Aromatase Inhibitors in the Treatment of Breast Cancer

http://www.100md.com 内分泌进展 2005年第3期

     Medicinal Chemistry and Pharmacognosy, College of Pharmacy, and Hormones and Cancer Program, Ohio State University Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210

    Abstract

    Estradiol, the most potent endogenous estrogen, is biosynthesized from androgens by the cytochrome P450 enzyme complex called aromatase. Aromatase is present in breast tissue, and intratumoral aromatase is the source of local estrogen production in breast cancer tissues. Inhibition of aromatase is an important approach for reducing growth-stimulatory effects of estrogens in estrogen-dependent breast cancer. Steroidal inhibitors that have been developed to date build upon the basic androstenedione nucleus and incorporate chemical substituents at varying positions on the steroid. Nonsteroidal aromatase inhibitors can be divided into three classes: aminoglutethimide-like molecules, imidazole/triazole derivatives, and flavonoid analogs. Mechanism-based aromatase inhibitors are steroidal inhibitors that mimic the substrate, are converted by the enzyme to a reactive intermediate, and result in the inactivation of aromatase. Both steroidal and nonsteroidal aromatase inhibitors have shown clinical efficacy in the treatment of breast cancer. The potent and selective third-generation aromatase inhibitors, anastrozole, letrozole, and exemestane, were introduced into the market as endocrine therapy in postmenopausal patients failing antiestrogen therapy alone or multiple hormonal therapies. These agents are currently approved as first-line therapy for the treatment of postmenopausal women with metastatic estrogen-dependent breast cancer. Several clinical studies of aromatase inhibitors are currently focusing on the use of these agents in the adjuvant setting for the treatment of early breast cancer. Use of an aromatase inhibitor as initial therapy or after treatment with tamoxifen is now recommended as adjuvant hormonal therapy for a postmenopausal woman with hormone-dependent breast cancer.

    I. Introduction

    II. Aromatase and Estrogen Biosynthesis

    A. Biochemistry of aromatase

    B. Aromatase gene expression

    C. Aromatase in breast cancer tissues

    III. Development of Aromatase Inhibitors

    A. Steroidal inhibitors

    B. Nonsteroidal inhibitors

    IV. Aromatase Inhibitors in Breast Cancer

    A. First- and second-generation aromatase inhibitors

    B. Third-generation aromatase inhibitors

    C. Clinical studies

    V. Conclusions

    I. Introduction

    ESTROGENS ARE INVOLVED in numerous physiological processes including the development and maintenance of the female sexual organs, the reproductive cycle, reproduction, and various neuroendocrine functions. These hormones also have crucial roles in certain disease states, particularly in mammary and endometrial carcinomas. Cancer is the leading cause of death among women between the ages of 30 and 54, with breast and uterine cancers comprising 28% and 10%, respectively, of all cancers in females per year. An estimated 217,440 new cases of breast cancer will be diagnosed, and 40,580 women in the United States were projected to die from breast cancer in 2004 (1). Currently, one of eight American women will develop breast cancer in her lifetime. Approximately two thirds of postmenopausal breast cancer patients have hormone-dependent (estrogen-dependent) breast cancer, which contains estrogen receptors and requires estrogen for tumor growth. Estrogens produce normal physiological effects by binding to specific nuclear receptor proteins, estrogen receptor- and estrogen receptor-? (2). The predominant estrogen receptor in the female reproductive tract and mammary glands is estrogen receptor-. Following the binding of estrogen to its receptor, the estrogen-receptor complexes form homodimers and interact with sequence-specific estrogen response elements present in the promoter region of responsive genes in target cell chromatin. Binding of the nuclear steroid-receptor complexes to DNA and interaction with various nuclear transcriptional factors, such as steroid receptor coactivator proteins, initiate the transcription of the relevant gene to produce mRNA. The elevated mRNA levels result in increased protein synthesis in the endoplasmic reticulum. These proteins include enzymes, receptors, and secreted factors that subsequently result in the steroid hormonal response regulating cell function, growth, and differentiation. Estrogens enhance growth and proliferation of certain target cells, such as breast epithelial cells and estrogen-dependent mammary carcinoma cells, and induce the formation and secretion of various growth factors in established cell lines such as MCF-7, T47D, and ZR-75–1 human mammary carcinoma lines (3).

