cys were chemically synthesized as previously described (Luo et al., 2018b). The inhibition screening assays included 125 µg of S9 protein from the aromatase-overexpressing cell line, 1 µM or 10 µM of inhibitor (EXE, 17β-DHE, or their metabolites), 5 µM androstenedione, 100 mM potassium phosphate buffer (pH 7.4), and 3 mM of magnesium chloride, in a final volume of 50 µL. The concentration of 5 µM androstenedione was chosen since it is significantly above the known Km of aromatase for androstenedione (Gibb and Lavoie, 1980; Sohl and Guengerich, 2010). Linear reaction conditions were tested with respect to both protein concentration and time, with 125 μg S9 protein and a 45-minute incubation time both well within the linear range of estrone formation. Methanol and DMSO concentrations (the vehicles for androstenedione and EXE and its metabolites) in the final assays were 1.2% and 0.4%, respectively. The NAPDH regeneration system was added to initiate the reaction, which was incubated for 45 minutes at 37°C. An assay without EXE, 17β-DHE, or EXE metabolite was used as the reference for uninhibited (100%) enzyme activity. Negative controls included 1) an incubation with S9 fraction from the parent HEK293 cell line without the overexpressed aromatase enzyme and 2) an incubation with no NADPH regeneration system. After incubation, reactions were quenched with 50 µL cold acetonitrile spiked with 0.4 ppm of internal standard (estrone-2,3,4-13C3) and centrifuged at 4°C for 15 minutes at 13,200g. Supernatants were transferred to a glass vial for analysis by ultraperformance LC-MS. Estrone formation (in ppm) was quantified using a standard curve with serial dilutions of known estrone concentrations. All experiments were performed in triplicate.

Anti-aromatase Activity Determinations in Ovarian Tissue

Three normal ovarian tissue specimens were obtained from the Cooperative Human Tissue Network. All three specimens were from Caucasian female donors (30–40 years old) and were flash frozen within 2 hours of removal. Each frozen tissue (250 mg) was placed in a 2 mL tube with 750 µL of homogenizing buffer (25 mM Tris base, 138 mM NaCl, 2.7 mM KCl, pH 7.4) and a 5 mm metal bead. Tissues were homogenized with a TissueLyzer for 2 minutes at 20 Hz, followed by five freeze/thaw cycles (frozen in liquid nitrogen followed by thawing in a 37°C water bath). Cell homogenate was further processed on ice in a Dounce homogenizer for 30 strokes. An S9 fraction was prepared by collecting supernatant after centrifugation of the cell homogenate at 9,000g for 30 minutes at 4°C. Total protein content in the S9 fraction was analyzed with the Pierce BCA kit. A pooled (n = 3) ovarian S9 fraction was prepared by combining aliquot containing 2 mg protein of each ovarian S9 fraction, and inhibition screening assays were performed with 1 µM and 10 µM EXE, 17β-DHE, or EXE-cys as described above, using a total of 300 µg of pooled ovarian S9 protein in each assay.

Ultraperformance LC-MS Methods for Estrone Detection

The method for estrone detection was optimized from a previously published study (Peterson et al., 2017a). Samples (3 µL) were injected onto an Acquity UPLC BEH C18 column (1.7 µm 2.1 × 50 mm) and analyzed using a XEVO G2-S QToF mass spectrometer coupled to an Acquity UPLC (Waters). An elution gradient of mobile phase A (0.1% formic acid in water) and mobile phase B (100% methanol) was used under the following conditions: 0.0–2.5 minutes at 57% mobile phase B (43% mobile phase A), followed by a linear gradient to 100% B from 2.5–4.0 minutes, and a re-equilibriation at 57% B from 4.0–6.5 minutes. The flow rate was set at 0.4 mL/min and the column temperature at 35°C, whereas the sample temperature was kept at 10°C. Estrone was detected with the XEVO G2-S QToF operating in the MS/MS mode, with an ESI probe operating in positive ion mode with a capillary voltage of 0.6 kV. Nitrogen was used for both the cone and desolvation gases, with flow rates of 50 and 800 L/h, respectively. The collision energy was tuned at 20V and the cone voltage at 20V for both estrone and estrone-2,3,4-13C3 detection. The following mass transitions were used for metabolite detection: androstenedione, m/z 287.20→287.20; estrone, m/z 271.17→133.07; and estrone-2,3,4-13C3, m/z 273.17→136.08. Androstenedione was monitored to make sure that it was added to all incubations and, more importantly, to make sure that it was observed at a different retention time than estrone, assuring that any measured estrone peaks were not potentially contaminated by signal interference from the androstenedione peak in the instrument.

IC50 and IC50 Shift Determinations for Aromatase Activity Inhibition

IC50 values were determined for EXE-cys in the S9 fraction of the HEK293 aromatase-overexpressing cell line. Multiple concentrations of EXE-cys, ranging from 0.01 to 100 µM, were analyzed in incubations containing androstenedione as described above. EXE and 17β-DHE IC50 values were also determined as positive controls and validation of the assay system, using EXE in concentrations ranging from 0.01 to 30 µM and 17β-DHE in concentrations ranging from 0.01 to 100 µM.

