Surgical issues surrounding use of aromatase inhibitors

There are important surgical issues related to the use of the third generation aromatase inhibitors in both the neoadjuvant and adjuvant settings. Neoadjuvant hormone therapy is effective at downstaging tumours, particularly large tumours initially thought to be inoperable or requiring mastectomy. Randomised trials have shown that the newer aromatase inhibitors letrozole and anastrozole increase the numbers of women who are suitable for breast-conservation compared with tamoxifen, and that letrozole is superior to tamoxifen in terms of clinical response.

Aromatase inhibitors are most effective in ER-rich tumours and are clinically and biologically effective in both HER2 positive and negative tumours, whereas HER2 positive tumours show a level of resistance to tamoxifen.

In neoadjuvant studies comparing aromatase inhibitors with tamoxifen, the duration of use has been 3–4 months, by which time any response is usually evident but longer treatment periods produce continued shrinkage and response. The re-excision rate following breast conservation surgery after neoadjuvant hormone therapy is favourable compared with the rates following immediate wide local excision. Local recurrence rates are acceptable in patients undergoing neoadjuvant therapy and breast-conserving surgery providing post-operative radiotherapy is given.

Adjuvant aromatase inhibitors, as well as having an effect on metastatic disease and survival, reduce local and regional recurrence.     

Endocrine effects of aromatase inhibitors and inactivators in vivo: review of data and method limitations

The so-called “third-generation” aromatase inhibitors/inactivators have become standard first-line endocrine therapy for postmenopausal women in the metastatic setting. In addition, these compounds, administered as monotherapy or in sequence with tamoxifen, are likely to become standard adjuvant therapy in most countries in the near future. In contrast to the SERMs, aromatase inhibitors may be assessed for their biochemical efficacy in vivo either by measuring their ability to suppress plasma and tissue estrogen levels or, alternatively, by measuring their ability to inhibit the conversion of tracer-labelled androstenedione into estrone. While contemporary methods for estrogen measurement (with the exception of estrone sulphate) lack the sensitivity to measure plasma estrogen levels during treatment with the most potent compounds, in vivo aromatase inhibition can be determined with a much better sensitivity. Thus, in a joint program conducted by the Royal Marsden Hospital, London and our team in Bergen, we were able to reveal profound differences between first- and second-generation aromatase inhibitors, causing 50–90% aromatase inhibition, and the three third-generation compounds, causing >98% inhibition of total body aromatization.     

Breast cancer tissue estrogens and their manipulation with aromatase inhibitors and inactivators

Despite the dramatic fall in plasma estrogen levels at menopause, only minor differences in breast tissue estrogen levels have been reported comparing pre- and postmenopausal women. Thus, postmenopausal breast tissue has the ability to maintain concentrations of estrone (E₁) and estradiol (E₂) that are 2–10- and 10–20-fold higher than the corresponding plasma estrogen levels. This finding may be explained by uptake of estrogens from the circulation and/or local estrogen production. Local aromatase activity in breast tissue seems to be of crucial importance for the local estrogen production in some patients while uptake from the circulation may be more important in other patients. Beside aromatase, breast tissue expresses estrogen sulfotransferase and sulfatase as well as dehydrogenase activity, allowing estrogen storage and release in the cells as well as conversions between estrone and estradiol. The activity of the enzyme network in breast cancer tissue is modified by a variety of factors like growth factors and cytokines. Aromatase inhibitors have been used for more than two decades in the treatment of postmenopausal metastatic breast cancer and are currently investigated in the adjuvant treatment and even prevention of breast cancer. Novel aromatase inhibitors and inactivators have been shown to suppress plasma estrogen levels effectively in postmenopausal breast cancer patients. However, knowledge about the influence of these drugs on estrogen levels in breast cancer tissue is limited. Using a novel HPLC-RIA method developed for the determination of breast tissue estrogen concentrations, we measured tissue E₁, E₂ and estrone sulfate (E₁S) levels in postmenopausal breast cancer patients before and during treatment with anastrozole. Our findings revealed high breast tumor tissue estrogen concentrations that were effectively decreased by anastrozole. While E₁S was the dominating estrogen fraction in the plasma, estradiol was the estrogen fraction with the highest concentration in tumor tissue. Moreover, plasma estrogen levels did not correlate with tissue estrogen concentrations. The overall experience with aromatase inhibitors and inactivators concerning their influences on breast tissue estrogen concentrations is summarized.     

