Aromatase and its inhibitors--an overview

Estrogen synthesis by aromatase occurs in a number of tissues throughout the body. Strategies which reduce production of estrogen offer useful means of treating hormone-dependent breast cancer. Initially, several steroidal compounds were determined to be selective inhibitors of aromatase. The most potent of these, 4-hydroxyandrostenedione (4-OHA) inhibits aromatase competitively but also causes inactivation of the enzyme. A number of other steroidal inhibitors appear to act by this mechanism also. In contrast, the newer imidazole compounds are reversible, competitive inhibitors. In vivo studies demonstrated that 4-OHA inhibited aromatase activity in ovarian and peripheral tissues and reduced plasma estrogen levels in rat and non-human primate species. In rats with mammary tumors, reduction in ovarian estrogen production was correlated with tumor regression. 4-OHA was also found to inhibit gonadotropin levels in animals in a dose-dependent manner. The mechanism of this effect appears to be associated with the weak androgenic activity of the compound. Together with aromatase inhibition, this action may contribute to reducing the growth stimulating effects of estrogen. A series of studies have now been completed in postmenopausal breast cancer patients treated with 4-OHA either 500 mg/2 weeks or weekly, or 250 mg/2 weeks. These doses did not affect gonadotropin levels. Plasma estrogen concentrations were significantly reduced. Complete or partial tumor regression occurred in 26% of the patients and the disease was stabilized in 25% of the patients. The results suggest that 4-OHA is of benefit to postmenopausal patients who have relapsed from prior hormonal therapies. Several of the steroidal inhibitors are now entering clinical trials as well as non-steroidal compounds which are more potent and selective than aminoglutethimide. Aromatase inhibitors should provide several useful additions to the treatment of breast cancer.     

Aromatase inhibitors as adjuvant therapies in patients with breast cancer

There is increasing evidence that endocrine therapy has an important role in patients with oestrogen receptor positive breast cancer. Several large meta-analyses have reinforced the value of both ovarian ablation and tamoxifen in improving survival. Over the past decade, aromatase inhibitors have become the treatment of choice for second-line therapy of metastatic breast cancer, and the third generation inhibitors have now an established reputation for good patient tolerability. Early studies indicated that aminoglutethimide/hydrocortisone could benefit postmenopausal patients with primary breast cancer, and in 2001, the ATAC study showed that the third generation aromatase inhibitor, anastrozole, seemed superior to tamoxifen in that anastrozole-treated patients had a longer disease-free survival. Other studies will report on the relative merits of the steroidal inhibitor exemestane as well as non-steroidal letrozole. The exact duration and sequencing of treatment, together with the long-term effects on bone are at present, unknown.     

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.     

A summary of second-line randomized studies of aromatase inhibitors

The new generation of selective aromatase inhibitors (anastrozole, letrozole and exemestane) offer a significant efficacy and safety advantage over both older agents in this class (aminoglutethimide) and the progestins (megestrol acetate (MA)), as second-line treatment for postmenopausal women with advanced hormone-dependent breast cancer who have failed on tamoxifen therapy. Exemestane, a steroidal aromatase inhibitor, has been shown to have activity after failure with the non-steroidal aromatase inhibitors, anastrozole and letrozole, and could be used as third-line treatment. Although the newer aromatase inhibitors belong to the same class and appear, from indirect comparisons, to have similar efficacy compared with the older therapies, they have different pharmacokinetic and pharmacodynamic profiles, suggesting the potential for clinical differences. Compared with exemestane and letrozole, anastrozole shows greater selectivity for aromatase, as it lacks any evidence of an effect on adrenal steroidogenesis and has no androgenic effects. Therefore, it is clear that these agents should not be considered to be similar in all respects. In summary, the introduction of the aromatase inhibitors represents a significant step forward in the treatment of advanced breast cancer in postmenopausal women. Studies in the adjuvant setting will ultimately determine whether the differences in pharmacokinetics and phamacodynamics will be of clinical relevance.     

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.     