    II. Aromatase and Estrogen Biosynthesis

    A. Biochemistry of aromatase

    Estradiol is the most potent endogenous estrogen. Estradiol is biosynthesized from androgens by the cytochrome P450 enzyme complex called "aromatase" (4), with the highest levels of enzyme present in the ovaries of premenopausal women, in the placenta of pregnant women, and in the peripheral adipose tissues of postmenopausal women and of men. Aromatase activity has also been demonstrated in breast tissue in vitro (5, 6, 7). Furthermore, expression of aromatase is highest in or near breast tumor sites (6, 8).

    The enzyme complex is bound in the endoplasmic reticulum of the cell and is comprised of two major proteins (4, 9). One protein is cytochrome P450arom, a hemoprotein that converts C19 steroids (androgens) into C18 steroids (estrogens) containing a phenolic A ring (4, 10). The second protein is NADPH-cytochrome P450 reductase, which transfers reducing equivalents to cytochrome P450arom. Three moles of NADPH and three moles of oxygen are used in the conversion of one mole of substrate into one mole of estrogen product (Fig. 1). Aromatization of androstenedione, the preferred substrate, proceeds via three successive oxidation steps, with the first two being hydroxylations of the angular C-19 methyl group. The final oxidation step proceeds with the aromatization of the A ring of the steroid and loss of the C-19 carbon atom as formic acid.

    This third and final step in the aromatase reaction oxidatively cleaves the C10-C19 bond, although the mechanism of this step remains to be elucidated. A number of mechanisms have been proposed, and one mechanism for the oxidative deformylation step that has received significant favor involves nucleophilic attack of the 19-aldehyde by the reduced ferrous dioxygen, or peroxy, intermediate (Fig. 2A). The resulting peroxo hemiacetal is suggested to decay via a process by which the proximal oxygen atom removes the 1?-hydrogen, resulting in aromatization of the steroid A ring and formic acid release (11). Model reaction studies have suggested that formation of the 2,3-enol is a prerequisite for aromatization. A homology model of the aromatase enzyme suggests the presence of an aspartic acid residue near the 2?-hydrogen and lysine or histidine residues near the 3-ketone of androstenedione (12). The orientation of these residues is supportive of an enzyme acid-base-catalyzed enolization process to selectively remove the 2?-hydrogen. Recently, our laboratory has employed computational chemistry approaches to unravel the mysterious third step of aromatase catalysis (13). Recent advances in computational chemistry, including density functional theory alone or in combination with molecular mechanical methods, have provided better tools that enable study of the active species in their native protein environment, such as the cytochrome P450 oxidant Compound I (14). A model system of the cytochrome P450 active site and truncated steroid substrates has been studied using density functional theory calculations and ab initio molecular dynamics. Analysis for the reduced ferrous dioxygen (peroxy) intermediate suggests that 1?-hydrogen atom abstraction by the proximal oxygen of the peroxo hemiacetal intermediates encounters a high energetic barrier (>60 kcal/mol) that is enzymatically inaccessible. Also, the resulting species do not directly fragment to the experimentally observed formic acid and aromatized steroid products. A novel, alternative mechanism was examined, in which the widely accepted cytochrome P450 oxidant Compound I, the iron oxene catalytic intermediate (Fig. 2B), abstracts the 1?-hydrogen atom initiating the aromatization and deformylation cascade. The steroid models that contain the 2,3-enol moiety have a strikingly low barrier for 1?-hydrogen atom abstraction (<7 kcal/mol) due to the ability of the enolized A ring to delocalize the impending radical. The transition states containing the 2,3-enol moiety and the 19-gem diol decay directly to the aromatized product, formic acid, and the aqua-bound model cytochrome P450 enzyme. Analysis of the reaction vectors indicate that the second hydrogen transfer occurs with a concerted, nonsynchronous mechanism without an energetic barrier. Thus, these calculations support a final catalytic step of aromatase involving the cytochrome P450 oxene intermediate, 1?-hydrogen atom abstraction, and release of formic acid (Fig. 2).

