Steroidal Aromatase Inhibitors Research Articles

Localization and regulation of reproductive steroid receptors in the raphe serotonin system of male macaques

We previously showed that tryptophan hydroxylase 2 (TPH2) and serotonin reuptake transporter (SERT) mRNAs are increased by the androgens, testosterone (T) and dihydrotestosterone (DHT) in serotonin neurons of male macaques. In addition, we observed that serotonin in axons of a terminal region were markedly decreased by aromatase inhibition and lack of estradiol (E) from metabolism of T. These observations implicated androgen receptors (AR) and estrogen receptors (ER) in the transduction of steroid hormone actions in serotonin neurons. Due to the longer treatment period employed, the expression of the cognate nuclear receptors was sought. We used single and double immunohistochemistry to quantitate and phenotypically localize AR, ERα and ERβ in the dorsal raphe of male macaques. Male Japanese macaques (Macaca fuscata) were castrated for 5–7 months and then treated for 3 months with [1] placebo, [2] T, [3] DHT (non-aromatizable androgen) plus ATD (steroidal aromatase inhibitor), or [4] Flutamide (FLUT; androgen antagonist) plus ATD (n=5/group). After single labeling of each receptor, quantitative image analysis was applied and receptor positive neurons were counted. Double-label of raphe neurons for each receptor plus TPH2 determined whether the receptors were localized in serotonin neurons. There were significantly more AR-positive neurons in T- and DHT+ATD-treated groups (p=0.0014) compared to placebo or FLUT+ATD-treated groups. There was no difference in the number of positive-neurons stained for ERα or ERβ⋅ Double-immunohistochemistry revealed that serotonin neurons did not contain AR. Rather, AR-positive nuclei were found in neighboring cells that are likely neurons. However, approximately 40% of dorsal raphe serotonin neurons contained ERα or ERβ⋅ In conclusion, the stimulatory effect of androgens on TPH2 and SERT mRNA expression is mediated indirectly by neighboring neurons contain AR. The stimulatory effect of E, derived from T metabolism, on serotonin transport is partially mediated directly via nuclear ERs.

Prediction of aromatase inhibitory activity using the efficient linear method (ELM).

Aromatase inhibition is an effective treatment strategy for breast cancer. Currently, several in silico methods have been developed for the prediction of aromatase inhibitors (AIs) using artificial neural network (ANN) or support vector machine (SVM). In spite of this, there are ample opportunities for further improvements by developing a simple and interpretable quantitative structure-activity relationship (QSAR) method. Herein, an efficient linear method (ELM) is proposed for constructing a highly predictive QSAR model containing a spontaneous feature importance estimator. Briefly, ELM is a linear-based model with optimal parameters derived from genetic algorithm. Results showed that the simple ELM method displayed robust performance with 10-fold cross-validation MCC values of 0.64 and 0.56 for steroidal and non-steroidal AIs, respectively. Comparative analyses with other machine learning methods (i.e. ANN, SVM and decision tree) were also performed. A thorough analysis of informative molecular descriptors for both steroidal and non-steroidal AIs provided insights into the mechanism of action of compounds. Our findings suggest that the shape and polarizability of compounds may govern the inhibitory activity of both steroidal and non-steroidal types whereas the terminal primary C(sp3) functional group and electronegativity may be required for non-steroidal AIs. The R code of the ELM method is available at http://dx.doi.org/10.6084/m9.figshare.1274030.

Clinical benefit of sequential use of endocrine therapies for metastatic breast cancer.

Sequential use of endocrine agents is common in estrogen receptor (ER)-positive metastatic breast cancer (MBC). In premenopausal women with MBC, tamoxifen and/or a luteinizing hormone-releasing hormone (LH-RH) agonist are options for endocrine treatment. Meta-analysis showed that the combination of the two agents is superior to either monotherapy. Under ovarian ablation or function-suppression, an agent used to treat postmenopausal women, such as the aromatase inhibitor (AI), fulvestrant, becomes possible as a subsequent therapy. In postmenopausal women, endocrine treatment options are widely available and an optimal sequence has not yet to be determined. Options for first-line therapy of metastatic disease include an AI for women who have received adjuvant tamoxifen, or tamoxifen for patients who have received adjuvant AI. In addition, data suggest that fulvestrant is a promising therapeutic option that has proven efficacy in the treatment of postmenopausal women with MBC. Other agents that may be used in the sequence include a steroidal AI or a progestin. Furthermore, estrogen additive therapy by with diethylstilbestrol or ethinylestradiol has recently been revived and showed a high response rate as a salvage endocrine therapy, although its mechanism of action is still unclear. Thus, sequential use of endocrine therapies, especially the use of a subsequent therapy with a different mechanism of action to the prior therapy, has a beneficial effect in maintaining a good quality of life and extending survival for MBC with hormone responsiveness.