To determine if the inhibition occurred in a time-dependent manner, an IC50 shift assay was performed for all three compounds (EXE, 17β-DHE, and EXE-cys). S9 protein from the aromatase-overexpressing cell line was preincubated with NADPH and inhibitor (0.01 to 100 µM for 17β-DHE and EXE-cys, and 0.01 to 30 µM for EXE) for 30 minutes at 37°C. Androstenedione (5 µM) was then added, and the reaction was incubated for 45 minutes at 37°C. IC50 values were determined for incubations that contained the 30-minute preincubation step, as well as for incubations that contained no preincubation step. The ratio between the IC50 values with a 30-minute preincubation step and those with no preincubation step was calculated and presented as the IC50 shift. Per Food and Drug Administration (FDA) guidelines, if this ratio is greater than 1.5, the inhibition is considered time-dependent. All IC50 assays were performed in triplicate.

Data Analysis

Estrone formation was quantified in ppm using TargetLynx software (Waters). Values were exported and presented as percent relative activity = (estrone formation with EXE or EXE metabolite/uninhibited estrone formation) × 100. IC50 values were calculated using a three-parameter IC50 model using GraphPad Prism 7.04 software (GraphPad Software Inc., San Diego, CA) by plotting percent relative activity versus log concentration of inhibitor. The IC50 values were calculated in μM for each of the triplicate experiments and then averaged to give a mean IC50. cLogP values were obtained using ChemDraw software.

Results

The three major phase II metabolites of EXE (EXE-cys, 17β-DHE-cys, and 17β-DHE-Gluc) were assayed for potential inhibition of estrone formation in a HEK293 cell line overexpressing the aromatase enzyme (Peterson et al., 2017a). The parent drug, EXE, and its active phase I metabolite 17β-DHE were also assayed as known inhibitors of aromatase activity. Screening results (Fig. 1) showed that at 1 µM final concentration, EXE was the most potent inhibitor of estrone formation, resulting in a 51% decrease in aromatase activity (49% relative activity) as compared with incubations without inhibitor (i.e., EXE or its metabolites). At 1 µM, EXE-cys, 17β-DHE, and 17β-DHE-cys exhibited similar levels of inhibition of estrone formation (28, 28, and 22%, respectively) but at levels lower than that observed for EXE. One µM of 17β-DHE-Gluc exhibited minimal inhibition of estrone activity (<5%). At a 10 µM final concentration, EXE, 17β-DHE, and EXE-cys each exhibited a decrease in estrone formation as compared with incubations without inhibitor (87%, 63%, and 63%, respectively). In comparison, 10 µM 17β-DHE-cys exhibited only a 35% decrease in estrone formation and 17β-DHE-Gluc again exhibited minimal inhibition of estrone formation (i.e., <10%).

Fig. 1.
Fig. 1.

Inhibition of estrone formation by major phase I and phase II metabolites of EXE in S9 fractions of HEK293 cells overexpressing recombinant aromatase enzyme. Parent drug and metabolites were screened at 1 µM and 10 µM in assays containing 125 µg of S9 fraction from the aromatase-overexpressing cell line. Data (plus or minus S.D.) are presented as the percent relative activity = (estrone formation with metabolite)/(uninhibited estrone formation) × 100%, with assays performed in triplicate experiments.

According to the human protein atlas [http://www.proteinatlas.org (Uhlén et al., 2015)], ovaries have one of the highest levels of expression of the aromatase enzyme. Utilizing the S9 fraction from pooled ovarian tissues, anti-aromatase screening assays utilizing EXE, 17β-DHE, and EXE-cys as potential inhibitors showed similar inhibition patterns to those observed in the aromatase cell line (Fig. 2). At 1 µM and 10 µM, EXE inhibited estrone formation by 46% and 78%, respectively (54% and 22% relative activity) in pooled ovarian S9. Similarly, 17β-DHE and EXE-cys inhibited estrone formation in ovarian tissue S9 fractions by 21% and 29%, respectively, at 1 µM and 49% and 47%, respectively, at 10 µM.

Fig. 2.
Fig. 2.

Ex vivo screening of EXE, 17β-DHE, and EXE-cys in pooled S9 fraction from ovarian tissue. Parent drug and metabolites were screened at 1 µM and 10 µM in assays containing 300 µg of S9 fraction from ovarian tissue. Data (plus or minus S.D.) are presented as the percent relative activity = (estrone formation with metabolite)/(uninhibited estrone formation) × 100%, with assays performed in triplicate experiments.

Based on the screening results, IC50 values were determined for EXE and its metabolites that exhibited ≥50% inhibition (≤50% relative activity) of aromatase activity in S9 fractions of aromatase-overexpressing cells in the screening assays (EXE, 17β-DHE, and EXE-cys; Table 1). The major phase II metabolite EXE-cys exhibited an IC50 value of 16 ± 10 µM, which is approximately 12-fold higher than EXE (1.3 ± 0.28 µM), but <1.8-fold higher than the active phase I metabolite 17β-DHE (9.2 ± 2.7 µM). The previously reported IC50 values for EXE and 17β-DHE [0.92 ± 0.17 µM and 4.3 ± 0.56 µM, respectively (Peterson et al., 2017a)] are similar to the values reported in the present study. Representative IC50 curves of all three compounds are shown in Fig. 3.

