Computer-aided structure analysis of an epimerized dehydroepiandrosterone derivative and its biological effect in a model of reactive gliosis

The naturally occurring steroid dehydroepiandrosterone (DHEA) is reported to reduce glial fibrillary acidic protein (GFAP) overexpression in a model of reactive gliosis due to its conversion to estradiol by the enzyme aromatase. In the present study we examined the biological effect of a new epimerized derivative of DHEA, 16α-iodomethyl-13α-dehydroepiandrosterone derivative (16α-iodomethyl-13α-DHEAd, 16α-iodomethyl-13α-androst-5-en-3β,17β-diol), using the same model system, and compared the 3D structure of this molecule with that of DHEA and two steroidal type aromatase inhibitors, formestane and exemestane. The synthetic compound, in contrast to the reported effect of DHEA, was able to reduce GFAP overexpression only if the enzyme aromatase was inhibited. Data obtained from computational calculations fortified by X-ray crystallography revealed that contrary to the nearly planar sterane framework of DHEA, the synthetic derivative 16α-iodomethyl-13α-DHEAd has a bent sterane skeleton, resulting in a 3D structure that is similar to that of formestane or exemestane. Moreover, 16α-iodomethyl-13α-DHEAd resulted to be metabolically more stable than DHEA.The results suggest that epimerization of the sterane skeleton of DHEA inclines the plane of the D ring, leading to a significantly altered biological activity. The synthetic molecule has a DHEA-like effect on GFAP overexpression when the enzyme aromatase is inhibited and the naturally occurring DHEA is ineffective in this respect. On the other hand, based on their structural similarity it can be hypothesized that 16α-iodomethyl-13α-DHEAd applied alone might have a biological effect similar to that of formestane or exemestane.     

Celecoxib anti-aromatase neoadjuvant (CAAN) trial for locally advanced breast cancer

OBJECTIVES: To evaluate the efficacy and safety of combing aromatase inhibitor (AI) and cyclooxygenase-2 (COX-2) inhibitor neoadjuvantly in postmenopausal patients with invasive hormone-sensitive breast cancer. METHODS: Eighty-two patients were randomly assigned to receive exemestane 25mg daily and celecoxib 400mg twice daily (group A, n=30), exemestane 25mg daily (group B, n=24) and letrozole 2.5mg daily (group C, n=28). RESULTS: All groups showed clinical responses (58.6% for group A, 54.5% for group B and 62.0% for group C) and decrease in tumor area (61.8% for group A, 58.1% for group B and 55.7% for group C). 3 out of 5 patients with complete clinical response were observed from group A and 2 out of 69 patients operated with pathologic complete response were observed in group C. The mean microscopic tumor size was 2.53 cm for group A, 3.05 cm for group B and 2.10 cm for group C. The differences were only statistically significant when group C was compared with group B (P=0.025). The toxicity profiles among groups were satisfactory. CONCLUSION: AI is effective in treating breast cancer and may be safely used preoperatively. The addition of COX-2 inhibitor may provide additional benefit.     

Preclinical evaluation of exemestane as a novel chemotherapy for gastric cancer

CYP19A1/aromatase (Ar) is a prognostic biomarker of gastric cancer (GCa). Ar is a critical enzyme for converting androstenedione to oestradiol in the steroidogenesis cascade. For decades, Ar has been targeted with Ar inhibitors (ARIs) in gynaecologic malignancies; however, it is unexplored in GCa. A single‐cohort tissue microarray examination was conducted to study the association between Ar expression and disease outcome in Asian patients with GCa. The results revealed that Ar was a prognostic promoter. Bioinformatics analyses conducted on a Caucasian‐based cDNA microarray databank showed Ar to be positively associated with GCa prognosis for multiple clinical modalities, including surgery, 5‐Fluorouracil (5‐FU) for adjuvant chemotherapy, or HER2 positivity. These findings imply that targeting Ar expression exhibits a potential for fulfilling unmet medical needs. Hence, Ar‐targeting compounds were tested, and the results showed that exemestane exhibited superior cancer‐suppressing efficacy to other ARIs. In addition, exemestane down‐regulated Ar expression. Ablating Ar abundance with short hairpin (sh)Ar could also suppress GCa cell growth, and adding 5‐FU could facilitate this effect. Notably, adding oestradiol could not prevent exemestane or shAr effects, implicating a nonenzymatic mechanism of Ar in cancer growth. Regarding translational research, treatment with exemestane alone exhibited tumour suppression efficacy in a dose‐dependent manner. Combining subminimal doses of 5‐FU and exemestane exerted an excellent tumour suppression effect without influencing bodyweight. This study validated the therapeutic potentials of exemestane in GCa. Combination of metronomic 5‐FU and exemestane for GCa therapy is recommended.     