A Novel Validated Stability-Indicating RP-HPLC Method for the Determination of Exemestane (Steroidal Aromatase Inhibitor)

Background: Exemestane is an active irreversible lipophilic steroidal aromatase inhibitor used to treat breast cancer in addition to surgery and/or radiation in post-menopausal women. It is a white to slightly yellow crystalline powder with a molecular weight of 296.41. Exemestane is freely soluble in N, N-dimethyl formamide, soluble in methanol, and practically insoluble in water. The present robust RP-HPLC method supports the quantitative analysis of Exemestane in pharmaceutical formulations and for carrying out the forced degradation studies. Methods: A novel stability indicating liquid chromatographic method was developed for the determination of Exemestane using HPLC system of Shimadzu Model CBM-20A/20 Alite, equipped with SPD M20A prominence PDA and Zorbax SB C18 (150 mm × 4.6 mm i.d., 3.5 μm particle size) column. A mixture of sodium acetate buffer and acetonitrile (30:70, v/v) was used as a mobile phase with 1.0 ml/min flow rate and the method was validated as per ICH guidelines. Forced degradation studies were performed in different stress conditions such as acidic, basic, oxidation and thermal degradations. Results: The proposed liquid chromatographic method has shown linearity over a concentration range 0.1–200 μg/ml with regression equation y = 59411x - 7316 with correlation coefficient 0.999. During the validation process i.e. the intra-day and inter-day precision studies, accuracy and robustness studies the method has shown an RSD of less than 2.0 %. Exemestane is found to be more stable during all the degradation studies because the percentage of degradation was reported to be less than 10. Conclusions: The proposed method was found to be precise, accurate and robust and it can be applied for the determination of Exemestane in any formulations.

Current medical treatment of estrogen receptor-positive breast cancer.

Approximately 80% of breast cancers (BC) are estrogen receptor (ER)-positive and thus endocrine therapy (ET) should be considered complementary to surgery in the majority of patients. The advantages of oophorectomy, adrenalectomy and hypophysectomy in women with advanced BC have been demonstrated many years ago, and currently ET consist of (1) ovarian function suppression (OFS), usually obtained using gonadotropin-releasing hormone agonists (GnRHa); (2) selective estrogen receptor modulators or down-regulators (SERMs or SERDs); and (3) aromatase inhibitors (AIs), or a combination of two or more drugs. For patients aged less than 50 years and ER+ BC, there is no conclusive evidence that the combination of OFS and SERMs (i.e., tamoxifen) or chemotherapy is superior to OFS alone. Tamoxifen users exhibit a reduced risk of BC, both invasive and in situ, especially during the first 5 years of therapy, and extending the treatment to 10 years further reduced the risk of recurrences. SERDs (i.e., fulvestrant) are especially useful in the neoadjuvant treatment of advanced BC, alone or in combination with either cytotoxic agents or AIs. There are two types of AIs: type I are permanent steroidal inhibitors of aromatase, while type II are reversible nonsteroidal inhibitors. Several studies demonstrated the superiority of the third-generation AIs (i.e., anastrozole and letrozole) compared with tamoxifen, and adjuvant therapy with AIs reduces the recurrence risk especially in patients with advanced BC. Unfortunately, some cancers are or became ET-resistant, and thus other drugs have been suggested in combination with SERMs or AIs, including cyclin-dependent kinase 4/6 inhibitors (palbociclib) and mammalian target of rapamycin (mTOR) inhibitors, such as everolimus. Further studies are required to confirm their real usefulness.

3D QSAR studies, pharmacophore modeling and virtual screening on a series of steroidal aromatase inhibitors.

Aromatase inhibitors are the most important targets in treatment of estrogen-dependent cancers. In order to search for potent steroidal aromatase inhibitors (SAIs) with lower side effects and overcome cellular resistance, comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were performed on a series of SAIs to build 3D QSAR models. The reliable and predictive CoMFA and CoMSIA models were obtained with statistical results (CoMFA: q2 = 0.636, r2ncv = 0.988, r2pred = 0.658; CoMSIA: q2 = 0.843, r2ncv = 0.989, r2pred = 0.601). This 3D QSAR approach provides significant insights that can be used to develop novel and potent SAIs. In addition, Genetic algorithm with linear assignment of hypermolecular alignment of database (GALAHAD) was used to derive 3D pharmacophore models. The selected pharmacophore model contains two acceptor atoms and four hydrophobic centers, which was used as a 3D query for virtual screening against NCI2000 database. Six hit compounds were obtained and their biological activities were further predicted by the CoMFA and CoMSIA models, which are expected to design potent and novel SAIs.