Fig. 3.
Fig. 3.

Representative IC50 inhibition curves. Estrone formation was measured at varying concentrations of EXE, 17β-DHE, and EXE-cys. Transformed log (µM) concentrations of inhibitor are plotted against the percent relative estrone formation activity.

View this table:
TABLE 1

IC50 (µM) and IC50 shift values of EXE and EXE metabolites using S9 fractions of HEK293 cells overexpressing recombinant aromatase enzyme

All three compounds (EXE, 17β-DHE, and EXE-cys) were further evaluated for TDI in an IC50 shift assay. An IC50 shift of greater than 1.5-fold is indicative of TDI (Berry and Zhao, 2008). In the present study, EXE, a known time-dependent irreversible inhibitor of aromatase (Giudici et al., 1988; Zilembo et al., 1995; Lombardi, 2002), exhibited an IC50 shift of 36 ± 12, well above the suggested 1.5-fold cutoff (Table 1). The major metabolites 17β-DHE and EXE-cys exhibited IC50 shifts of 5.0 ± 2.5 and 13 ± 10, respectively, indicating that these inhibitors also act in a time-dependent manner (Table 1). Representative curves for the IC50 shift assays are shown in Fig. 4.

Fig. 4.
Fig. 4.

Representative IC50 shift curves. Red lines represent the IC50 curves following a 30-minute preincubation with inhibitor (i.e., activation step). Black lines represent IC50 curves with no preincubation (i.e., no activation step).

Discussion

The present study is the first to comprehensively examine the major metabolites of EXE (17β-DHE, EXE-cys, 17β-DHE-cys, and 17β-DHE-Gluc) for inhibition potential of the aromatase enzyme, a key target for the treatment and prevention of ER+ breast cancer. Previous studies have focused mainly on the inhibition kinetics of the parent drug, EXE, and its phase I metabolite 17β-DHE (Buzzetti et al., 1993; Hong et al., 2007; Peterson et al., 2017a). However, the phase II metabolites (EXE-cys, 17β-DHE-cys, and 17β-DHE-Gluc) combine for a total of 71% of total EXE constituents (i.e., EXE plus its metabolites) observed in the plasma of patients taking EXE as compared with 29% for the parent EXE and its phase I active metabolite 17β-DHE (Luo et al., 2018b). According to FDA guidelines for the safety testing of drug metabolites, any metabolite that is present in the plasma at greater than 10% at steady state should be further tested for pharmacologic activity at the therapeutic target receptor or enzyme (Schadt et al., 2018). The three phase II metabolites EXE-cys, 17β-DHE-cys, and 17β-DHE-Gluc are present in the plasma at 23%, 12%, and 36%, respectively, warranting a comprehensive aromatase inhibition study of these metabolites.

EXE-cys, a phase II metabolite, was shown to inhibit estrogen formation by 63% at 10 µM, which was similar to the level of inhibition observed for the phase I metabolite 17β-DHE. The remaining major phase II metabolites did not reach inhibition levels of greater than 50% at 10 µM concentrations. By comparison, the level of estrogen formation inhibition observed for the parent drug, EXE, at this concentration was 1.4-fold higher than EXE-cys or 17β-DHE, and 2.5- and 11-fold higher than 17β-DHE-cys and 17β-DHE-Gluc, respectively. This pattern was similar to that observed in S9 fractions of ovarian tissue, where EXE-cys inhibited estrone formation to a similar extent as 17β-DHE, with both metabolites approaching 50% inhibition at 10 µM. Inhibition of estrone formation in ovary S9 fractions by the parent drug, EXE, was also similar to that observed in the aromatase-overexpressing cell line and was 1.6-fold higher than the inhibition observed with EXE-cys. Although the calculated IC50 values for EXE-cys were 12-fold higher than the parent EXE, it was only marginally higher than that observed for 17β-DHE (1.77-fold). Together, these data suggest that EXE-cys exhibits significant anti-aromatase activity.

The mean circulating levels of EXE-cys in the plasma of patients with ER+ breast cancer taking EXE (22 nM) is approximately 9-fold higher than 17β-DHE and 1.6-fold higher than the parent EXE [14 nM (Luo et al., 2018b)]. Although in vitro and in vivo values cannot be directly compared, the ratio of the steady-state plasma concentrations (Css) of 17β-DHE, EXE, and EXE-cys quantified in our previous studies (Luo et al., 2018b) versus the in vitro IC50 values calculated in the present study (i.e., Css/IC50; Table 2) indicates that EXE-cys is approximately 7-fold lower than EXE but nearly 5-fold higher than that observed for 17β-DHE. Although the Css/IC50 ratio does not take into account the unbound in vivo concentration, the cLogP value observed for EXE-cys (0.31) is ∼10-fold lower than that observed for EXE or 17β-DHE, which suggests that the cysteine conjugate is less lipophilic than either the parent drug or 17β-DHE and that the unbound fraction of EXE-cys is likely higher than EXE or 17β-DHE (Ghafourian and Amin, 2013). Therefore, our in vitro study suggests that EXE-cys may possess a similar in vivo inhibitory potency as compared with EXE, further supporting an important role for EXE-cys in the overall inhibition of aromatase by EXE. Experiments designed to examine the free fraction of EXE versus its major metabolites will be required to establish more definitively their individual contribution to overall EXE clinical efficacy.