Celecoxib anti-aromatase neoadjuvant (CAAN) trial for locally advanced breast cancer: preliminary report

Anti-aromatase therapy is important in the treatment of breast cancer in postmenopausal women. Cyclooxygenase-2 (COX-2) inhibitors have been shown to be effective in chemoprevention in animal and clinical studies. A proof of principle study was performed to investigate the efficacy of combing anti-aromatase therapy (exemestane) and COX-2 inhibitors neoadjuvantly in hormone-sensitive postmenopausal breast cancers. The initial results are reported. The patients were randomly assigned to receive exemestane 25 mg daily and celecoxib 400 mg twice daily (group A), exemestane 25 mg daily (group B) and letrozole 2.5 mg daily (group C). The analysis was based on 20 patients who received at least one cycle of treatment. Fourteen patients completed two cycles and 12 patients three cycles. All groups showed clinical response and there was decrease in tumor area in each group. However, complete clinical response was only observed for group A patients. There was also progressive decline in blood CEA and CA15.3 levels but the differences between the three groups were not significant. The results of the preliminary analysis are encouraging but definitive conclusion could only be drawn after the completion of the study.     

Oral Bioavailability Enhancement of Exemestane from Self-Microemulsifying Drug Delivery System (SMEDDS)

Limited aqueous solubility of exemestane leads to high variability in absorption after oral administration. To improve the solubility and bioavailability of exemestane, the self-microemulsifying drug delivery system (SMEDDS) was developed. SMEDDS comprises of isotropic mixture of natural or synthetic oil, surfactant, and cosurfactant, which, upon dilution with aqueous media, spontaneously form fine o/w microemulsion with less than 100 nm in droplet size. Solubility of exemestane were determined in various vehicles. Ternary phase diagrams were plotted to identify the efficient self-emulsification region. Dilution studies, droplet size, and zeta potential of the formulations were investigated. The release of exemestane from SMEDDS capsules was studied using USP dissolution apparatus in different dissolution media and compared the release of exemestane from a conventional tablet. Oral pharmacokinetic study was performed in female Wistar rats (n = 8) at the dose of 30 mg kg⁻¹. The absorption of exemestane from SMEDDS form resulted in about 2.9-fold increase in bioavailability compared with the suspension. Our studies illustrated the potential use of SMEDDS for the delivery of hydrophobic compounds, such as exemestane by the oral route.Key words: bioavailability enhancement, exemestane, microemulsion, SMEDDS     

Detection of new exemestane metabolites by liquid chromatography interfaced to electrospray-tandem mass spectrometry

Exemestane is an irreversible aromatase inhibitor used for anticancer therapy. Unfortunately, this drug is also misused in sports to avoid some adverse effects caused by steroids administration. For this reason exemestane has been included in World Anti-Doping Agency prohibited list. Usually, doping control laboratories monitor prohibited substances through their metabolites, because parent compounds are readily metabolized. Thus metabolism studies of these substances are very important. Metabolism of exemestane in humans is not clearly reported and this drug is detected indirectly through analysis of its only known metabolite: 17β-hydroxyexemestane using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and gas chromatography coupled to mass spectrometry (GC-MS). This drug is extensively metabolized to several unknown oxidized metabolites. For this purpose LC-MS/MS has been used to propose new urinary exemestane metabolites, mainly oxidized in C6-exomethylene and simultaneously reduced in 17-keto group. Urine samples from four volunteers obtained after administration of a 25mg dose of exemestane were analyzed separately by LC-MS/MS. Urine samples of each volunteer were hydrolyzed followed by liquid-liquid extraction and injected into a LC-MS/MS system. Three unreported metabolites were detected in all urine samples by LC-MS/MS. The postulated structures of the detected metabolites were based on molecular formulae composition obtained through high accuracy mass determination by liquid chromatography coupled to hybrid quadrupole-time of flight mass spectrometry (LC-QTOF MS) (all mass errors below 2ppm), electrospray (ESI) product ion spectra and chromatographic behavior.     