    B. Aromatase gene expression

    Over the past two decades, knowledge of the biochemistry, molecular biology, and regulation of aromatase has increased greatly. The aromatase gene, designated CYP19, encodes the cytochrome P450arom, and this gene is located on chromosome 15q21.1. The coding region is approximately 30 kb in size, and the regulatory region is approximately 93 kb (9, 15). The aromatase gene consists of 10 exons, and its full-length cDNA of 3.4 kb encodes for a protein of 503 amino acids. The aromatase protein is a glycosylated cytochrome P450 protein with a molecular mass of approximately 58,000 Da (16). The regulation of aromatase is complex in various tissues, and several tissue-specific promoter regions have been identified upstream from the CYP19 gene (9, 17, 18). These tissue-specific promoters include promoter PI.1, PI.3, PI.4, PI.6, PI.7, and PII (Fig. 3). Promoter PI.1 is the major promoter used in placental tissues and is the farthest upstream. The PII promoter is used in the ovary and in breast cancer tissues, and it contains a cAMP response element. Promoters PI.3, PI.4, PI.6, and PI.7 are the promoters used in extraglandular sites. Promoter PI.4 is the primary promoter used in normal adipose tissue and is responsive to glucocorticoids and cytokines such as IL-1?, IL-6, and TNF. Promoter PI.3 is also present in adipose tissues such as normal breast tissue and is elevated in breast cancer tissues.

    C. Aromatase in breast cancer tissues

    Aromatase is found in breast tissue, and the importance of intratumoral aromatase and local estrogen production is being unraveled (6, 19, 20). Aromatase has been measured in the stromal cell component of normal breast and breast tumors, but the enzyme has also been detected in the breast epithelial cells in vitro (5, 8, 20, 21, 22). Furthermore, expression of aromatase is highest in or near breast tumor sites (8, 20). The exact cellular location(s) of aromatase must await more rigorous analysis by several laboratories with a new monoclonal antibody now being developed and evaluated (23).

    The increased expression of aromatase cytochrome P450arom observed in breast cancer tissues is associated with a switch in the major promoter region used in gene expression, with promoter PII being the predominant promoter used in breast cancer tissues (24). As a result of the use of the alternate promoter, the regulation of estrogen biosynthesis switches from one controlled primarily by glucocorticoids and cytokines to a promoter regulated through cAMP-mediated pathways (24). Prostaglandin E2 (PGE2) increases intracellular cAMP levels and stimulates estrogen biosynthesis (24), whereas other autocrine factors such as IL-1? do not appear to act via PGE2 (25).

    Local production of PGE2 via the cyclooxygenase isozymes (constitutive COX-1 isozyme and inducible COX-2 isozyme) can influence estrogen biosynthesis and estrogen-dependent breast cancer. This biochemical mechanism may explain epidemiological observations of the beneficial effects of nonsteroidal antiinflammatory drugs (NSAIDs) on breast cancer (26, 27, 28, 29). Investigations using human breast cancer patient specimens demonstrated a strong linear correlation between CYP19 expression and the sum of COX-1 and COX-2 expression (30). Gene expression analysis for CYP19, COX-1, and COX-2 were performed in 20 human breast cancer specimens and in five normal control breast tissue samples. A positive correlation was observed between CYP19 expression and the greater extent of breast cancer cellularity (Fig. 4A), in agreement with literature reports showing that aromatase levels were higher in tumors than in normal tissue. Furthermore, a positive linear correlation was observed between COX-2 expression breast cancer cellularity in each sample. Linear regression analysis using a bivariate model shows a strong linear association between CYP19 expression and the sum of COX-1 and COX-2 expression (Fig. 4B). Similar correlations between CYP19 expression and COX-2 expression in breast cancer patient specimens have been confirmed in other laboratories (31). This significant relationship between the aromatase and COX enzyme systems suggests that autocrine and paracrine mechanisms may be involved in hormone-dependent breast cancer development via growth stimulation from local estrogen biosynthesis. In human breast stromal cells, PGE2 acts via two G protein-coupled receptors, EP1 and EP2 receptors, to stimulate aromatase gene expression via protein kinase A and protein kinase C signaling pathways (32). NSAIDs and COX-1- and COX-2-selective inhibitors produce dose-dependent decreases in aromatase activity in breast cancer tissues (Fig. 5) (33, 34). Real time PCR analysis of aromatase gene expression showed a significant decrease in mRNA levels by these agents, and the effect of COX inhibitors on aromatase expression occurs through suppression at the tissue-specific promoters PI.3, PI.4, and PII. This significant relationship between the aromatase and COX enzyme systems suggests that autocrine and paracrine mechanisms may be involved in hormone-dependent breast cancer development via growth stimulation from local estrogen biosynthesis (Fig. 6).