View this table:
TABLE 2

Ratios of steady state plasma concentrations observed in vivoa versus IC50 values observed in the present study for EXE, 17β-DHE, and EXE-cys

Because EXE is a known time-dependent inhibitor, both EXE-cys and 17β-DHE were screened for TDI (Lombardi, 2002). The IC50 shift assays show a 13- and 5-fold decrease in the IC50 for EXE-cys and 17β-DHE, respectively, which far exceeds the FDA’s 1.5-fold threshold for a TDI, confirming that both EXE-cys and 17β-DHE also act as time-dependent inhibitors of aromatase (Berry and Zhao, 2008). These results suggest that EXE-cys is a phase II metabolite that contributes to the inhibitory pharmacologic effect on the target aromatase enzyme through a similar mechanism as the parent drug, EXE.

The mechanism of action for the parent EXE against aromatase is suicide inhibition. Structurally, EXE resembles hormonal substrates of the aromataselike androstenedione , with the key difference of an additional 1,2 double bond that prevents full aromatization by the formation of a covalent bond with the enzyme (Lombardi, 2002). All three phase II metabolites, EXE-cys, 17β-DHE-cys, and 17β-DHE-Gluc, have 1,2 double bonds that are essential for the inactivation of aromatase. An explanation for why enzyme inhibition was observed for cysteine conjugates but not the glucuronide conjugate may be conjugate location on the steroid ring of the EXE molecule. Previous studies have shown that C-6 substitutions on the substrate androstenedione do not typically interfere with the binding site of aromatase but protrude outside the aromatase cavity, whereas the C-17 position is important for hydrogen bond formation in the hydrophobic binding pocket of aromatase (Ghosh et al., 2012; Yadav et al., 2015). This is consistent with cysteine and glucuronide conjugate structures, with the cysteine conjugates of EXE and 17β-DHE being located on the C-6 position, whereas the glucuronide conjugate is located on the C-17 position (Luo et al., 2018b).

The EXE-cys metabolite is formed via conjugation of EXE with the tripeptide GSH (γ-glu-cys-gly), catalyzed mainly by the GSTA1 enzyme, with subsequent removal of the glutamyl and glycyl groups by γ-glutamyl transferase and dipeptidases (Teslenko et al., 2021). Phase II biotransformation reactions are generally detoxifying steps in the metabolism of drugs, which make most drugs more soluble and yield pharmacologically inactive metabolites (Jancova et al., 2010). However, in some cases, conjugated products may be pharmacologically active (Obach, 2013). One example of biotransformation that leads to pharmacologically active phase II metabolites is the glucuronidation of morphine to morphine-6-glucuronide (M6G), a metabolite with a similar affinity to the target μ-opioid receptor and results in similar analgesic effects (Mulder, 1992; Kilpatrick and Smith, 2005). Interestingly, M6G has a better safety profile then its parent drug, morphine (Mulder, 1992). In clinical studies, healthy volunteers who received M6G intravenously had fewer side effects, such as nausea and vomiting, compared with volunteers who received morphine (Romberg et al., 2004), and M6G was found to be 19–50 times less likely to cause respiratory depression (Romberg et al., 2003).

However, not all active metabolites lead to improved efficacy with fewer severe adverse events. Reactive metabolites are a major concern in drug discovery and development as well as in the clinic. Most commonly, these metabolites contain electrophilic moieties that can form covalent bonds with nonspecific cellular proteins, causing idiosyncratic drug-induced liver injury (Dahal et al., 2013; Gómez-Lechón et al., 2016). The marker for a parent drug or reactive metabolite’s potential to form nonspecific covalent bonds is its ability to form conjugates with GSH or cysteine since electrophiles are trapped by the thiol residues of GSH or L-cysteine (Gómez-Lechón et al., 2016). Acetaminophen (APAP) hepatotoxicity is one of the most well known examples, in which the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQ) forms the glutathione conjugate APAP-GSH. When APAP is taken at more than 4 g/day, the excess of NAPQ leads to the depletion of GSH stores. NAPQ starts forming protein adducts through nonspecific binding with the cysteine groups on the mitochondrial proteins, leading to mitochondrial oxidative stress and programmed necrosis (Albano et al., 1985; Mazaleuskaya et al., 2015; Ramachandran and Jaeschke, 2018). In contrast, despite the presence of cysteine conjugates as two of its major phase II metabolites, EXE has a low covalent binding burden (under 10 mg/day), meaning this drug has low nonspecific binding in vivo (Dahal et al., 2013). In addition, EXE is well tolerated, with mild to moderate adverse events and few hepatotoxicity events (Bao et al., 2010; Goss et al., 2011). However, EXE is taken as an adjuvant therapy in combination with chemotherapy and as a chronic treatment [up to 10 years for the prevention of breast cancer recurrence (Burstein et al., 2019; Wazir et al., 2019)]. Long-term EXE treatment and chemotherapy have been shown to induce GST expression and can lead to GSH depletion and potential toxicities or adverse events that were not previously elucidated (Townsend and Tew, 2003; Pljesa-Ercegovac et al., 2018). Interestingly, the discovery of cysteine conjugates as the major metabolite of EXE occurred in patients undergoing chronic dosing regiments (Luo et al., 2018b). Earlier studies with acute dosing did not identify cysteine conjugates, suggesting a possibility for metabolite accumulation and the potential for significant therapeutic or adverse effects and drug-drug interactions that have not yet been elucidated (http://www.pfizer.com/files/products/uspi_aromasin.pdf).