Abstract 2110: Molecular characterization of everolimus-resistant cell lines established from estrogen depletion-resistant MCF-7

Abstract mTOR inhibitor everolimus has been considered as a promising agent for breast cancer treatment especially among estrogen receptor (ER) positive postmenopausal advanced breast cancer. As expected, combination therapy of everolimus and steroidal aromatase inhibitor (AI) showed remarkable clinical efficacy for the first line nonsteroidal AI treatment failure patients in BOLERO-2 trial. However, the subsequent treatment after refractory to that therapy remains a great matter. We already reported several types of long term estrogen-depletion resistant (EDR) cell lines from MCF-7-E10, which has been constitutively ERE-GFP-transfected to monitor ER activity in living cells (Fujiki N et al., J Steroid Biochem Mol Biol. 2013 Oct 13;139C:61-72). They mimic AI resistance and these EDR cells have been characterized as the followings. 1) Type 1 as ER positive (ERE-GFP positive), constitutive ER overexpression without estrogen, and PI3K/Akt/mTOR pathway upregulated. 2) Type 2 as ER negative conversion (ERE-GFP negative) and IGF-1R/JNK signaling pathway upregulated. Then with those two types of sub-cell lines, we generated everolimus resistant cells after long term exposure to everolimus in estrogen depleted medium. Those everolimus resistant (EVR) cells are equivalent to resistance to the combination therapy of AI and everolimus as the second line endocrine treatment after the first line AI treatment failure. Though everolimus extremely inhibited cell proliferation of two EDR cell lines, everolimus resistant cells named as Type 1-EVR, Type 2-EVR no longer responded to everolimus. ER expression and estrogen sensitivity of two types of EVR cells were similar to those of the parental cells, respectively. Pure anti-estrogen fulvestrant inhibited cell proliferation of Type1-EVR cells. On the other hand, Type2-EVR did not respond to fulvestrant but JNK inhibitor inhibited the cell growth. Phosphorylation status of p70S6K and 4E-BP1 were different among individual parental EDR cells and EVR cells. Type1-EVR showed much higher expression of phosphorylation of MAP-kinase than the parental cells. In contrast, Type2-EVR expressed phosphorylation of MAP-kinase less than the parental cells. As above, the mechanism of acquired resistance to everolimus was likely to be different between those two sub-cell lines. This finding implies that different mechanisms of AI resistance follow different types of everolimus resistance. Citation Format: Mariko Kimura, Toru Hanamura, Toshifumi Niwa, Yuri Yamaguchi, Itaru Endo, Shin-ichi Hayashi. Molecular characterization of everolimus-resistant cell lines established from estrogen depletion-resistant MCF-7. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2110. doi:10.1158/1538-7445.AM2014-2110

Exemestane metabolites: Synthesis, stereochemical elucidation, biochemical activity and anti-proliferative effects in a hormone-dependent breast cancer cell line

Exemestane is a third-generation steroidal aromatase inhibitor that has been used in clinic for hormone-dependent breast cancer treatment in post-menopausal women. It is known that exemestane undergoes a complex metabolization, giving rise to some already identified metabolites, the 17β-hydroxy-6-methylenandrosta-1,4-dien-3-one (17-βHE) and the 6-(hydroxymethyl)androsta-1,4,6-triene-3,17-dione (6-HME). In this study, four metabolites of exemestane have been analyzed, three of them were synthesized (6β-spirooxiranandrosta-1,4-diene-3,17-dione (2), 1α,2α-epoxy-6-methylenandrost-4-ene-3,17-dione (3) and 17-βHE (4)) while one was acquired, the 6-HME (6). The stereochemistry of the epoxide group of 2 and 3 has been unequivocally elucidated for the first time on the basis of NOESY experiments. New structure–activity relationships (SAR) have been established through the observation that the substitution of the double bonds by epoxide groups led to less potent derivatives in microsomes. However, the reduction of the C-17 carbonyl group to a hydroxyl group originating 17-βHE (4) resulted in a significant increase in activity in MCF-7aro cells when compared to exemestane (IC50 0.25 μM vs 0.90 μM, respectively). All the studied metabolites reduced MCF-7aro cells viability in a dose and time-dependent manner, and metabolite 3 was the most potent one. Altogether our results showed that not only exemestane but also its main metabolites are potent aromatase inhibitors and reduce breast cancer cells viability. This suggests that exemestane efficacy may also be due to the active metabolites that result from its metabolic transformation. Our results emphasize the importance of performing further studies to expand our understanding of exemestane actions in breast cancer cells.

Mechanism of inhibition of estrogen biosynthesis by azole fungicides.