Aromatase and its inhibitors

Inhibitors of aromatase (estrogen synthetase) have been developed as treatment for postmenopausal breast cancer. Both steroidal substrate analogs, type I inhibitors, which inactivate the enzyme and non-steroidal competitive reversible, type II inhibitors, are now available. 4-hydroxyandrostenedione (4-OHA), the first selective aromatase inhibitor, has been shown to reduce serum estrogen concentrations and cause complete and partial responses in approximately 25% of patients with hormone responsive disease who have relapsed from previous endocrine treatment. Letrozole (CGS 20, 269) and anastrozole (ZN 1033) have been recently approved for treatment. Both suppress serum estrogen levels to the limit of assay detection. Letrozole has been shown to be significantly superior to megace in overall response rates and time to treatment failure, whereas anastrozole was found to improve survival in comparison to megace. Both were better tolerated than the latter. The potential of aromatase within the breast as a significant source of estrogen mediating tumor proliferation and which might determine the outcome of inhibitor treatment was explored. Using immunocytochemistry and in situ hybridization, aromatase and mRNA_arom was detected mainly in the epithelial cells of the terminal ductal lobular units (TDLU) of the normal breast and also in breast tumor epithelial cells as well as some stromal cells. Increase in proliferation, measured by increased thymidine incorporation into DNA and by PCNA immunostaining in response to testosterone was observed in histocultures of breast cancer samples. This effect could be inhibited by 4-OHA and implies that intratumoral aromatase has functional significance. An intratumoral aromatase model in the ovariectomized nude mouse was developed which simulated the hormone responsive postmenopausal breast cancer patient. This model also allows evaluation of the efficacy of aromatase inhibitors and antiestrogens in tumors of estrogen receptor positive, human breast carcinoma cells transfected with the human aromatase gene. Thus, the cells synthesized estrogen which stimulated tumor formation. Both aromatase inhibitors and antiestrogens were effective in suppressing tumor growth in this model. However, letrozole was more effective than tamoxifen. When the aromatase inhibitors were combined with tamoxifen, tumor growth was suppressed to about the same extent as with the aromatase inhibitors alone. Thus, there was no additive or synergistic effects of combining tamoxifen with aromatase inhibitors. This suggests that sequential treatment with these agents is likely to be more beneficial to the patient in terms of longer response to treatment.     

Aromatase inhibitors and their application in breast cancer treatment*

Estrogens are known to be important in the growth of breast cancers in both pre- and postmenopausal women. The number of breast cancer patients with hormone-dependent disease increases with age, as does the incidence of breast cancer. Although estrogens are no longer made in the ovaries after menopause, peripheral tissues produce sufficient concentrations to stimulate tumor growth. Because aromatase catalyzes the rate-limiting step in the biosynthesis of estrogen, inhibitors of this enzyme have been developed in the last few years as a logical treatment strategy. Two classes of aromatase inhibitors, steroidal and nonsteroidal compounds, are now in use. Among the steroid substrate analogs, formestane and examestane have been shown to be effective in breast cancer patients with advanced disease. Highly potent and selective nonsteroidal inhibitors have recently been found to suppress plasma and urinary estrogens by more than 95% in breast cancer patients. Two of these compounds recently were approved in the United States and have been shown to be more effective than other second-line agents in terms of overall response rates and treatment failure, as well as better tolerated. Although studies of the efficacy of these agents in earlier stage disease are awaited, it is evident that aromatase inhibitors can extend the duration of treatment in breast cancer patients.     

Molecular pharmacology of aromatase and its regulation by endogenous and exogenous agents