Local endocrine effects of aromatase inhibitors within the breast

To determine the effects of aromatase inhibitors on oestrogen uptake, in situ aromatase activity and endogenous oestrogens in the breast, postmenopausal women with large primary ER-rich breast cancers have been treated neoadjuvantly for 3 months with either letrozole (2.5 or 10 mg daily) or anastrozole (1 or 10 mg daily) or exemestane (25 mg daily). Patients were given an infusion of ³H-androstenedione and ¹⁴C-oestrone for 18 h before and at the end of the study period. Blood, tumour and non-malignant breast were taken immediately after each infusion; oestrogens were extracted and purified. Tumour volume was measured before and during treatment at monthly intervals so that endocrinological changes could be related to clinical response. Treatment with each of the aromatase inhibitors was associated with a profound reduction in peripheral aromatase (as monitored by the level of plasma ³H-oestrone). There was no consistent effect on uptake of radioactively labelled oestrogen into breast tumours but a tendency for levels to increase after treatment in non-malignant breast. Conversely, therapy was associated with a marked inhibition of in situ oestrogen synthesis in both tumour and non-malignant breast (in occasional tissues, inhibitors appeared to be less effective but the effect was not related to clinical or pathological responses). Similar decreases were apparent in endogenous levels of oestrone and oestradiol. The absence of in situ aromatase activity tended to be associated with lack of clinical response to aromatase inhibition but the relationship was not absolute, limiting the utility of measurements of tumour aromatase as a predictive indices. Ex vivo studies of tissue aromatase indicated that such measurements consistently underestimate the inhibitory potential of reversible non-steroidal agents (and occasionally paradoxical in vitro increases in aromatase activity were seen with treatment). However, in situ assays demonstrate that new aromatase inhibitors such as anastrozole, exemestane and letrozole have profound effects on the local endocrinology within the postmenopausal breast, these being compatible with the clinico-pathological changes which occur with treatment.     

Comparative clinical pharmacology and pharmacokinetic interactions of aromatase inhibitors

The clinical development of aromatase inhibitors (AIs) has been closely guided by clinical pharmacological investigations. During the early phases of development studies were focused on dose-related pharmacological effectiveness and specificity. More recently attention has been given to the metabolic changes which AIs elicit, with particular regard to their potential use in early breast cancer and the prophylactic setting. Pharmacological effectiveness has been studied with plasma oestrogen assays but primary oestrogens (E1 and E2) are not helpful in comparing the third generation inhibitors: anastrozole, letrozole, exemestane. All three of these compounds suppress whole body aromatisation by >96%. Most recently, we have established that significantly greater inhibition is achieved by letrozole than anastrozole at their clinically used dosages. This more complete inhibition is paralleled by significantly greater suppression of E1S.

A broad panel of endocrine investigations has indicated that these compounds have essentially complete specificity at their clinical dosages. A minor androgenic effect of exemestane is revealed by a significant suppression of sex hormone binding globulin (SHBG). Lipid and bone biomarker data are being collected in many current studies. A pharmacokinetic interaction has been established between letrozole and tamoxifen, whereby reduced circulating levels of letrozole are found with combined application. Neither anastrozole nor letrozole have any effect on plasma concentrations of tamoxifen when given in combination with it.     