Biosynthesis of estrogens from androgens is catalyzed by cytochrome P450 aromatase. Aromatase inhibition by the triazole compounds letrozole (LTZ) and anastrozole is a prevalent therapy for estrogen-dependent postmenopausal breast cancer. Azoles are widely used as agricultural fungicides and antimycotic drugs that target 14α-demethylase. Some were previously shown to inhibit aromatase, thereby raising the possibility of endocrine disruptive effects. However, mechanistic analysis of their inhibition has never been undertaken. We have evaluated the inhibitory effects of 3 common fungicides, bifonazole, imazalil, and flusilazole, in human aromatase purified from placenta and compared them with LTZ, the most potent inhibitor of aromatase. Bifonazole exhibits strong inhibitory effects with an IC50 of 270nM and Ki (Michaeles-Menten inhibition constant) of 68nM, compared with 10nM and 13nM, respectively, for LTZ. The IC50 and Ki are 1100nM and 278nM for imazilil and 3200nM and 547nM for flusilazole, respectively. Analyses of inhibition kinetics suggest that the modes of inhibition by azole fungicides are mixed or competitive, whereas LTZ inhibition could be noncompetitive or mixed. We interpret the inhibition mechanism in the context of the x-ray structure of aromatase-androstenedione complex. Structural data show that aromatase has 3 binding pockets in relation to the heme. The substrate-binding cavity at the heme-distal site closely compliments the structures of the natural substrate, androstenedione, and steroidal aromatase inhibitors. Because the structures of LTZ and the azole fungicides are entirely dissimilar to the androstenedione backbone, the azoles possibly inhibit by binding to a structurally rearranged active site, the 2 other catalytically important sites, or both, in agreement with the kinetics data.

Relationships between androgens, serotonin gene expression and innervation in male macaques

Androgen administration to castrated individuals was purported to decrease activity in the serotonin system. However, we found that androgen administration to castrated male macaques increased fenfluramine-induced serotonin release as reflected by increased prolactin secretion. In this study, we sought to define the effects of androgens and aromatase inhibition on serotonin-related gene expression in the dorsal raphe, as well as serotonergic innervation of the LC. Male Japanese macaques (Macaca fuscata) were castrated for 5–7months and then treated for 3months with (1) placebo, (2) testosterone (T), (3) dihydrotestosterone (DHT; non-aromatizable androgen) and ATD (steroidal aromatase inhibitor), or (4) Flutamide (FLUT; androgen antagonist) and ATD (n=5/group). This study reports the expression of serotonin-related genes: tryptophan hydroxylase 2 (TPH2), serotonin reuptake transporter (SERT) and the serotonin 1A autoreceptor (5HT1A) using digoxigenin-ISH and image analysis. To examine the production of serotonin and the serotonergic innervation of a target area underlying arousal and vigilance, we measured the serotonin axon density entering the LC with ICC and image analysis. TPH2 and SERT expression were significantly elevated in T- and DHT+ATD-treated groups over placebo- and FLUT+ATD-treated groups in the dorsal raphe (p<0.007). There was no difference in 5HT1A expression between the groups. There was a significant decrease in the pixel area of serotonin axons and in the number of varicosities in the LC across the treatment groups with T>placebo>DHT+ATD=FLUT+ATD treatments. Comparatively, T- and DHT+ATD-treated groups had elevated TPH2 and SERT gene expression, but the DHT+ATD group had markedly suppressed serotonin axon density relative to the T-treated group. Further comparison with previously published data indicated that TPH2 and SERT expression reflected yawning and basal prolactin secretion. The serotonin axon density in the LC agreed with the area under the fenfluramine-stimulated prolactin curve, providing a morphological basis for the pharmacological results. This suggested that androgen activity increased TPH2 and SERT gene expression but, aromatase activity, and neural production of estradiol (E), may subserve axonal serotonin and determination of the compartment acted upon by fenfluramine. In summary, androgens stimulated serotonin-related gene expression, but aromatase inhibition dissociated gene expression from the serotonin innervation of the LC terminal field and fenfluramine-stimulated prolactin secretion.

Quality of Life in MAP.3 (Mammary Prevention 3): A Randomized, Placebo-Controlled Trial Evaluating Exemestane for Prevention of Breast Cancer