The present study suggests that the biotransformation of EXE to EXE-cys results in an active metabolite capable of TDI of the target aromatase enzyme. Since EXE is extensively metabolized by first-pass metabolism, the EXE-cys metabolite may play an important role in contributing to the observed 95% decrease in the circulating levels of estrogen in vivo in patients taking EXE (Singh et al., 2009; Kittaneh and Glück, 2011). Further studies examining the off-target effects of EXE-cys, the potential for drug-drug interactions with major chemotherapeutic agents prescribed in combination with EXE, and the contribution of EXE-cys to overall patient response to EXE will be important to more fully understand EXE’s mechanism of action and drug efficacy.

Acknowledgments

The authors would like to thank Shaman Luo and Bhagwat Prasad for their helpful insight and contributions to the study.

Authorship Contributions

Participated in research design: Teslenko, Lazarus.

Conducted experiments: Teslenko.

Performed data analysis: Teslenko.

Wrote or contributed to the writing of the manuscript: Teslenko, Watson, Chen, Lazarus.

Footnotes

    • Received April 20, 2022.
    • Accepted August 5, 2022.

  • These studies were made possible through the gracious support of the Public Health Service, the US Department of Health and Human Services, and National Institutes of Health National Cancer Institute [Grant 1R01-CA164366-01A1] (to P.L.).

  • There is no conflict of interest for any of the authors for this work.

  • dx.doi.org/10.1124/molpharm.122.000545.

Abbreviations

17β-DHE
17β-dihydroexemestane
17β-DHE-cysteine
17β-DHE-cys
17β-DHE-Gluc
17β-hydroxy-EXE-17-O-b-D-glucuronide
APAP
acetaminophen
Css
steady-state plasma concentration
ER
estrogen receptor
EXE
exemestane
EXE-cys
6-methylcysteinylandrosta-1, 4-diene-3, 17-dione, FDA, Food and Drug Administration
GSH
glutathione
GST
glutathione S-transferase
LC-MS
liquid chromatography–mass spectrometry
M6G
morphine-6-glucuronide
NAPQ
N-acetyl-p-benzoquinone imine
TDI
time-dependent inhibition

This is an open access article distributed under the CC BY-NC Attribution 4.0 International license.