Validation of a Rapid and Sensitive LC-MS/MS Method for Determination of Exemestane and Its Metabolites, 17β-Hydroxyexemestane and 17β-Hydroxyexemestane-17-O-β-D-Glucuronide: Application to Human Pharmacokinetics Study

A novel, rapid and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the evaluation of exemestane pharmacokinetics and its metabolites, 17β-dihydroexemestane (active metabolite) and 17β-dihydroexemestane-17-O-β-D-glucuronide (inactive metabolite) in human plasma. Their respective D3 isotopes were used as internal standards. Chromatographic separation of analytes was achieved using Thermo Fisher BDS Hypersil C18 analytic HPLC column (100 × 2.1 mm, 5 μm). The mobile phase was delivered at a rate of 0.5 mL/min by gradient elution with 0.1 % aqueous formic acid and acetonitrile. The column effluents were detected by API 4000 triple quadrupole mass spectrometer using electrospray ionisation (ESI) and monitored by multiple reaction monitoring (MRM) in positive mode. Mass transitions 297 > 121 m/z, 300 > 121 m/z, 299 > 135 m/z, 302 > 135 m/z, 475 > 281 m/z, and 478 > 284 m/z were monitored for exemestane, exemestane-d3, 17β-dihydroexemestane, 17β-dihydroexemestane-d3, 17β-dihydroexemestane-17-O-β-D-glucuronide, and 17β-dihydroexemestane-17-O-β-D-glucuronide-d3 respectively. The assay demonstrated linear ranges of 0.4 – 40.0 ng/mL, for exemestane; and 0.2 – 15.0 ng/mL, for 17β-dihydroexemestane and 17β-dihydroexemestane-17-O-β-D-glucuronide, with coefficient of determination (r²) of > 0.998. The precision (coefficient of variation) were ≤10.7%, 7.7% and 9.5% and the accuracies ranged from 88.8 to 103.1% for exemestane, 98.5 to 106.1% for 17β-dihydroexemestane and 92.0 to 103.2% for 17β-dihydroexemestane-17-O-β-D-glucuronide. The method was successfully applied to a pharmacokinetics/dynamics study in breast cancer patients receiving exemestane 25mg daily orally. For a representative patient, 20.7% of exemestane in plasma was converted into 17β-dihydroexemestane and 29.0% of 17β-dihydroexemestane was inactivated as 17β-dihydroexemestane-17-O-β-D-glucuronide 24 hours after ingestion of exemestane, suggesting that altered 17-dihydroexemestane glucuronidation may play an important role in determining effect of exemestane against breast cancer cells.     

Apoptosis and Autophagy in Breast Cancer Cells following Exemestane Treatment

Aromatase inhibitors (AIs), which block the conversion of androgens to estrogens, are used for hormone-dependent breast cancer treatment. Exemestane, a steroidal that belongs to the third-generation of AIs, is a mechanism-based inhibitor that binds covalently and irreversibly, inactivating and destabilizing aromatase. Since the biological effects of exemestane in breast cancer cells are not totally understood, its effects on cell viability, cell proliferation and mechanisms of cell death were studied in an ER-positive aromatase-overexpressing breast cancer cell line (MCF-7aro). The effects of 3-methyladenine (3-MA), an inhibitor of autophagy and of ZVAD-FMK, an apoptotic inhibitor, in exemestane treated cells were also investigated. Our results indicate that exemestane induces a strong inhibition in MCF-7aro cell proliferation in a dose- and time-dependent manner, promoting a significant cell cycle arrest in G(0)/G1 or in G(2)/M phases after 3 and 6 days of treatment, respectively. This was accompanied by a decrease in cell viability due to activation of cell death by apoptosis, via mitochondrial pathway and the occurrence of autophagy. Inhibition of autophagy by the autophagic inhibitor, 3-MA, resulted in a reduction of cell viability and activation of caspases. All together the results obtained suggest that exemestane induced mitochondrial-mediated apoptosis and autophagy, which act as a pro-survival process regulating breast cancer cell apoptosis.     