Exemestane, a steroidal aromatase inhibitor, reduced invasive breast cancer incidence by 65% among 4,560 postmenopausal women randomly assigned to exemestane (25 mg per day) compared with placebo in the National Cancer Institute of Canada (NCIC) Clinical Trials Group MAP.3 (Mammary Prevention 3) trial, but effects on quality of life (QOL) were not fully described. Menopause-specific and health-related QOL were assessed by using the four Menopause-Specific Quality of Life Questionnaire (MENQOL) domains and the eight Medical Outcomes Study Short Form Health Survey (SF-36) scales at baseline, 6 months, and yearly thereafter. MENQOL questionnaire completion was high (88% to 98%) in both groups at each follow-up visit. Change scores for each MENQOL and SF-36 scale, calculated at each assessment time relative to baseline, were compared by using the Wilcoxon rank-sum test. Clinically important worsened QOL was defined as a MENQOL change score increase of more than 0.5 (of 8) points and an SF-36 change score decrease of more than 5 (of 100) points from baseline. Exemestane had small negative effects on women's self-reported vasomotor symptoms, sexual symptoms, and pain, which occurred mainly in the first 6 months to 2 years after random assignment. However, these changes represented only a small excess number of women being given exemestane with clinically important worsening of QOL at one time or another; specifically, 8% more in the vasomotor domain and 4% more each in the sexual domain and for pain. No other between-group differences were observed. Overall, slightly more women in the exemestane arm (32%) than in the placebo arm (28%) discontinued assigned treatment. Exemestane given for prevention has limited negative impact on menopause-specific and health-related QOL in healthy postmenopausal women at risk for breast cancer.

Aromatase inhibitors in the breast cancer clinic: focus on exemestane

Breast cancer is the most prevalent type of cancer in women and responsible for significant female cancer-related mortality worldwide. In the Western world, over 80% of breast cancers are hormone-receptor positive for which endocrine therapy is administered. The main anti-estrogen treatments in use consist of selective estrogen-receptor modulators, such as tamoxifen, and third-generation aromatase inhibitors (AIs), such as exemestane, letrozole, and anastrozole. In this review, the focus will lie on exemestane, its clinical use, and its side-effect profile. Exemestane is the only third-generation steroidal AI. Its efficacy as a first-line treatment in metastatic breast cancer has been demonstrated. Therefore, exemestane could be considered a valid first-line therapeutic option, but it also can be used in second-line or further situations. Exemestane is mostly used as part of sequential adjuvant treatment following tamoxifen, but in this setting it is also active in monotherapy. Furthermore, this AI has been studied in the neoadjuvant setting as presurgical treatment, and even as chemoprevention in high-risk healthy postmenopausal women. It may reverse side effects of tamoxifen, such as endometrial changes and thromboembolic disease but may also cause some inconvenient side effects itself. Additionally, there is a lack of total cross-resistance between exemestane and nonsteroidal AIs as far as their anti-tumoral efficacy is concerned; moreover the two classes of AIs display a nontotal overlapping toxicity profile. Taking together, exemestane can be considered as a useful treatment option at all stages of breast cancer.

Long-term complete remission of metastatic breast cancer induced by a steroidal aromatase inhibitor after failure of a non-steroidal aromatase inhibitor

Patient: Female, 56Final Diagnosis: Breast cancerSymptoms: Solid mass in the right breastMedication: ExemestaneClinical Procedure: —Specialty: OncologyObjective:Unusual clinical courseBackground:The efficacy of third-generation aromatase inhibitors for hormone receptor-positive postmenopausal metastatic breast cancer is well established. Although several clinical trials have reported incomplete cross-resistance between different aromatase inhibitors, few cases of complete responses of recurrent metastatic breast cancer occurring after substituting a second aromatase inhibitor have been reported. We here present a rare case of non-steroidal aromatase inhibitor-tolerant metastatic breast cancer with long-term complete remission following substitution of a steroidal aromatase inhibitor.Case Report:We present the case of a 56-year-old Japanese woman who underwent right breast-conserving surgery for breast cancer, TNM staging T1, N0, M0, Stage I. She received adjuvant chemotherapy with 6 cycles of FEC100 and radiation therapy, and then began hormonal therapy with anastrozole. Twelve months postoperatively, computed tomography (CT) revealed multiple lung metastases. Exemestane was substituted for anastrozole. After 3 months of exemestane, CT showed that all lung metastases had completely resolved. Her complete response was maintained for 5 years: she died during a tsunami 6 years after the initial surgery.Conclusions:Substitution of a steroidal for a non-steroidal aromatase inhibitor produced a sustained complete remission in a patient with hormonal receptor-positive postmenopausal recurrent breast cancer.Achieving complete response after switching from a non-steroidal to a steroidal aromatase inhibitor in a hormonal receptor-positive postmenopausal recurrent breast cancer contributed to a higher quality of life for the patient. Further investigation is needed to identify the predictors of long-term remission following such a switch.