Synthesis of some novel androstanes as potential aromatase inhibitors

Novel pyrazole and isoxazole derivatives (6-9) were synthesized as a aromatase inhibitors. Pyrazole was synthesized from hydrazine hydrate and isoxazoles from hydroxylamine hydrochloride under different conditions. Molecular docking studies were carried out for the synthesized compounds. The best score was obtained for the compound (9) followed by compound (6) while compound (8) afforded poorest of the score. Aromatase inhibitory activity for compound (6) having pyrazole ring at 2,3 position showed highest activity followed by nitrile derivative (9). Isomeric forms of isoxazole (7 and 8) showed very poor activity compared to fadrozole and aminoglutethimide. Preliminary kinetic studies have shown that both of the active compounds (6 and 9) are reversible inhibitors of the enzyme.     

Breast cancer prevention--clinical trials strategies involving aromatase inhibitors

Estrogens and their metabolites have been implicated in both the initiation and the prevention of breast cancer. The reduction in breast cancer incidence seen in the tamoxifen arms of the four prospective trials to date has established the proof of principle that antagonizing estrogen is a potential means of reducing breast cancer risk. However, the areas to improve on these results include: (a) enhanced efficacy, (b) reduction in the incidence of receptor-negative tumors, (c) improved overall and endocrinological side effects, and (d) improved function on end-organs other than the breast. The aromatase inhibitors offer the potential to achieve these goals in part in the following ways: (a) greater reduction in risk of disease as evidenced by superior efficacy in advanced breast cancer and by inhibition of both initiation and promotion of breast cancer, (b) reduction in receptor-negative tumors by synergy with COX-2 inhibitors resulting in growth factor inhibition, anti-angiogenesis and inhibition of tumor-associated aromatase expression, (c) fewer vasomotor and urogenital abnormalities, and (d) reduced thromboembolism and cardiovascular complications and satisfactory effects on bone metabolism. Important differences may exist between non-steroidal reversible inhibitors and steroidal irreversible inactivators in particular related to the androgenic/anabolic effects of the steroidal inactivators. Pilot studies of aromatase inhibitors described elsewhere in this session have begun in healthy women with dense mammography, or a high-risk genetic and/or histocytopathologic profile, to determine potential efficacy, as well as effects on end-organ function. A number of phase three trials with aromatase inhibitors are also underway or in planning. Among these are the BRCA 1 and 2 study of exemestane versus placebo in unaffected postmenopausal carriers, the International Breast Intervention Study 2 (IBIS 2) of anastrozole versus placebo in women with a high-risk profile, and the National Cancer Institute of Canada’s Clinical Trial Group (NCIC CTG) study of exemestane with or without celecoxib versus placebo in women at risk of the disease. For premenopausal women, combination strategies of gonadotrophin agonists and aromatase inhibitors are being investigated. The potential of using low doses of aromatase inhibitors to lower “in breast” estrogen levels without unduly perturbing plasma concentrations is also being explored. The potential of the aromatase gene functioning as an oncogene within the breast may be tied to breast density which in turn may represent both a selection tool for elevated risk and an intermediate marker of prevention. The strong link between postmenopausal estrogen levels and breast cancer risk suggests the possibility that plasma estrogen levels may be a useful intermediate marker of prevention. The aromatase inhibitors offer us the first ever tool to render women virtually free of estrogen and are potentially an exciting tool for the prevention of breast cancer.     

Adjuvant trials of aromatase inhibitors: determining the future landscape of adjuvant endocrine therapy