    III. Development of Aromatase Inhibitors

    Two primary approaches have been developed to reduce the growth-stimulatory effects of estrogens in breast cancer: 1) interfering with the ability of estrogen to bind to its receptor; and 2) decreasing circulating levels of estrogen. Antiestrogens compete for binding to the estrogen receptors and reduce the number of receptors available for binding to endogenous estrogen. This approach has proven effective as an anticancer strategy (35, 36) and has led to the development of efficacious antiestrogens, such as the drug tamoxifen, that exhibit minimal toxicity. Inhibition of aromatase is the second approach for reducing growth-stimulatory effects of estrogens. Effective aromatase inhibitors have been developed as therapeutic agents for controlling estrogen-dependent breast cancer. Investigations on the development of aromatase inhibitors began in the 1970s and have expanded greatly in the past three decades. Research summaries of aromatase inhibitors have been presented at international aromatase conferences (37, 38, 39, 40, 41), and several reviews have also been published (42, 43, 44, 45, 46, 47, 48, 49, 50, 51).

    A. Steroidal inhibitors

    1. Competitive enzyme inhibitors.

    Investigations on the development of aromatase inhibitors began with the synthesis and biochemical evaluation of competitive inhibitors (52, 53, 54). Competitive inhibitors are molecules that compete with the substrate androstenedione for noncovalent binding to the active site of the enzyme to decrease the amount of product formed. The term "apparent Ki" represents the equilibrium constant for the reversible binding of the enzyme and the inhibitor. The values are used in comparisons of inhibitors, and the smaller the Ki value, the better the inhibitor. Steroidal inhibitors that have been developed to date build upon this basic androstenedione nucleus and incorporate chemical substituents at varying positions on the steroid (Fig. 7). These inhibitors bind to the aromatase cytochrome P450 enzyme in the same manner as the substrate androstenedione. Initial structure-activity relationships were developed with these early investigations on competitive aromatase inhibitors. In summary, effective inhibition is observed with an androstane steroid molecule possessing an A/B trans ring junction, a ketone functionality at the C-3 position, unsaturation in the steroid nucleus (4-ene, 4,6-diene, or 1,4,6-diene functions), and either a 17-ketone or 17-hydroxyl moiety.

    A limited number of effective inhibitors with substituents on the A ring have been reported. Several steroidal aromatase inhibitors contain modifications at the C-4 position, with 4-hydroxyandrostenedione being the prototype agent. Initially, 4-hydroxyandrostenedione was thought to be a competitive inhibitor with an apparent Ki of approximately 170 nM, but the compound was later shown to produce enzyme-mediated inactivation. The spacial requirements of the A ring for binding of the steroidal inhibitor to aromatase are rather restrictive, permitting only small structural modifications to be made. Incorporation of the polar hydroxyl group at C-4 enhances inhibitory activity. 1-Methylandrosta-1,4-diene-3,17-dione is a potent inhibitor of aromatase in vitro and in vivo (55); on the other hand, bulky substitutents at the 1-position are poor inhibitors (54).