Abstract ES03-2: Clinical 101: Clinical care of breast cancer in 2013

Abstract The clinical care of breast cancer continues to evolve at a rapid pace. While many traditional principles still hold true, advances in the local and systemic management of breast cancer are being incorporated into individualized care of breast cancer patients. In the curative setting (stage I – III), tri-modality therapy with surgery, radiation and systemic therapy continues to be standard of care. In the absence of an absolute contraindication to breast conserving therapy (i.e. inflammatory or multi-centric breast cancer), overall survival remains similar for mastectomy and lumpectomy/radiation therapy. Following mastectomy, radiation to the chest wall and regional nodal lymphatics is generally reserved for patients with T3 (tumor &amp;gt;5 cm) and/or lymph node positive disease. The decision to incorporate systemic therapy is largely driven by breast cancer subtype (i.e. hormone receptor [HR] and HER2 status), stage of breast cancer and general health of the patient. Systemic therapy can either be prescribed pre-operatively (neo-adjuvantly) or post-operatively (adjuvantly) and studies have not shown a survival advantage by order of therapy. Neoadjuvant therapy is commonly reserved for locally-advanced, node positive and/or inflammatory (T4d) breast cancer. Anthracycline and/or taxane-based therapies are recommended for patients with larger tumors (i.e. &amp;gt;2cm) and lymph node positive breast cancer. Chemotherapy and chemotherapy plus HER2-directed (i.e. trastuzumab) therapy is recommended for patients with triple negative and HER2-positive breast cancer of all stages &amp;gt; T1aN0, respectively. In patients with stage I - II HR positive breast cancer, genomic tests (i.e. Oncotype® and Mammoprint®) can be employed to help guide the decision to add chemotherapy to standard endocrine therapy. At least five years of an adjuvant aromatase inhibitor (AI, or combination of an AI and Tamoxifen) is recommended for post-menopausal women with early stage HR-positive breast cancer, while five to ten years of tamoxifen is now recommended for pre-menopausal women with this subtype of breast cancer. Therapy for stage IV breast cancer is palliative and is heavily reliant on systemic therapy, while radiation and surgery are commonly reserved for palliation. In the absence of a visceral crisis, endocrine therapy is mainstay of treatment for patients with advanced HR-positive breast cancer. The addition of mTOR inhibition plus a steroidal aromatase inhibitor has recently been approved following progression on a non-steroidal aromatase inhibitor in this setting. Once patients are refractory to endocrine therapy, chemotherapy is usually prescribed. Presently, stage IV triple negative breast cancer is treated with chemotherapy. While many targeted agents have been investigated (i.e. bevacizumab, PARP and EGFR inhibitors), none are currently FDA-approved for this indication. Finally, the landscape for advanced HER2-positive breast cancer has changed dramatically over the past few years with the approval of lapatinib, pertuzumab, and TDM-1. In closing, the clinical care of breast cancer continues to mature at a rapid pace and advances in care will only continue to improve outcome. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr ES03-2.

Abstract P2-16-17: Characterization of response to everolimus (EVE) in BOLERO-2: A phase 3 trial of EVE plus exemestane (EXE) in postmenopausal women with HR+, HER2- advanced breast cancer

Abstract Introduction: The BOLERO-2 trial demonstrated that combining the oral mammalian target of rapamycin (mTOR) inhibitor, EVE, with the steroidal aromatase inhibitor, EXE, more than doubled median progression-free survival (PFS) compared with placebo (PBO) plus EXE in postmenopausal women with hormone-receptor-positive (HR+), human epidermal growth factor receptor-2-negative (HER2−) breast cancer (BC) who relapsed or progressed following a nonsteroidal aromatase inhibitor (NSAI). Patients also achieved responses per Response Evaluation Criteria in Solid Tumors (RECIST) during treatment with EVE+EXE. Methods: The phase 3, double-blind, BOLERO-2 trial randomized postmenopausal women with HR+ BC progressing or recurring after NSAIs in a 2:1 manner to EVE 10 mg once daily plus EXE 25 mg once daily (EVE+EXE; n = 485) or placebo (PBO) plus EXE (PBO+EXE; n = 239). The primary endpoint was PFS by local assessment. Overall response rate (ORR; complete + partial response per investigator assessment based on RECIST 1.0) and duration of overall response were secondary endpoints. In addition, best percentage change from baseline in sum of longest diameters of target lesions was assessed. Results: At the time of final PFS analyses at 18 months’ median follow-up, ORR was significantly higher in the EVE+EXE arm compared with the PBO+EXE arm (12.6% vs 1.7%, respectively, by local assessment; P&amp;lt;.0001). Among patients with measurable disease at baseline, 71% in the EVE+EXE arm had a decrease in the sum of longest diameters of target lesions compared with baseline vs 30% in the PBO+EXE arm. Median duration of overall response was 10.5 months (95% confidence interval [CI]: 8.2, 21.9 months) for EVE+EXE and 6.9 months (95% CI: 4.2, 6.9 months) for PBO+EXE. Of note, only 4 patients in the PBO+EXE arm had an objective response to treatment. Conclusions: In addition to PFS, the combination of EVE plus EXE significantly improved ORR vs PBO+EXE in patients with HR+, HER2− advanced BC progressing during or after NSAI therapy. Furthermore, greater than two-thirds of patients treated with EVE+EXE experienced tumor shrinkage during treatment. These results further support the rationale for combining EVE with EXE to improve clinical outcomes in HR+, HER2− advanced BC progressing after NSAI therapy. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-16-17.