This review will discuss the role of aromatase inhibitors (AIs) in the adjuvant setting, and will summarize major strategies behind individual adjuvant trials using aromatase inhibitors. Studies with the third generation AIs including anastrozole, letrozole and exemestane, have shown better outcome and improved therapeutic ratio over second line hormonal approaches (i.e. progestins or aminoglutethimide) and, more recently, over tamoxifen also. These promising results have led recently to testing of AIs in the adjuvant setting for postmenopausal patients. Most trials now in progress are evaluating the role of new AIs versus tamoxifen (T) given×5 years, which in most institutions is currently the standard hormonal adjuvant therapy for breast cancer. Three adjuvant approaches are being tested. First is the use of AI+T×5 years in combination versus each agent alone, as reflected in the recently completed ATAC trial. Second is a sequential approach T first×2–3 years followed by AIs×2–3 years, or the other way round; and third, T×5 years followed by AIs for additional 5 years (i.e. total duration of adjuvant hormones of 10 years). Many patients in the above trials will survive their first cancer. Hence, the non-oncological outcomes known to be affected by hormones are of rising importance. Therefore, the assessment of lipids as surrogates for cardiovascular morbidity, and of bone mineral status, as a marker for osteoporosis/bone fractures, is an important component of these trials. Also discussed in this review are proposals for future studies of AIs with focus on hormone resistance, such as early alteration of multiple hormonal agents or their intermittent use, the impact of the new generation of SERMs or ‘pure’ antiestrogens on activity of AIs, and the rising importance of AIs interacting with biologicals, cytokines or hormone modulators.     

Chemoprevention with aromatase inhibitors--trial strategies

Estrogen and its catechol metabolites from both the circulation and synthesized within the breast are important in the pathogenesis of breast cancer. Blocking estrogen's effects on the breast with selective estrogen receptor modulators (SERMS) is an ongoing strategy. Thus, tamoxifen and raloxifene reduce risk as monotherapy. Aromatase (estrogen synthetase) inhibitors are a logical alternative to SERMS. To date, SERMS have demonstrated reduction only in estrogen–progesterone receptor positive cancers without reduction in receptor negative tumors. By inhibiting the parent estrogens and their catechol metabolites, true prevention of cancer initiation might occur and reduction not only in the receptor positive but also negative tumors might result. Ongoing adjuvant breast cancer trials are exploring aromatase inhibitors as alternatives to tamoxifen, or in sequence or in combination with tamoxifen. Relative efficacies including reduction in contralateral breast cancer, toxicities and end-organ effects and impact on quality of life, are being explored. Data from these trials will help to guide future chemoprevention strategies. Proof of principal trials in ‘high risk’ cohorts such as premalignant breast lesions, dense screening mammograms, high plasma estradiol levels or increased bone density are already ongoing. Issues such as dose, schedule, therapeutic index and mono versus combination therapy are important to define.     

DCIS and aromatase inhibitors

In patients with hormone receptor positive DCIS tamoxifen reduces recurrence rates by almost 50%. Few data are available with aromatase inhibitors from randomised studies. In the ATAC study there were three DCIS lesions in the anastrozole arm and four in the tamoxifen arm in the women with ER positive invasive cancer. In the MA17 study which randomised patients to up to 5 years of letrozole or placebo there was only one DCIS event in the contralateral breast in patients taking letrozole and five on placebo. There were also four patients in this study who had DCIS in the conserved breast on placebo and none in the letrozole treated group. The few clinical data that are available therefore suggest the aromatase inhibitors are likely to be effective in DCIS. A histological review of a study of 206 postmenopausal women with invasive oestrogen receptor positive breast cancer who were randomised as part of a 14 day preoperative study to receive 2.5 mg of letrozole or 1 mg of anastrozole identified 27 patients with 28 pairs of tumours in whom there was sufficient ER positive DCIS in invasive cancer in the initial core biopsy and in the subsequent surgery specimen, to evaluate for PgR activity and proliferation. Within the DCIS both aromatase inhibitors significantly reduced PgR expression and both drugs also produced a significant fall in proliferation. There was a moderate degree of agreement between the fall in PgR in both the invasive cancer and DCIS (Kappa = 0.5; p = 0.0013) and between the fall in proliferation and between the invasive and in situ components (correlation coefficient = 0.68; p < 0.001). This study has shown significant effects of aromatase inhibitors on DCIS indicating that these agents are therapeutically active in this condition.     