References

  1. ↵
    1. Albano E,
    2. Rundgren M,
    3. Harvison PJ,
    4. Nelson SD, and
    5. Moldéus P
    (1985) Mechanisms of N-acetyl-p-benzoquinone imine cytotoxicity. Mol Pharmacol 28:306–311.
    OpenUrlAbstract
  2. ↵
    1. Bao T,
    2. Fetting J,
    3. Mumford L,
    4. Zorzi J,
    5. Shahverdi K,
    6. Jeter S,
    7. Herlong F,
    8. Stearns V, and
    9. Lee L
    (2010) Severe prolonged cholestatic hepatitis caused by exemestane. Breast Cancer Res Treat 121:789–791.
    OpenUrlPubMed
  3. ↵
    1. Berry LM and
    2. Zhao Z
    (2008) An examination of IC50 and IC50-shift experiments in assessing time-dependent inhibition of CYP3A4, CYP2D6 and CYP2C9 in human liver microsomes. Drug Metab Lett 2:51–59.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Burstein HJ,
    2. Lacchetti C,
    3. Anderson H,
    4. Buchholz TA,
    5. Davidson NE,
    6. Gelmon KA,
    7. Giordano SH,
    8. Hudis CA,
    9. Solky AJ,
    10. Stearns V, et al.
    (2019) Adjuvant Endocrine Therapy for Women With Hormone Receptor-Positive Breast Cancer: ASCO Clinical Practice Guideline Focused Update. J Clin Oncol 37:423–438.
    OpenUrlCrossRefPubMed
  5. ↵
    1. Buzzetti F,
    2. Di Salle E,
    3. Longo A, and
    4. Briatico G
    (1993) Synthesis and aromatase inhibition by potential metabolites of exemestane (6-methylenandrosta-1,4-diene-3,17-dione). Steroids 58:527–532.
    OpenUrlCrossRefPubMed
  6. ↵
    1. Campos SM
    (2004) Aromatase inhibitors for breast cancer in postmenopausal women. Oncologist 9:126–136.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    1. Coombes RC,
    2. Kilburn LS,
    3. Snowdon CF,
    4. Paridaens R,
    5. Coleman RE,
    6. Jones SE,
    7. Jassem J,
    8. Van de Velde CJ,
    9. Delozier T,
    10. Alvarez I, et al
    ; Intergroup Exemestane Study (2007) Survival and safety of exemestane versus tamoxifen after 2-3 years’ tamoxifen treatment (Intergroup Exemestane Study): a randomised controlled trial. Lancet 369:559–570.
    OpenUrlCrossRefPubMed
  8. ↵
    1. Dahal UP,
    2. Obach RS, and
    3. Gilbert AM
    (2013) Benchmarking in vitro covalent binding burden as a tool to assess potential toxicity caused by nonspecific covalent binding of covalent drugs. Chem Res Toxicol 26:1739–1745.
    OpenUrlCrossRefPubMed
  9. ↵
    Early Breast Cancer Trialists’ Collaborative Group (EBCTCG) (2015) Aromatase inhibitors versus tamoxifen in early breast cancer: patient-level meta-analysis of the randomised trials. Lancet 386:1341–1352.
    OpenUrlCrossRefPubMed
  10. ↵
    1. Ghafourian T and
    2. Amin Z
    (2013) QSAR models for the prediction of plasma protein binding. Bioimpacts 3:21–27.
    OpenUrl
  11. ↵
    1. Ghosh D,
    2. Lo J,
    3. Morton D,
    4. Valette D,
    5. Xi J,
    6. Griswold J,
    7. Hubbell S,
    8. Egbuta C,
    9. Jiang W,
    10. An J, et al.
    (2012) Novel aromatase inhibitors by structure-guided design. J Med Chem 55:8464–8476.
    OpenUrlCrossRefPubMed
  12. ↵
    1. Gibb W and
    2. Lavoie JC
    (1980) Substrate specificity of the placental microsomal aromatase. Steroids 36:507–519.
    OpenUrlCrossRefPubMed
  13. ↵
    1. Giudici D,
    2. Ornati G,
    3. Briatico G,
    4. Buzzetti F,
    5. Lombardi P, and
    6. di Salle E
    (1988) 6-Methylenandrosta-1,4-diene-3,17-dione (FCE 24304): a new irreversible aromatase inhibitor. J Steroid Biochem 30:391–394.
    OpenUrlCrossRefPubMed
  14. ↵
    1. Gómez-Lechón MJ,
    2. Tolosa L, and
    3. Donato MT
    (2016) Metabolic activation and drug-induced liver injury: in vitro approaches for the safety risk assessment of new drugs. J Appl Toxicol 36:752–768.
    OpenUrl
  15. ↵
    1. Goss PE,
    2. Ingle JN,
    3. Alés-Martínez JE,
    4. Cheung AM,
    5. Chlebowski RT,
    6. Wactawski-Wende J,
    7. McTiernan A,
    8. Robbins J,
    9. Johnson KC,
    10. Martin LW, et al
    ; NCIC CTG MAP.3 Study Investigators (2011) Exemestane for breast-cancer prevention in postmenopausal women. N Engl J Med 364:2381–2391.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Hayes JD,
    2. Flanagan JU, and
    3. Jowsey IR
    (2005) Glutathione transferases. Annu Rev Pharmacol Toxicol 45:51–88.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Hinchman CA and
    2. Ballatori N
    (1994) Glutathione conjugation and conversion to mercapturic acids can occur as an intrahepatic process. J Toxicol Environ Health 41:387–409.