Abstract P1-14-01: Randomized controlled trial of toremifene 120 mg compared with exemestane 25 mg after prior non-steroidal aromatase inhibitor treatment in postmenopausal women with hormone receptor-positive metastatic breast cancer (Hi-FAIR ex study)

Abstract (Background) After the failure of a non-steroidal aromatase inhibitor (nsAI) for postmenopausal patients with metastatic breast cancer (mBC), it is uncertain which of endocrine therapy is the most appropriate. (Methods) A randomized, open-label, multicenter phase II study was conducted to compare the efficacy and safety of daily toremifene 120 mg (TOR120), a selective estrogen receptor modulator, with exemestane 25 mg (EXE), a steroidal aromatase inhibitor. After disease progressed with assigned drug, the patients were subsequently treated with the other drug if patients were suitable for continuation with endocrine treatment. The primary endpoint was clinical benefit rate (CBR). The secondary end points were objective response rate (ORR), progression-free survival (PFS), overall survival (OS), toxicity and antitumor effect of subsequent endocrine treatment. To prove a probability of 90% that TOR120 was superior to EXE, 41 patients were required for each group. To account for dropouts and protocol violation, we planned to recruit 90 patients (45 in each group). (Results) Initially, a total of 91women was registered in the study and randomly assigned to either TOR120 (n = 46) or EXE (n = 45) from October 2008 to November 2011. Three of the 46 patients in the TOR120 arm were not received treatment, 2 patients having withdrawn from the trial by their preference and one having been dropped due to administration of another SERM. When analyzed after a median observation period of 16.9 months, the intention-to-treat analysis showed that there was no statistical difference between TOR120 (n = 46) and EXE (n = 45) in terms of CBR (41.3% vs. 26.7%, p = 0.14), ORR (10.8% vs. 2.2%, p = 0.083), and OS (Hazard ratio (HR) 0.60, p = 0.22). The PFS of TOR120 was longer than that of EXE, the difference being statistically significant (HR 0.61, p = 0.045). Both treatments were well-tolerated with no severe adverse events, although the treatment of 3 of 43 women with TOR120 was stopped after a few days because of nausea, general fatigue, hot flash and night sweating. Twenty-four patients received subsequent TOR120 after failure of initial EXE treatment. Of these patients, ORR and CBR were 4.2% (1/24) and 33.3% (8/24), respectively. In 23 patients who received EXE after TOR120, ORR and CBR were 17.4% (4/23) and 30.4% (7/23), respectively. (Conclusion) TOR120, as a subsequent endocrine therapy for mBC patients who failed non-steroidal AI treatment, could be equal to or potentially be more beneficial than EXE. In addition, about one third of patients who progress on either TOR120 or EXE could obtain clinical benefit from subsequent EXE or TOR120. Trial registration number: UMIN000001841. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-14-01.

Synthesis and Bioconversions of Formestane

In an effort to generate new steroidal aromatase inhibitors, formestane (4-hydroxyandrost-4-ene-3,17-dione) (1) was biotransformed by Rhizopus oryzae to yield the known 4β,5α-dihydroxyandrostane-3,17-dione as the major product (5) and bioconverted by Beauveria bassiana to afford the known reduced 4,17β-dihydroxyandrost-4-en-3-one (6) and 3α,17β-dihydroxy-5β-androstan-4-one (7) and the new 4,11α,17β-trihydroxyandrost-4-en-3-one (8). All the metabolites showed more potent activities than their parent congener in the aromatase and MCF-7 breast cancer assays. The bioactivities and structural elucidation of these metabolites as well as the semisynthesis of formestane (1) from testosterone (2) are reported herein.