Exemestane Loaded Self-Microemulsifying Drug Delivery System (SMEDDS): Development and Optimization

The purpose of this research work was to formulate and characterize self-micro emulsifying drug delivery system containing exemestane. The solubility of exemestane was determined in various vehicles. Pseudo ternary phase diagram was used to evaluate the micro-emulsification existence area. SMEDDS formulations were tested for micro-emulsifying properties, and the resultant formulations loaded with exemestane (ME1, ME2, ME3, ME4 and ME5) were investigated for clarity, phase separation, globule size and shape, zeta potential, effect of various diluents and dilutions, thermodynamic and thermal stability. From the results it is concluded that increase in droplet size is proportional to the concentration of oil in SMEDDS formulation. Minor difference in the droplet size and zeta potential was observed by varying the diluents (deionized water and 0.1 N HCl) and dilutions (1:10, 1:50 and 1:100). Formulations, which were found to be thermodynamically stable (ME1, ME2, ME3 and ME4), were subjected to stability studies as per International Conference on Harmonization (ICH) guidelines. No significant variations were observed in the formulations over a period of 3 months at accelerated and long-term conditions. TEM photographs of microemulsions formulations further conformed the spherical shape of globules. Among the various SMEDDS formulations, ME4 offer the advantages of good clarity systems at high oil content and thus offer good solubilization of exemestane. Thus this study indicates that the SMEDDS can be used as a potential drug carrier for dissolution enhancement of exemestane and other lipophilic drug(s).     

Exemestane (FCE 24304), a new steroidal aromatase inhibitor.

Exemestane (FCE 24304; 6-methylenandrosta-1,4-diene-3,17-dione) is a novel orally active irreversible aromatase inhibitor. Its in vitro and in vivo pharmacological properties have been compared to 4-hydroxyandrostenedione (4-OHA). In preincubation studies with human placental aromatase, exemestane, like 4-OHA, showed enzyme inactivating properties with a similar affinity (Ki 26 vs 29 nM) and a lower rate of inactivation (t1/2 13.9 vs 2.1 min). Conversely, when tested in pregnant mares' serum gonadotropin-treated rats, exemestane was more potent in reducing microsomal ovarian aromatase activity than 4-OHA, after both subcutaneous (ED50 1.8 vs 3.1 mg/kg) and oral dosing (ED50 3.7 vs greater than 100 mg/kg). No interference of exemestane on desmolase or 5 alpha-reductase activity was found. The compound did not show any relevant binding affinity to steroidal receptors, but slight binding to the androgen receptor (approximately 0.2% of dihydrotestosterone), like 4-OHA. In the first phase I trial, healthy postmenopausal volunteers were given single oral doses of exemestane, ranging from 0.5 to 800 mg, and plasma [estrone (E1), estradiol (E2) and estrone sulphate (E1S)] and urinary estrogens (E1 and E2) were measured up to 5-8 days. The minimal effective dose in decreasing estrogens was 5 mg. At 25 mg the maximal suppression was observed at day 3: plasma estrogens fell to 35 (E1), 39 (E2) and 28% (E1S), and urinary estrogens fell to 20 (E1) and 25% (E2) of basal values, these effects still persisting on day 5. No effects on plasma levels of cortisol, aldosterone, 17-hydroxyprogesterone, DHEAS, LH and FSH, and no significant adverse events were observed up to the highest tested dose of 800 mg exemestane.     

Development and validation of a sensitive liquid chromatography/tandem mass spectrometry method for the determination of exemestane in human plasma

Exemestane, irreversible steroidal aromatase inhibitor, acts as a false substrate for aromatase enzyme and significantly lowers circulating estrogen concentrations in postmenopausal women with hormone-sensitive breast cancer. A sensitive bioanalytical method was developed and validated to study pharmacokinetics of exemestane. The method was based on liquid-liquid extraction of exemestane with methyl t-butyl ether followed by reversed-phase liquid chromatography. Positive electrospray ionization tandem mass spectrometry in multiple reaction monitoring mode was applied for detection of exemestane. Anastrozole was used as internal standard. Calibration curve, fitted to 1/x2 weighted linear regression model, was linear in the range of 0.1-40.0 ng/mL. Intra-run precision and accuracy were 1.80-3.17% and 103.4-111.5%, respectively. Inter-run precision and accuracy measured within 3 days were 3.37-4.19% and 101.8-109.6%, respectively. Extraction recoveries of exemestane and internal standard were 79.7-86.2% and 82.9-83.6%, respectively. The method was fully validated and may be applied to pharmacokinetic studies in humans after a single dose administration of 25mg exemestane tablets.     