    Extensive structural modifications may be made on the B ring of the steroid nucleus. Bulky substitutions at the C-7 position of the B ring have provided several very potent aromatase inhibitors (54). One of the more potent inhibitors, 7-(4'-amino)phenylthio-4-androstene-3,17-dione with an apparent Ki of 18 nM, demonstrated effectiveness in inhibiting aromatase in cell cultures and in treating hormone-dependent rat mammary tumors (56, 57, 58). Evaluation of various substituted aromatic analogs of 7-(4'-amino)phenylthio-4-androstene-3,17-dione provided no correlation between the electronic character of the substituents and inhibitory activity. Investigations of various 7-substituted androsta-4,6-diene-3,17-dione derivatives suggest that only those derivatives that can project the 7-aryl substitutent into the 7-pocket are effective inhibitors (59). Several compounds with substituents at the C-6 position have been described which exhibit irreversible inhibition; these are discussed in a later section. Overall, the most effective B ring-modified aromatase inhibitors are those with 7-aryl derivatives, with several analogs having 2–10 times greater affinity for the enzyme than the substrate. These results suggest that additional interactions occur between the phenyl ring at the 7-position and amino acids at or near the enzymatic site of aromatase, resulting in enhanced affinity.

    The other position of androstenedione that has received considerable investigation and has resulted in effective inhibitors is the C-19 methyl position, the site of enzymatic oxidation. Competitive inhibitors have been designed to enhance affinity through noncovalent binding of a heteroatom at C-19 with the heme iron of the cytochrome P450arom. 19-Substituted aromatase inhibitors include thiiranes and oxiranes (60, 61), epoxysteroids (62), and thiol and amino analogs (63, 64). The 19R-isomers of the thiiranes and oxiranes were potent inhibitors with apparent Ki values ranging from 1–7 nM, showed affinity 36- to 80-fold greater than the corresponding 19S-isomers, and demonstrated binding to the heme iron in spectroscopic studies. Unique 2,19-bridged androstenediones have also been reported (65, 66, 67). These A ring-bridged steroids consist of both five-membered and six-membered ring analogs containing carbon, oxygen, nitrogen, or sulfur atoms. Several of these compounds exhibited apparent Ki values in the low nanomolar range and demonstrated tight-binding competitive inhibition. Thus, effective inhibitors have been prepared with geometrically small functionalities at the C-19 position, suggesting that the enzyme active site can accommodate small changes in structure.

    2. Mechanism-based enzyme inhibitors.

    A mechanism-based inhibitor is an inhibitor that mimics the substrate, is converted by the enzyme to a reactive intermediate, and results in the inactivation of the enzyme. The term "mechanism-based" is used because the inhibitor contains a chemical functionality that is acted upon by the enzyme during the normal catalytic process. A mechanism-based inhibitor produces time-dependent inactivation of the enzyme only in the presence of catalytically active enzyme; omission of a cofactor, such as NADPH, does not produce inactivation. Other terms that are used for these inhibitors are "enzyme-activated irreversible inhibitors," "suicide substrates," and "suicide inactivators." Several mechanism-based aromatase inhibitors, structurally related to the natural substrate androstenedione (Fig. 8), are initially recognized by the aromatase enzyme as alternate substrates and are then transformed (through an NADPH-dependent mechanism) to reactive intermediates, which bind irreversibly to the enzyme and produce inactivation. Inactivation kinetic values are determined from plots of the half-time of inactivation, i.e., the time required to decrease the enzymatic activity by 50%, vs. the reciprocal of the inhibitor concentration. The y-intercept of the resulting line is the half-time of inactivation at infinite inhibitor concentration (t1/2) and the rate of inactivation, apparent kinact, is equal to 0.693/t1/2. The most effective mechanism-based inhibitors exhibit short half-times of inactivation (t1/2) and rapid rates of inactivation. Mechanism-based inhibitors have distinct advantages in drug design, because these inhibitors are highly enzyme specific, produce prolonged inhibition, and often exhibit minimal toxicities.

    The first compound designed as a mechanism-based inhibitor of aromatase was 10-propargyl-4-estrene-3,17-dione (MDL 18,962); it was synthesized and studied independently by three research groups (68, 69, 70). MDL 18,962 has an electron-rich alkynyl function on the C-19 carbon atom, the site of aromatase-mediated oxidation of the substrate, and is an effective inhibitor in vitro and in vivo (71, 72, 73, 74). In biochemical assays, MDL 18,962 exhibited a half-time of inactivation at infinite inhibitor concentration (t1/2) of 10.41 min and an apparent kinact of 1.11 x 10–3 sec–1. Although the identity of the reactive intermediate formed is not known, an oxirene and a Michael acceptor have been suggested.