Aromatase (estrogen synthase) is the cytochrome P450 enzyme complex that converts C₁₉ androgens to C₁₈ estrogens. Aromatase activity has been demonstrated in breast tissue in vitro, and expression of aromatase is highest in or near breast tumor sites. Thus, local regulation of aromatase by both endogenous factors as well as exogenous medicinal agents will influence the levels of estrogen available for breast cancer growth. The prostaglandin E₂ (PGE₂) increases intracellular cAMP levels and stimulates estrogen biosynthesis, and our recent studies have shown a strong linear association between CYP19 expression and the sum of COX-1 and COX-2 expression in breast cancer specimens. PGE₂ can bind to four receptor subtypes, EP₁–EP₄, which are coupled to different intracellular signaling pathways. In primary human breast stromal cell cultures, aromatase activity was significantly induced by PGE₂, dexamethasone, and agonists for the EP₁ and EP₂ receptor subtypes. An EP₁ antagonist, SC-19220, inhibited the induction of enzyme activity by PGE₂ or 17-phenyltrinor-PGE₂, an EP₁ agonist. Sulprostone, an EP₃ agonist, did not alter aromatase activity levels. Investigations are also underway on the regulation of aromatase by exogenous medicinal agents. Selective steroidal and nonsteroidal agents are effective in inhibiting breast tissue aromatase. The benzopyranone ring system is a molecular scaffold of considerable interest, and this scaffold is found in certain flavonoid natural products that have weak aromatase inhibitory activity. Our novel synthetic route for benzopyranones utilizes readily available salicylic acids and terminal alkynes as starting materials. The synthesis of flavones with diversity on the benzopyranone moiety and at the C-2 position occurs with good to excellent yields using these reaction conditions, resulting in an initial benzopyranone library of thirty compounds exhibiting enhanced and differential aromatase inhibition. Current medicinal chemistry efforts focus on diversifying the benzopyranone scaffold and utilizing combinatorial chemistry approaches to construct small benzopyranone libraries as potential aromatase inhibitors.     

Aromatase inhibitors and inactivators in the treatment of advanced breast cancer

Advanced breast cancer remains incurable. For these patients, durable response and minimal toxicity are the main goals of current therapy. The antiestrogen tamoxifen has proved to be a significant advance in the treatment of breast cancer. Due to its partial estrogen activity, long term medication with tamoxifen has been found to cause endometrium proliferation wich can result in cancer in some patients. Reduction of estrogen production identified the aromatase inhibitors. Both steroidal substrate analog, type I inactivator, wich inactivate the enzyme and non-steroidal competitive reversible, type II inhibitors, are now avaiable. Two new 3(rd) generation aromatase inactivators have recently completed phase III evaluation (anastrozole and letrozole) and we have some results investigating one of the new 3(rd) generation aromatase inhibitors (exemestane). The 3(rd) generation aromatase inhibitors and inactivators are better tolerated and more effective than each of our current standard 2(nd) line endocrin therapies. These agents are being directly compared with standard adjuvant medication, tamoxifen, or are being evaluated in different sequences.     

The control and biological importance of intratumoural aromatase in breast cancer

The existence of aromatase activity in human breast carcinomas has been established for about 20 years but the clinical and biological importance of this remains unclear. A number of studies in clinical material suggest that aromatase activity may be a prerequisite of response to aromatase inhibitors and that aromatase activity may be enhanced in those tumours relapsing during treatment with one such inhibitor, aminoglutethimide. These results would carry more significance, however, if it was demonstrable that the growth of breast carcinomas is affected by the conversion of androgens to oestrogens by intratumoural aromatase. We have tried to address this by establishing model systems with aromatase-transfected MCF7 breast cancer cells. We have demonstrated that these cells can be stimulated mitogenically with androgen and that this proliferation is suppressible with aromatase inhibitors. Similarly the growth of aromatase transfected cells but not wild type cells as xenografts is supported by androstenedione and inhibitable by both the steroidal aromatase inhibitor, 4-hydroxyandrostenedione and the non-steroidal inhibitor, CGS 20267. Work with the former of these, which is a suicide inhibitor allowed us to demonstrate that growth can proceed with aromatase activity approximating to the highest level seen in breast carcinomas indicating that at least at this extreme level the intratumoural conversion of androgens to oestrogens may indeed be able to support tumour growth. Further work with this model system should allow us to define the minimal amount of intratumoural activity which can support tumour growth.     

Endocrine approaches for the treatment of early and advanced breast cancer in postmenopausal women

Preventing clinical progression is the major treatment goal for both early and advanced breast cancer. For hormone-responsive cases (about 70% of the total), this can necessitate the use of sequential hormone therapies at various points during the patient's life. Newer hormonal therapies, such as the third-generation aromatase inhibitor anastrozole, are now competing with tamoxifen as first choice endocrine therapy in breast cancer. In addition, a further non-steroidal aromatase inhibitor letrozole has been shown to be beneficial when given at completion of 5 years adjuvant tamoxifen. In light of these new data, current treatment paradigms need to be reviewed. Already well established as second-line treatments for advanced breast cancer, the improved risk:benefit profiles of anastrozole and letrozole compared with tamoxifen mean that these agents are now also recognised alternative treatments in the first-line relapse setting. More recent studies demonstrate that anastrozole may also have an improved risk:benefit profile compared with tamoxifen when used as initial adjuvant therapy in early breast cancer. Anastrozole is also being evaluated as a preventative treatment in women at high risk of developing breast cancer. A new addition to the endocrine treatment armamentarium is the oestrogen receptor antagonist fulvestrant, which, unlike tamoxifen, has no agonist effects. Fulvestrant is at least as effective as anastrozole in the second-line treatment of advanced breast cancer, and provides similar benefits to tamoxifen when used as first-line therapy in patients with advanced, hormone receptor-positive tumours.     