Exemestane metabolites suppress growth of estrogen receptor-positive breast cancer cells by inducing apoptosis and autophagy: A comparative study with Exemestane

Around 60–80% of all breast tumors are estrogen receptor-positive. One of the several therapeutic approaches used for this type of cancers is the use of aromatase inhibitors. Exemestane is a third-generation steroidal aromatase inhibitor that undergoes a complex and extensive metabolism, being catalytically converted into chemically active metabolites. Recently, our group showed that the major exemestane metabolites, 17β-hydroxy-6-methylenandrosta-1,4-dien-3-one and 6-(hydroxymethyl)androsta-1,4,6-triene-3,17-dione, as well as, the intermediary metabolite 6β-Spirooxiranandrosta-1,4-diene-3,17-dione, are potent aromatase inhibitors in breast cancer cells. In this work, in order to better understand the biological mechanisms of exemestane in breast cancer and the effectiveness of its metabolites, it was investigated their effects in sensitive and acquired-resistant estrogen receptor-positive breast cancer cells. Our results indicate that metabolites induced, in sensitive breast cancer cells, cell cycle arrest and apoptosis via mitochondrial pathway, involving caspase-8 activation. Moreover, metabolites also induced autophagy as a promoter mechanism of apoptosis. In addition, it was demonstrated that metabolites can sensitize aromatase inhibitors-resistant cancer cells, by inducing apoptosis. Therefore, this study indicates that exemestane after metabolization originates active metabolites that suppress the growth of sensitive and resistant breast cancer cells. It was also concluded that, in both cell lines, the biological effects of metabolites are different from the ones of exemestane, which suggests that exemestane efficacy in breast cancer treatment may also be dependent on its metabolites.     

Design, synthesis and evaluation of novel 16-imidazolyl substituted steroidal derivatives possessing potent diversified pharmacological properties

As a part of our investigations into the structural-activity relationship studies of a novel class of medicinally active 16-substituted steroids, several new 16-imidazolyl substituted steroidal derivatives have been synthesized and pharmacologically evaluated in the current study. The new steroidal analogues 5, 6, 8, 9, 11 and 12 exhibited moderate cytotoxic effects in sixty cancer cell lines derived from nine cancers types. The imidazolyl substituted steroidal derivatives 6 (DPJ-RG-1241) and 7 (RB-401) were obtained as the powerful inhibitors of aromatase with IC50=0.18 μM and IC50=0.168 μM, respectively, approximately 1.2 and 1.4 times more potent in comparison to standard drug exemestane. The bis-quaternary steroids 13 and 14 displayed potent skeletal muscle relaxant properties. An affinity constant of 0.007 μM was observed for compound 14 on frog rectus abdominis muscle preparation and 13 displayed a very high anticholinesterase activity K(i)=25 nM, approximately 115-fold higher in comparison to standard drug galanthamine (K(i)=2.9 μM).     

New experimental models for aromatase inhibitor resistance

Clinical trials have demonstrated the importance of aromatase inhibitor (AI) therapy in the effective treatment of hormone-dependent breast cancers. In contrast to tamoxifen, an antagonist of the estrogen receptor (ER), AIs have shown to be better tolerated along with decreased recurrence rates of the disease. Currently, three third-generation AIs are being used: exemestane, letrozole, and anastrozole. Our laboratory is attempting to understand several aspects of AI functionality. In this paper, we first review recent findings from our structure–function studies of aromatase as well as the molecular characterization of the interaction between AIs and aromatase. Based on these studies, we propose new evidence for the interaction of letrozole and exemestane with aromatase. In addition, we will discuss recent results generated from our AI-resistant cell lines. Our laboratory has generated MCF-7aro cells that are resistant to letrozole, anastrozole, exemestane, and tamoxifen. Basic functional characterization of aromatase and ER in these resistant cell lines has been done and microarray analysis has been employed in order to better understand the mechanism responsible for AI resistance on a genome-wide scale. The results generated so far suggest the presence of at least four types of resistant cell lines. Overall, the information presented in this paper supplements our understanding of AI function, and such information can be valuable for the development of treatment strategies against AI resistant breast cancers.     