Steroidal aromatase inhibitors inhibit growth of hormone-dependent breast cancer cells by inducing cell cycle arrest and apoptosis

Different hormonal therapies are used for estrogen receptor positive (ER(+)) breast cancers, being the third-generation of aromatase inhibitors (AIs), an effective alternative to the classical tamoxifen. AIs inhibit the enzyme aromatase, which is responsible for catalyzing the conversion of androgens to estrogens. In this study, it was evaluated the effects of several steroidal AIs, namely 3β-hydroxyandrost-4-en-17-one (1), androst-4-en-17-one (12), 4α,5α-epoxyandrostan-17-one (13a) and 5α-androst-2-en-17-one (16), on cell proliferation, cell cycle progression and cell death in an ER(+) aromatase-overexpressing human breast cancer cell line (MCF-7aro). All AIs induced a decrease in cell proliferation and these anti-proliferative effects were due to a disruption in cell cycle progression and cell death, by apoptosis. AIs 1 and 16 caused cell cycle arrest in G0/G1, while AIs 12 and 13a induced an arrest in G2/M. Moreover, it was observed that these AIs induced apoptosis by different pathways, since AIs 1, 12 and 13a activated the apoptotic mitochondrial pathway, while AI 16 induced apoptosis through activation of caspase-8. These results are important for the elucidation of the cellular effects of steroidal AIs on breast cancer cells and will also highlight the importance of AIs as inducers of apoptosis in hormone-dependent breast cancers.

Randomized controlled trial of toremifene 120 mg compared with exemestane 25 mg after prior treatment with a non-steroidal aromatase inhibitor in postmenopausal women with hormone receptor-positive metastatic breast cancer

BackgroundAfter the failure of a non-steroidal aromatase inhibitor (nsAI) for postmenopausal patients with metastatic breast cancer (mBC), it is unclear which of various kinds of endocrine therapy is the most appropriate. A randomized controlled trial was performed to compare the efficacy and safety of daily toremifene 120 mg (TOR120), a selective estrogen receptor modulator, and exemestane 25 mg (EXE), a steroidal aromatase inhibitor. The primary end point was the clinical benefit rate (CBR). The secondary end points were objective response rate (ORR), progression-free survival (PFS), overall survival (OS) and toxicity.MethodsInitially, a total of 91 women was registered in the study and randomly assigned to either TOR120 (n = 46) or EXE (n = 45) from October 2008 to November 2011. Three of the 46 patients in the TOR120 arm were not received treatment, 2 patients having withdrawn from the trial by their preference and one having been dropped due to administration of another SERM.ResultsWhen analyzed after a median observation period of 16.9 months, the intention-to-treat analysis showed that there were no statistical difference between TOR120 (N = 46) and EXE (n = 45) in terms of CBR (41.3% vs. 26.7%; P = 0.14), ORR (10.8% vs. 2.2%; P = 0.083), and OS (Hazard ratio, 0.60; P = 0.22). The PFS of TOR120 was longer than that of EXE, the difference being statistically significant (Hazard ratio, 0.61, P = 0.045). The results in treatment-received cohort (N = 88) were similar to those in ITT cohort. Both treatments were well-tolerated with no severe adverse events, although the treatment of 3 of 43 women administered TOR120 was stopped after a few days because of nausea, general fatigue, hot flush and night sweating.ConclusionsTOR120, as a subsequent endocrine therapy for mBC patients who failed non-steroidal AI treatment, could potentially be more beneficial than EXE.Trial registration numberUMIN000001841

Encapsulation of exemestane in polycaprolactone nanoparticles: optimization, characterization, and release kinetics.

This study was aimed at developing a polymeric drug delivery system for a steroidal aromatase inhibitor, exemestane (exe) intended for sustained targeted delivery of drug through intravenous route. Carboxylated polycaprolactone (cPCL) was synthesized by ring opening polymerization of caprolactone. Exe-loaded cPCL nanoparticles (NPs) were prepared by interfacial deposition of preformed polymer and characterized. A 3-factor, 3-level Box–Behnken design was used to derive a second-order polynomial equation and construct contour and response plots for maximized response of percentage drug entrapment (PDE) with constraints on particle size (PS). The independent variables selected were ratio of exe/cPCL, amount of cPCL, and volume of organic phase. Polymerization of caprolactone to cPCL was confirmed by Fourier transform infrared (FTIR) and gel permeation chromatography. The prepared NPs were evaluated for differential scanning calorimetry (DSC), transmission electron microscopy (TEM), and in vitro release studies. Optimum formulation based on desirability (1.0) exhibited PDE of 83.96 % and PS of 180.5 nm. Check point analysis confirmed the role of the derived polynomial equation and contour plots in predicting the responses. Zeta potential of optimized formulation was −33.8 ± 2.1 mV. DSC studies confirmed the absence of any interaction between drug and polymer. TEM image showed non-aggregated and spherical shaped NPs. Drug release from NPs showed sustained release and followed Korsmeyer–Peppas model, indicating Fickian drug release. Thus, preparation of exe-loaded cPCL NPs with high PDE and desired PS suitable for providing passive targeting could be statistically optimized using Box–Behnken design.Electronic supplementary materialThe online version of this article (doi:10.1007/s12645-013-0037-4) contains supplementary material, which is available to authorized users.