Development and evolution of therapies targeted to the estrogen receptor for the treatment and prevention of breast cancer

This article describes the origins and evolution of "antiestrogenic" medicines for the treatment and prevention of breast cancer. Developing drugs that target the estrogen receptor (ER) either directly (tamoxifen) or indirectly (aromatase inhibitors) has improved the prognosis of breast cancer and significantly advanced healthcare. The development of the principles for treatment and the success of the concept, in practice, has become a model for molecular medicine and presaged the current testing of numerous targeted therapies for all forms of cancer. The translational research with tamoxifen to target the ER with the appropriate duration (5 years) of adjuvant therapy has contributed to the falling national death rates from breast cancer. Additionally, exploration of the endocrine pharmacology of tamoxifen and related nonsteroidal antiestrogen (e.g. keoxifene now known as raloxifene) resulted in the laboratory recognition of selective ER modulation and the translation of the concept to use raloxifene for the prevention of osteoporosis and breast cancer. However, the extensive evaluation of tamoxifen treatment revealed small but significant side effects such as endometrial cancer, blood clots and the development of acquired resistance. The solution was to develop drugs that targeted the aromatase enzyme specifically to prevent the conversion of androstenedione to estrone and subsequently estradiol. The successful translational research with the suicide inhibitor 4-hydroxyandrostenedione (known as formestane) pioneered the development of a range of oral aromatase inhibitors that are either suicide inhibitors (exemestane) or competitive inhibitors (letrozole and anastrozole) of the aromatase enzyme. Treatment with aromatase inhibitors is proving effective and is associated with reduction in the incidence of endometrial cancer and blood clots when compared with tamoxifen and there is also limited cross resistance so treatment can be sequential. Current clinical trials are addressing the value of aromatase inhibitors as chemopreventive agents for postmenopausal women.     

Aromatase inhibitors in gynecologic cancers

The female genital tract is hormonally responsive, and consequently some tumors, which arise within in it, may be treated at least in part, with hormonal manipulation. The range of responses in clinical trials and case reports will be reviewed. Many of these diseases are too rare for clinical trial testing, and in some cases evidence is anecdotal at best. Recurrences of ovarian cancer have been treated with tamoxifen and megesterol acetate with variable response rates from 0 to 56%. The favorable toxicity profile of aromatase inhibitors led to trials of these agents for the treatment of relapsed epithelial ovarian cancer. These agents have proved tolerable with minor response rates but a significant disease stabilization rate, which may be prolonged in a minority of cases. It is unclear if these responses may be predicted by estrogen receptor expression or aromatase expression. Anastrazole has also been tried in combination with an EGFR receptor-inhibitor, again showing minor responses but possibly an increase in TTT in some patients. Granulosa cell tumors of the ovary are rare, hormonally sensitive tumors, with reported responses to a variety of hormonal manipulations, including aromatase inhibition. In addition, combined endocrine blockade, including aromatase inhibition, has been tried with reports of success. Endometrial cancers, particularly type I lesions, are often treated with hormonal manipulation, most commonly with progestins, but also with antiestrogens such as tamoxifen. A trial of aromatase inhibition in the treatment of recurrent endometrial cancer showed minimal responses. Endometrial stromal sarcoma, an uncommon uterine malignancy, has shown response to hormonal treatments, with multiple case reports of efficacy of aromatase inhibition. Despite the rarity of some of these tumor types, rare tumor study groups, such as within the Gynecologic Oncology Group, should make an effort to prospectively define the utility of these treatments.     