Exemestane experience in breast cancer treatment

Exemestane is a very potent, orally active, selective and long-lasting steroidal irreversible inhibitor of aromatase. It is 150 times more potent than aminoglutethimide (AG) in inhibiting human placental aromatase (K_i of 4.3 and 671 nM, respectively). The compound is presently under phase III evaluation in Europe and the U.S.A. for the treatment of postmenopausal advanced breast cancer (ABC). Clinical pharmacology studies have been carried out with single doses ranging from 0.5 to 800 mg and repeated doses of up to 600 mg a day, in 132 postmenopausal healthy volunteers and in 185 post-menopausal women with ABC. Results obtained using a very specific and sensitive analytical method (high performance liquid chromatography—radioimmunoassay; HPLC—RIA) indicated that exemestane is extremely effective in inhibiting plasma estrogens levels. Estrogen inhibition is clearly evident at 5 mg a day and maximal suppression for E₂, E₁ and E₁S (>85%, >90% and>90%, respectively) is obtained at 10–25 mg a day. Data from non-controlled phase II studies involving more than 400 patients indicated a clear anti-tumour activity in postmenopausal ABC patients failing multiple hormonal treatments. In 62 patients progressing on AG (≥500 mg a day) exemestane treatment resulted in an objective response rate of approximately 24%; disease stabilization ≥24 weeks was observed in an additional 24% of cases. With regard to safety, although daily doses up to 600 mg were administered, the maximal tolerated dose was not achieved; reported symptoms were mainly related to the pharmacological action of the compound and were usually mild to moderate in severity, resulting in the discontinuation of therapy in less than 3% of cases. In conclusion, the available results suggest that exemestane treatment is associated with minimal toxicity, and may be of significant benefit for ABC women who have exhausted conventional therapy.     

Is there a benefit by the sequence anastrozole-formestane for postmenopausal metastatic breast cancer women?

To explore the different sequence interactions between reversible non-steroidal (anastrozole, ANZ and letrozole, LTZ) and non-reversible steroidal aromatase inhibitors (formestane, FOR and exemestane, EXE), we evaluated the clinical benefit (CB) in postmenopausal breast cancer patients, who had previously received anastrozole and subsequently formestane. In 19 out of 21 patients (90.5%), a clinical benefit response was achieved by anastrozole, with a median duration of 12 months. Out of the 21 women progressing on anastrozole, 12 achieved stable disease (SD)>/=6 months by formestane only. The overall clinical benefit was 66.5%. The median duration of clinical benefit was 11 months with a time to progression of 6.5 months. The median duration of clinical benefit in our series is similar to that reported in two phase II trials with the sequence aminogluthetimide-->formestane and aminogluthetimide-->exemestane as third-line hormonal therapy, suggesting a non-cross-resistance between the two classes of inhibitors.     

Benefit with aromatase inhibitors in the adjuvant setting for postmenopausal women with breast cancer