Pharmacological profile of MEN 11066, a novel potent and selective aromatase inhibitor

MEN 11066 is a new non-steroidal compound which potently inhibits human placenta (K_i=0.5 nM) and rat ovarian (K_i=0.2 nM) aromatase in vitro. In vivo, a single oral dose of 0.3 mg kg⁻¹ significantly decreased uterus weight in immature rats after stimulation of uterus growth by androstenedione. MEN 11066 reduced in a dose-dependent manner plasma estradiol levels in adult female rats treated with pregnant mare serum gonadotropin (PMSG). After 2 weeks of repeated daily treatment in adult rats, a significant decrease in uterine weight was observed together with a 65% decrease in plasma estradiol, whereas plasma levels of testosterone, progesterone, aldosterone, corticosterone, cholesterol, LH and FSH were not affected. The lack of any effect by MEN 11066 on adrenal steroids was confirmed by the unchanged plasma corticosterone and aldosterone levels in immature rats and also in adult rats when the repeated treatment with MEN 11066 (15 days) was followed by the administration of a synthetic ACTH analogue. No change in 11β-hydroxylase or 21-hydroxylase activities was produced in vitro by the addition of 10 μM MEN 11066. Fifteen-day treatment with MEN 11066 did not produce changes in several rat hepatic enzymatic activities involved in the metabolism of xenobiotics. These results demonstrated that MEN 11066 is a potent inhibitor of aromatase which does not interfere with the cytochrome P450 involved in the synthesis of other steroids or in the metabolism of xenobiotics.     

Novel treatment of short stature with aromatase inhibitors

Estrogens have an essential role in the regulation of bone maturation and importantly in the closure of growth plates in both sexes. This prospective, randomized, placebo-controlled study was undertaken to evaluate whether suppression of estrogen synthesis in pubertal boys delays bone maturation and ultimately results in increased adult height.

A total of 23 boys with constitutional delay of puberty (CDP) received a conventional, low-dose testosterone treatment for inducing progression of puberty. Eleven of these 23 boys were randomized to receive a specific and potent P450-aromatase inhibitor, letrozole, for suppression of estrogen action, and 12 boys were randomized to receive placebo. Estradiol concentrations in the letrozole-treated boys remained at the pretreatment level during the administration of letrozole, whereas the concentrations increased during the treatment with testosterone alone and during spontaneous progression of puberty. Testosterone concentrations increased in all groups, but during the letrozole treatment, the increase was more than fivefold higher than in the group treated with testosterone alone.

The inhibition of estrogen synthesis delayed bone maturation. The slower bone maturation in the boys treated with testosterone and letrozole, despite higher androgen concentrations, than in the boys treated with testosterone indicate that estrogens are more important than androgens in regulation of bone maturation in pubertal boys. During the 18 months follow-up, an increase of 5.1 cm in predicted adult height was observed in the boys who received testosterone and letrozole, but no change was seen in the boys who received testosterone alone or in the untreated boys. This finding indicates that an increase in adult height can be attained in growing adolescent boys by inhibiting of estrogen action.     

Recent data on intratumor estrogens in breast cancer

The contribution of local synthesis versus circulatory delivery of normal breast as well as breast cancer tissue estrogens has remained a controversial area for decades. Novel data on tissue estrogen levels confirm a positive normal breast tissue to plasma concentration gradient for estrone, and to a smaller extent estradiol. Remarkably, this gradient is similar for pre- and post-menopausal women. Together with pharmacokinetic data on estrogen disposition, these findings suggest plasma and breast tissue estrogens to rapidly equilibrate, with circulating estrogens being a major contributor to breast tissue estrone levels. A likely explanation to the concentration gradient could be the fact that non-polar estrogens easily dissolve into tissue fat compartments as compared to plasma. While intratumor estrone levels are low as compared to benign tissue concentrations, intratumor estradiol is elevated in ER+ tumors. The correlation between intratumor estradiol levels and expression levels of dehydrogenases reducing estrone into estradiol but also intratumor ER concentrations are consistent with intratumor estrogen activation but also a scavenger effect of the ER.     