    OpenUrlCrossRefPubMed
  18. ↵
    1. Hong Y,
    2. Yu B,
    3. Sherman M,
    4. Yuan Y-C,
    5. Zhou D, and
    6. Chen S
    (2007) Molecular basis for the aromatization reaction and exemestane-mediated irreversible inhibition of human aromatase. Mol Endocrinol 21:401–414.
    OpenUrlCrossRefPubMed
  19. ↵
    1. Jancova P,
    2. Anzenbacher P, and
    3. Anzenbacherova E
    (2010) Phase II drug metabolizing enzymes. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 154:103–116.
    OpenUrlCrossRefPubMed
  20. ↵
    1. Kamdem LK,
    2. Flockhart DA, and
    3. Desta Z
    (2011) In vitro cytochrome P450-mediated metabolism of exemestane. Drug Metab Dispos 39:98–105.
    OpenUrl
  21. ↵
    1. Kilpatrick GJ and
    2. Smith TW
    (2005) Morphine-6-glucuronide: actions and mechanisms. Med Res Rev 25:521–544.
    OpenUrlCrossRefPubMed
  22. ↵
    1. Kittaneh M and
    2. Glück S
    (2011) Exemestane in the adjuvant treatment of breast cancer in postmenopausal women. Breast Cancer (Auckl) 5:209–226.
    OpenUrlPubMed
  23. ↵
    1. Lombardi P
    (2002) Exemestane, a new steroidal aromatase inhibitor of clinical relevance. Biochim Biophys Acta 1587:326–337.
    OpenUrlPubMed
  24. ↵
    1. Luo S,
    2. Chen G,
    3. Truica C,
    4. Baird CC,
    5. Leitzel K, and
    6. Lazarus P
    (2018a) Role of the UGT2B17 deletion in exemestane pharmacogenetics. Pharmacogenomics J 18:295–300.
    OpenUrl
  25. ↵
    1. Luo S,
    2. Chen G,
    3. Truica CI,
    4. Baird CC,
    5. Xia Z, and
    6. Lazarus P
    (2018b) Identification and Quantification of Novel Major Metabolites of the Steroidal Aromatase Inhibitor, Exemestane. Drug Metab Dispos 46:1867–1878.
    OpenUrlAbstract/FREE Full Text
  26. ↵
    1. Mazaleuskaya LL,
    2. Sangkuhl K,
    3. Thorn CF,
    4. FitzGerald GA,
    5. Altman RB, and
    6. Klein TE
    (2015) PharmGKB summary: pathways of acetaminophen metabolism at the therapeutic versus toxic doses. Pharmacogenet Genomics 25:416–426.
    OpenUrlCrossRef
  27. ↵
    1. Mulder GJ
    (1992) Pharmacological effects of drug conjugates: is morphine 6-glucuronide an exception? Trends Pharmacol Sci 13:302–304.
    OpenUrlCrossRefPubMed
  28. ↵
    1. Obach RS
    (2013) Pharmacologically active drug metabolites: impact on drug discovery and pharmacotherapy. Pharmacol Rev 65:578–640.
    OpenUrlAbstract/FREE Full Text
  29. ↵
    1. Osborne CK and
    2. Schiff R
    (2011) Mechanisms of endocrine resistance in breast cancer. Annu Rev Med 62:233–247.
    OpenUrlCrossRefPubMed
  30. ↵
    1. Peterson A,
    2. Xia Z,
    3. Chen G, and
    4. Lazarus P
    (2017a) Exemestane potency is unchanged by common nonsynonymous polymorphisms in CYP19A1: results of a novel anti-aromatase activity assay examining exemestane and its derivatives. Pharmacol Res Perspect 5:e00313.
    OpenUrl
  31. ↵
    1. Peterson A,
    2. Xia Z,
    3. Chen G, and
    4. Lazarus P
    (2017b) In vitro metabolism of exemestane by hepatic cytochrome P450s: impact of nonsynonymous polymorphisms on formation of the active metabolite 17β-dihydroexemestane. Pharmacol Res Perspect 5:e00314.
    OpenUrl
  32. ↵
    1. Platt A,
    2. Xia Z,
    3. Liu Y,
    4. Chen G, and
    5. Lazarus P
    (2016) Impact of nonsynonymous single nucleotide polymorphisms on in-vitro metabolism of exemestane by hepatic cytosolic reductases. Pharmacogenet Genomics 26:370–380.
    OpenUrl
  33. ↵
    1. Pljesa-Ercegovac M,
    2. Savic-Radojevic A,
    3. Matic M,
    4. Coric V,
    5. Djukic T,
    6. Radic T, and
    7. Simic T
    (2018) Glutathione Transferases: Potential Targets to Overcome Chemoresistance in Solid Tumors. Int J Mol Sci 19:3785.
    OpenUrl
  34. ↵
    1. Ramachandran A and
    2. Jaeschke H
    (2018) Acetaminophen Toxicity: Novel Insights Into Mechanisms and Future Perspectives. Gene Expr 18:19–30.
    OpenUrl
  35. ↵
    1. Ramchand SK,
    2. Cheung YM,
    3. Yeo B, and
    4. Grossmann M
    (2019) The effects of adjuvant endocrine therapy on bone health in women with breast cancer. J Endocrinol 241:R111–R124.
    OpenUrl
  36. ↵
    1. Robertson JFR,
    2. Bondarenko IM,
    3. Trishkina E,
    4. Dvorkin M,
    5. Panasci L,
    6. Manikhas A,
    7. Shparyk Y,
    8. Cardona-Huerta S,
    9. Cheung KL,
    10. Philco-Salas MJ, et al.