Aromatase inhibitors and hormone-dependent cancers

Aromatase (estrogen synthetase) occurs in a variety of tissues. Using immunocytochemistry, we have recently located this enzyme in cellular compartments of several types of human tissue. Furthermore, we found the mRNA was located in the same structures where tested. As both gonadal and peripherally formed estrogen contribute to growth of hormone sensitive cancers, we have developed aromatase inhibitors to block synthesis of this hormone. We have determined that 4-hydroxyandrostenedione (4-OHA) selectively inhibits aromatase activity in ovarian and peripheral tissues and reduces plasma estrogen levels in rat and non-human primate species. 4-OHA was also found to inhibit gonadotropin levels and reduce estrogen and progesterone receptor levels in treated animals. The mechanism of these effects appear to be associated with the weak androgenic activity of the compound. These effects together with aromatase inhibition may result in a synergistic response reducing estrogen production and action. In postmenopausal women, estrogens are mainly of peripheral origin. When postmenopausal breast cancer patients were administered either daily oral or parenteral weekly treatment with 4-OHA at doses that did not affect their gonadotropin levels, plasma estrogen concentrations were significantly reduced. Complete or partial response to treatment occurred in 34% of 100 patients with advanced breast cancer, while the disease was stabilized in 12%. These results indicate that 4-OHA is of benefit in postmenopausal patients with advanced disease who have relapsed from prior hormonal therapies, and that steroidal inhibitors may be of value in premenopausal patients.     

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.     

Structure-activity relationships and binding model of novel aromatase inhibitors

The use of aromatase inhibitors is an established therapy for oestrogen-dependent breast cancer in postmenopausal women. However, the sole commercially available aromatase inhibitor, aminoglutethimide, is not very selective. We have therefore developed fadrozole hydrochloride and CGS 20 267, which are both currently under clinical evaluation. This report will present an analysis of structure-activity relationships in the azole series of inhibitors and give an account of the further optimization of our development compounds, starting from CGS 20 267 over CGP 45 688 and leading to CGP 47 645, the most potent aromatase inhibitors in vivo reported to date. In addition, on the basis of comparisons of these azole-type inhibitors with the most potent steroidal inhibitors published in the literature, we propose a CAMM-generated model describing the relative binding modes of these two classes of compounds at the active site of the enzyme.     

Control of aromatase activity in breast tumours: the role of the immune system

Cytokines such as interleukin-6 (IL-6) and tumour necrosis factor alpha (TNF), have been identified as important regulators of aromatase activity in fibroblasts derived from normal and malignant breast tissues, and may play an important role in controlling aromatase activity in breast tumours. The major source of such cytokines within breast tumours remains to be established but macrophages and lymphocytes, which can infiltrate tumours, have been identified as a potential source of aromatase stimulatory cytokines. To obtain further insight into the possible role played by the immune system in cancer development, and in particular its ability to regulate aromatase activity via cytokine production, we have obtained peripheral blood monocytes and lymphocytes from an immunosuppressed kidney transplant recipient, receiving cyclosporin A therapy, and a woman with breast cancer. Monocytes and lymphocytes were stimulated with lipopolysaccharide (LPS), and the conditioned medium (CM) collected from these cells was tested for its ability to stimulate aromatase activity in fibroblasts derived from normal breast tissue from a woman undergoing lumpectomy for the removal of a breast tumour. The white blood cell count was lower for the immunosuppressed patient, mainly because of the reduction in the number of monocytes and lymphocytes. The ability of CM from the monocytes and lymphocytes of the immunosuppressed patient to stimulate aromatase activity was significantly reduced (68% and 82% for monocytes and lymphocytes, respectively) compared with that of CM from the cells of the woman with breast cancer. It is possible, therefore, that immunosuppression, which has been found to be associated with a reduction in the incidence of de novo breast cancer in kidney transplant recipients, may exert its effect by inhibiting cytokine production by the cells of the immune system and thus oestrogen synthesis. In contrast to the stimulatory effects that TNF has on aromatase activity in breast fibroblasts, in MCF-7 breast cancer cells, which possess low aromatase activity, it reduced activity. However, the extent of inhibition of aromatase activity in these epithelial cells was much lower than the marked stimulation which it can induce in breast fibroblasts.     