Investigation of fluorinated and bifunctionalized 3-phenylchroman-4-one (isoflavanone) aromatase inhibitors

Fluorinated isoflavanones and bifunctionalized isoflavanones were synthesized through a one-step gold(I)-catalyzed annulation reaction. These compounds were evaluated for their in vitro inhibitory activities against aromatase in a fluorescence-based enzymatic assay. Selected compounds were tested for their anti-proliferative effects on human breast cancer cell line MCF-7. Compounds 6-methoxy-3-(pyridin-3-yl)chroman-4-one (3c) and 6-fluoro-3-(pyridin-3-yl)chroman-4-one (3e) were identified as the most potent aromatase inhibitors with IC₅₀ values of 2.5 μM and 0.8 μM. Therefore, these compounds have great potential for the development of pharmaceutical agents against breast cancer.     

An integrated view of aromatase and its inhibition

Aromatase inhibition has become a major treatment strategy for postmenopausal women with oestrogen-dependent breast cancer. Its optimal application is, however, dependent upon (i) the accurate identification of cancers which are ultimately dependent upon the activity of the aromatase enzyme, (ii) the use of the best method/inhibitor by which to blockade aromatase activity.

The single best predictor of response to aromatase inhibitors is the presence of tumour oestrogen receptors; receptor-negative cancers rarely respond whereas those with high levels seem particularly likely to benefit. However, there is a need for additional discriminatory markers. The use of microarray technology coupled with neoadjuvant therapy is likely to yield promising candidate genes. The finding that, amongst peripheral tissues, the tumour itself may have high activity has led to the suggestion that the tumour aromatase measurements may be predictive; however, in situ studies and the lack of robust assays for tumour aromatase suggest that tumour aromatase may not be an influential marker.

Whilst drugs such as anastrozole, exemestane, formestane and letrozole are all effective and specific inhibitors of aromatase, they differ in structure, potency and mechanism of action. Thus, differential sensitivity of tissues/tumours and non-cross resistance mean inhibitors are not equivalent and individual agents may have differing roles according to the setting in which they will be used. Aromatase inhibitors have evolved as key endocrine agents in the treatment of breast cancer. They offer the promise of rational treatment management based on the accurate identification of individual cohorts of tumours responsive to specific drugs.     

Investigation of aryl halides as ketone bioisosteres: Refinement of potent and selective inhibitors of human cytochrome P450 19A1 (aromatase)

Bioisosteric replacement of cyclic ketone functionality with aryl halides was investigated on a centrally-flexible, five-component 1,2,3-triazole-containing pharmacophore, resulting in enhanced inhibition of aromatase (CYP450 19A1). Structure–activity data generated from both syn- and anti-aldol precursors provides significant insights into the requirements for enhanced potency, validating this novel ketone-to-aryl halide bioisostere hypothesis.     

Hormonal effects of aromatase inhibitors: focus on premenopausal effects and interaction with tamoxifen

Third generation aromatase inhibitors have excellent specificity. Some reports indicate that letrozole may have a minor effect on cortisol synthesis but these were not confirmed: valid comparisons with other aromatase inhibitors requires randomised study.

The putative use of a third generation inhibitor as a single agent in premenopausal women has been investigated using YM511. It was hypothesised that in this situation site-specific suppression of estrogens in breast carcinomas, without systemic effects, may lead to a down-regulation of tumour proliferation. Plasma levels of androstenedione and testosterone were significantly increased by 2 weeks treatment with YM511. Mean plasma estrone levels were suppressed, but some plasma estradiol levels were abnormally high and others abnormally low. These differential effects of YM511 on circulating estrogens supported the concept that peripheral synthesis of estrogens might be suppressed while ovarian production remained high. However, YM511 did not demonstrate anti-proliferative effects in hormone sensitive breast carcinomas.

Consideration of the pharmacology of the estrogen receptor during tamoxifen therapy indicates that tamoxifen effectively saturates the receptor (>99.94% occupancy) in postmenopausal women. The addition of an aromatase inhibitor in this situation would be very unlikely to affect the biological activity of the estrogen receptor. This provides a possible explanation why the clinical efficacy of tamoxifen combined with an aromatase inhibitor appears to be equivalent to that of tamoxifen alone.     