    (2016) Fulvestrant 500 mg versus anastrozole 1 mg for hormone receptor-positive advanced breast cancer (FALCON): an international, randomised, double-blind, phase 3 trial. Lancet 388:2997–3005.
    OpenUrlCrossRefPubMed
  37. ↵
    1. Romberg R,
    2. Olofsen E,
    3. Sarton E,
    4. den Hartigh J,
    5. Taschner PE, and
    6. Dahan A
    (2004) Pharmacokinetic-pharmacodynamic modeling of morphine-6-glucuronide-induced analgesia in healthy volunteers: absence of sex differences. Anesthesiology 100:120–133.
    OpenUrlCrossRefPubMed
  38. ↵
    1. Romberg R,
    2. Olofsen E,
    3. Sarton E,
    4. Teppema L, and
    5. Dahan A
    (2003) Pharmacodynamic effect of morphine-6-glucuronide versus morphine on hypoxic and hypercapnic breathing in healthy volunteers. Anesthesiology 99:788–798.
    OpenUrlCrossRefPubMed
  39. ↵
    1. Schadt S,
    2. Bister B,
    3. Chowdhury SK,
    4. Funk C,
    5. Hop CECA,
    6. Humphreys WG,
    7. Igarashi F,
    8. James AD,
    9. Kagan M,
    10. Khojasteh SC, et al.
    (2018) A Decade in the MIST: Learnings from Investigations of Drug Metabolites in Drug Development under the “Metabolites in Safety Testing” Regulatory Guidance. Drug Metab Dispos 46:865–878.
    OpenUrlAbstract/FREE Full Text
  40. ↵
    1. Siegel RL,
    2. Miller KD,
    3. Fuchs HE, and
    4. Jemal A
    (2022) Cancer statistics, 2022. CA Cancer J Clin 72:7–33.
    OpenUrlCrossRefPubMed
  41. ↵
    1. Singh AK,
    2. Chaurasiya A,
    3. Awasthi A,
    4. Mishra G,
    5. Asati D,
    6. Khar RK, and
    7. Mukherjee R
    (2009) Oral bioavailability enhancement of exemestane from self-microemulsifying drug delivery system (SMEDDS). AAPS PharmSciTech 10:906–916.
    OpenUrlCrossRefPubMed
  42. ↵
    1. Sohl CD and
    2. Guengerich FP
    (2010) Kinetic analysis of the three-step steroid aromatase reaction of human cytochrome P450 19A1. J Biol Chem 285:17734–17743.
    OpenUrlAbstract/FREE Full Text
  43. ↵
    1. Sun D,
    2. Chen G,
    3. Dellinger RW,
    4. Sharma AK, and
    5. Lazarus P
    (2010) Characterization of 17-dihydroexemestane glucuronidation: potential role of the UGT2B17 deletion in exemestane pharmacogenetics. Pharmacogenet Genomics 20:575–585.
    OpenUrlCrossRefPubMed
  44. ↵
    1. Teslenko I,
    2. Watson CJW,
    3. Xia Z,
    4. Chen G, and
    5. Lazarus P
    (2021) Characterization of Cytosolic Glutathione S-Transferases Involved in the Metabolism of the Aromatase Inhibitor, Exemestane. Drug Metab Dispos 49:1047–1055.
    OpenUrlAbstract/FREE Full Text
  45. ↵
    1. Townsend DM and
    2. Tew KD
    (2003) The role of glutathione-S-transferase in anti-cancer drug resistance. Oncogene 22:7369–7375.
    OpenUrlCrossRefPubMed
  46. ↵
    1. Uhlén M,
    2. Fagerberg L,
    3. Hallström BM,
    4. Lindskog C,
    5. Oksvold P,
    6. Mardinoglu A,
    7. Sivertsson Å,
    8. Kampf C,
    9. Sjöstedt E,
    10. Asplund A, et al.
    (2015) Proteomics. Tissue-based map of the human proteome. Science 347:1260419.
    OpenUrlAbstract/FREE Full Text
  47. ↵
    1. Untch M and
    2. Thomssen C
    (2010) Clinical practice decisions in endocrine therapy. Cancer Invest 28 (Suppl 1):4–13.
    OpenUrl
  48. ↵
    1. Wazir U,
    2. Mokbel L,
    3. Wazir A, and
    4. Mokbel K
    (2019) Optimizing adjuvant endocrine therapy for early ER+ breast cancer: An update for surgeons. Am J Surg 217:152–155.
    OpenUrl
  49. ↵
    1. Yadav MR,
    2. Barmade MA,
    3. Tamboli RS, and
    4. Murumkar PR
    (2015) Developing steroidal aromatase inhibitors-an effective armament to win the battle against breast cancer. Eur J Med Chem 105:1–38.
    OpenUrl
  50. ↵
    1. Zilembo N,
    2. Noberasco C,
    3. Bajetta E,
    4. Martinetti A,
    5. Mariani L,
    6. Orefice S,
    7. Buzzoni R,
    8. Di Bartolomeo M,
    9. Di Leo A,
    10. Laffranchi A, et al.
    (1995) Endocrinological and clinical evaluation of exemestane, a new steroidal aromatase inhibitor. Br J Cancer 72:1007–1012.
    OpenUrlPubMed
Back to top

In this issue

Download PDF
Article Alerts
Email Article
Citation Tools

Research ArticleArticle

Anti-aromatase Activity of Exemestane Phase II Metabolites

Irina Teslenko, Christy J.W. Watson, Gang Chen and Philip Lazarus
Molecular Pharmacology November 1, 2022, 102 (5) 216-222; DOI: https://doi.org/10.1124/molpharm.122.000545
Reddit logo Twitter logo Facebook logo Mendeley logo

Jump to section

Advertisement