The third-generation aromatase inhibitors, letrozole, anastrozole, and exemestane, have been shown to be effective both as alternatives to tamoxifen in first-line treatment of hormone-sensitive advanced breast cancer in postmenopausal women and following failure of first-line tamoxifen for endocrine therapy. These 3 agents are now being investigated as adjuvant therapy of early breast cancer, as alternative or complementary treatments to the standard, tamoxifen. Three treatment strategies are under investigation: replacement of tamoxifen as adjuvant therapy for 5 years (early adjuvant therapy), sequencing of tamoxifen before or after an aromatase inhibitor during the first 5 years (early sequential adjuvant therapy), or following 5 years of tamoxifen (extended adjuvant therapy). In the first adjuvant trial (Arimidex, Tamoxifen Alone or in Combination [ATAC]), anastrozole was significantly superior to tamoxifen in reducing risk of disease recurrence, and recently, the Breast International Group (BIG) trial BIG 1-98 demonstrated the significant superiority of letrozole over tamoxifen in improving disease-free survival. A large trial (International Collaborative Cancer Group [ICCG] trial 96) investigated sequencing of 2 to 3 years of exemestane after 2 to 3 years of tamoxifen and found that switching to exemestane was significantly superior in disease-free survival compared with continuing on tamoxifen. The Arimidex or Nolvadex (ARNO) and the small ITA (Italian Tamoxifen Arimidex) trials similarly sequenced anastrozole after tamoxifen and also found that sequencing reduced the hazard of recurrence compared with remaining on tamoxifen. Trial MA.17 evaluated extended adjuvant therapy with letrozole vs placebo following 5 years of tamoxifen. Disease-free survival was significantly improved with letrozole vs placebo, irrespective of whether patients had lymph node-positive or node-negative tumors. All 3 aromatase inhibitors were generally well tolerated. Results of these trials indicate that aromatase inhibitors provide important benefits relative to tamoxifen in each of these adjuvant treatment settings, but the optimal approach still needs to be defined. Other trials continue to investigate some of these adjuvant treatment strategies.     

Binding features of steroidal and nonsteroidal inhibitors

Aromatase is the rate-limiting enzyme in estrogen biosynthesis. As a cytochrome P450, it utilizes electrons from NADPH-cytochrome P450 reductase (CPR) to produce estrogen from androgen. Estrogen is a key factor in the promotion of hormone-dependent breast cancer growth. Aromatase inhibitors (AIs) are drugs that block estrogen synthesis, and are widely used to treat estrogen-dependent breast cancer. Structure-function experiments have been performed to study how CPR and AIs interact with aromatase to further the understanding of how these drugs elicit their effects. Our studies have revealed a strong interaction between aromatase and CPR, and that the residue K108 is situated in a region important to the interaction of aromatase with CPR. The published X-ray structure of aromatase indicates that the F221, W224 and M374 residues are located in the active site. Our site-directed mutagenesis experiments confirm their importance in the binding of the androgen substrate as well as AIs, but these residues interact differently with steroidal inhibitors (exemestane) and non-steroidal inhibitors (letrozole and anastrozole). Furthermore, our results predict that the residue W224 also participates in the mechanism-based inhibition of exemestane, as time-dependent inhibition is eliminated with mutation on this residue. Together with previous research from our laboratory, this study confirms that W224, E302, D309 and S478 are important active site residues involved in the suicide mechanism of exemestane against aromatase.     

Novel aromatase and 5α-reductase inhibitors

Inhibitors of aromatase and 5α-reductase may be of use for the therapy of postmenopausal breast cancer and benign prostatic hyperplasia, respectively. FCE 27993 is a novel steroidal irreversible aromatase inhibitor structurally related to exemestane (FCE 24304). The compound was found to be a very potent competitive inhibitor of human placental aromatase, with a K_i of 7.2 nM (4.3 nM for exemestane). In preincubation studies with placental aromatase FCE 27993, like exemestane, was found to cause time-dependent inhibition with a higher rate of inactivation ( ) and a similar K_i(inact) (56 vs 66 nM). The compound was found to have a very low binding affinity to the androgen receptor (RBA 0.09% of dihydrotestosterone) and, in contrast to exemestane, no androgenic activity up to 100 mg/kg/day s.c. in immature castrated rats. Among a series of novel 4-azasteroids with fluoro-substituted-17β-amidic side chains, three compounds, namely FCE 28260, FCE 28175 and FCE 27837, were identified as potent in vitro and in vivo inhibitors of prostatic 5α-reductase. Their IC₅₀ values were found to be 16, 38 and 51 nM for the inhibition of the human enzyme, and 15, 20 and 60 nM for the inhibition of the rat enzyme, respectively. When given orally for 7 days in castrated and testosterone (Silastic implants) supplemented rats, the new compounds were very effective in reducing prostate growth. At a dose of 0.3 mg/kg/day inhibitions of 42, 36 and 41% were caused by FCE 28260, FCE 28175 and FCE 27837, respectively.