The intratumoral aromatase model: studies with aromatase inhibitors and antiestrogens

Aromatase inhibitors have now been approved as first-line treatment options for hormone-dependent advanced breast cancer. When compared to tamoxifen, these aromatase inhibitors provide significant survival and tolerability advantages. However, the optimal use of an aromatase inhibitor and tamoxifen remains to be established. To date, the intratumoral aromatase xenograft model has proved accurate in predicting the outcome of clinical trials. Utilizing this model, we performed long-term studies with tamoxifen and letrozole to determine time to disease progression with each of the treatment regimens. Aromatase-transfected MCF-7Ca human breast cancer cells were grown as tumor xenografts in female ovariectomized athymic nude mice in which androstenedione was converted to estrogen and stimulated tumor growth. When tumor volumes were approximately 300 mm³, the animals were grouped for continued supplementation with androstenedione only (control) or for treatment with letrozole 10 μg per day (long-term), tamoxifen 100 μg per day (long-term), letrozole alternating to tamoxifen (4-week rotation), tamoxifen alternating to letrozole (4-week rotation), or a combination of the two drugs. Tumors of control mice had doubled in volume in 3–4 weeks. In mice treated with tamoxifen and the combination, tumor doubling time was significantly shorter (16 and 18 weeks, respectively) than with letrozole (34 weeks). Furthermore, alternating letrozole and tamoxifen treatment every 4 weeks was less effective than letrozole alone. Tumors doubled in 17–18 weeks when the starting treatment was tamoxifen and in 22 weeks when the starting treatment was letrozole. Tumors progressing on tamoxifen remained sensitive to second-line therapy with letrozole (10 μg per day). However, when mice with letrozole-resistant tumors were switched to antiestrogen treatment, tumors did not respond to tamoxifen (100 μg per day) or faslodex (1 mg per day). This suggests that advanced breast cancers treated with letrozole may be insensitive to subsequent second-line hormonal agents. Thus, although letrozole was determined to be an effective second-line treatment option for tumors progressing on tamoxifen, antiestrogen therapy does not appear to be effective for tumors progressing on letrozole. However, response to second-line treatment was observed in a model where tumors that had progressed on letrozole were transplanted to new mice. These tumors had been allowed to grow in the presence of supplemented androstenedione but absence of letrozole. This suggests that resistance to letrozole may be reversible, allowing tumors to respond to subsequent antiestrogens and letrozole.     

Relationships between rapid changes in local aromatase activity and estradiol concentrations in male and female quail brain

Estradiol-17β (E₂) synthesized in the brain plays a critical role in the activation of sexual behavior in many vertebrate species. Because E₂ concentrations depend on aromatization of testosterone, changes in aromatase enzymatic activity (AA) are often utilized as a proxy to describe E₂ concentrations. Utilizing two types of stimuli (sexual interactions and acute restraint stress) that have been demonstrated to reliably alter AA within minutes in opposite directions (sexual interactions = decrease, stress = increase), we tested in Japanese quail whether rapid changes in AA are paralleled by changes in E₂ concentrations in discrete brain areas. In males, E₂ in the pooled medial preoptic nucleus/medial portion of the bed nucleus of the stria terminalis (POM/BST) positively correlated with AA following sexual interactions. However, following acute stress, E₂ decreased significantly (approximately 2-fold) in the male POM/BST despite a significant increase in AA. In females, AA positively correlated with E₂ in both the POM/BST and mediobasal hypothalamus supporting a role for local, as opposed to ovarian, production regulating brain E₂ concentrations. In addition, correlations of individual E₂ in POM/BST and measurements of female sexual behavior suggested a role for local E₂ synthesis in female receptivity. These data demonstrate that local E₂ in the male brain changes in response to stimuli on a time course suggestive of potential non-genomic effects on brain and behavior. Overall, this study highlights the complex mechanisms regulating local E₂ concentrations including rapid stimulus-driven changes in production and stress-induced changes in catabolism.