Aromatase inhibitors in the treatment of breast cancer

A number of inhibitors of estrogen synthesis are now becoming available which could be of value in the treatment of breast cancer. 4-Hydroxyandrostenedione (4-OHA), the first of these compounds to enter the clinic has been found to be effective in postmenopausal patients who have relapsed from tamoxifen. Thus, in studies of 240 patients, 26% patients experienced partial or complete response to treatment. An additional 25% patients had disease stabilization. 4-OHA is a potent selective, steroidal inhibitor which causes inactivation of aromatase in vitro. It is effective in reducing concentrations of ovarian estrogens in rats and of ovarian and peripheral estrogens in non-human primate species. The compound has been shown to lower serum estrogen levels in postmenopausal breast cancer patients. However, not all of these patients experienced disease remission, suggesting that their tumors were hormone insensitive rather than that the dose of 4-OHA was suboptimal. In trials of patients who had not received prior tamoxifen treatment, 4-OHA (250 mg i.m. every 2 weeks) was found to induce complete or partial tumor regression in 33% of patients. The response of patients was not significantly different from that observed in patients treated with tamoxifen (30 mg o.d) of 37%. No significant difference between treatments was observed for disease stabilization, the duration of response or median survival. Several other steroidal aromatase inhibitors have been studied, such as 7-substituted androstenedione derivatives. MDL 18962 [10-(2-propynyl)estr-4-ene-3,17-dione] and FCE 24304 (6-methylen-androsta-1,4-diene-3,17-dione) are currently in clinical trials. Non-steroidal inhibitors of cytochrome P-450 enzymes, such as imidazole and triazole derivatives have been developed which are highly selective for aromatase. Three triazoles which are very potent and selective inhibitors are vorazole (6-[(4-chlorophenyl)(1H-1,2,4-triazol-1-yl)-methyl]1-methyl-1H-benzotriazole R 76713, arimidex 2,2′[5-( -1,2,4-triazol-1-yl methyl)-1,3-phenylene]bis(2-methylpropiononitrile) (ZD1033) and letrozole 4-[1-(cyanophenyl)-1-(1,2,4-triazolyl)methyl]benzonitril (CGS 20267). These compounds reduce serum estradiol concentration to undetectable levels in breast cancer patients. These highly potent inhibitors provide the opportunity to determine whether a further degree of estrogen suppression will be important in producing greater clinical response. With the recent approval of 4-OHA in several countries and the introduction of the potent new compounds, aromatase inhibitors either alone or in combination with the antiestrogen are likely to improve the treatment of breast cancer.     

Structure-function studies of aromatase and its inhibitors: a progress report

The utilization of computer modeling, site-directed mutagenesis, inhibition kinetic analysis and reaction metabolite analysis allows us to better understand the structure–function relationship between aromatase and its inhibitors. Our results have helped in determining how steroidal and nonsteriodal aromatase inhibitors bind to the active site of the enzyme. This information has also aided in the understanding of the reaction mechanism of aromatase. Furthermore, our structure–function studies of aromatase have generated important information for predicting how environmental chemicals interact with the enzyme. During the last 2 years, a new aromatase computer model based on the X-ray structure of rabbit cytochrome P450 2C5 has been generated and used to evaluate the results obtained from new aromatase mutants produced in this laboratory. In addition, we have succeeded in the expression and purification of functionally active aromatase using an Escherichia coli expression method. The catalytic properties of this recombinant aromatase are similar to those properties exhibited by the human placental aromatase preparation and the mammalian cell-expressed enzyme. The E. coli expressed aromatase will be very useful for further structure–function studies of aromatase. Our laboratory has also evaluated the growth-inhibiting activity of aromatase inhibitors in estrogen receptor-positive breast cancer using three-dimensional cell cultures of aromatase-over expressing MCF-7 and T-47D cell lines (i.e. MCF-7aro and T-47Daro). Our results demonstrate that these three-dimensional cultures are valuable approaches to assess the growth-inhibiting activity of aromatase inhibitors. Finally, we have identified several phytochemicals to be potent inhibitors of aromatase. To demonstrate the impact of the phytochemicals on estrogen formation in vivo, we showed that the intake of anti-aromatase chemicals from red wine was capable of suppressing MCF-7aro-mediated tumor formation in nude mice and aromatase-induced hyperplasia in a transgenic mouse model in which aromatase is over-expressed in the mammary tissue.