    A larger number of mechanism-based inhibitors have developed from more detailed biochemical investigations of several inhibitors originally thought to be competitive inhibitors. These inhibitors can be grouped into the general categories of 4-substituted androst-4-ene-3,17-diones, substituted androsta-1,4-diene-3,17-diones, and 6-methylene- or 6-oxo-androst-4-ene-3,17-diones.

    4-Hydroxy-4-androstene-3,17-dione (4-hydroxyandrostenedione, formestane), originally thought to be a competitive inhibitor, produces enzyme-mediated inactivation (75). In enzyme assays, 4-hydroxyandrostenedione exhibited a half-time of inactivation at infinite inhibitor concentration (t1/2) of 2.57 min and an apparent kinact of 4.50 x 10–3 sec–1. In vivo, formestane inhibits reproductive processes (76) and causes regression of hormone-dependent mammary rat tumors (77, 78). Formestane is effective and well tolerated in the treatment of advanced breast cancer in postmenopausal women (79, 80); however, extensive first-pass metabolism of this agent in the liver necessitates im administration and limits its use (81, 82, 83).

    Numerous androsta-1,4-diene-3,17-diones have been prepared and have demonstrated mechanism-based or enzyme-mediated inactivation of aromatase in vitro. Androsta-1,4-diene-3,17-dione and androsta-1,4,6-triene-3,17-dione were initially thought to be competitive inhibitors, but further examination of the biochemistry of these inhibitors revealed that these compounds demonstrated inactivation of aromatase only in the presence of cofactors (84, 85). Introduction of substituents at the 7-position of both androsta-1,4-diene-3,17-dione and androsta-1,4,6-triene-3,17-dione have resulted in very effective mechanism-based inhibitors of aromatase (57, 86, 87, 88, 89). 7-(4'-Amino)phenylthioandrosta-1,4-diene-3,17-dione has high affinity for aromatase, with an apparent Ki of 9.9 nM, and has the most rapid rate of inactivation reported to date with a half-time of inactivation (t1/2) of 1.38 min and an apparent kinact of 8.40 x 10–3 sec–1. More metabolically stable inhibitors were synthesized by replacing the thioether linkage at the 7-position with a carbon-carbon linkage. The best inactivator of the series was the 7-phenpropylandrosta-1,4-diene-3,17-dione, which exhibited a t1/2 of 6.08 min and an apparent kinact of 1.90 x 10–3 sec–1.

    An exocyclic double bond at the C-6 carbon atom results in 6-methyleneandrost-1,4-diene-3,17-dione (exemestane), which produces aromatase inactivation in vitro and causes regression of hormone-dependent mammary rat tumors (90, 91, 92). Exemestane is a potent inhibitor of both human placental aromatase with apparent Ki of 26 nM, a t1/2 of 13.9 min, and an apparent kinact of 8.30 x 10–4 sec–1. Exemestane, when administered sc or orally, inhibits rat ovarian aromatase (ED50 of 1.8 and 3.7 mg/kg, respectively) (90, 93).

    Despite the large number of effective mechanism-based or enzyme-activated inhibitors that have been prepared, no detailed mechanism(s) of action have been identified. All agents produce time-dependent inactivation of aromatase activity only when incubated with catalytically active enzyme, no inactivation is observed in the absence of cofactors such as NADPH, and coincubations with the substrate androstenedione decrease the rates of inactivation. Investigations of radiolabeled mechanism-based inhibitors, [125I]-7-(4'-iodo)phenylthioandrosta-1,4-diene-3,17-dione and [14C]-MDL 18,962, provided the first evidence of irreversible binding of these inhibitors to the aromatase protein (94). Radiolabeled inhibitors were incubated with purified reconstituted aromatase preparations and NADPH, and the cytochrome P450arom protein was isolated by gel chromatography after the incubation. Subsequent treatments of the cytochrome P450arom protein fractions by precipitation, extensive washings, and SDS-PAGE demonstrated that the radioactive inhibitors remain bound to the cytochrome P450arom protein. Thus, the mechanism-based inactivation that occurs is due to irreversible, covalent binding of the inhibitors to the enzyme protein. The exact nature of the covalent bound(s), the chemical structures of the bound inhibitors, and the amino acids involved are yet to be elucidated. Additionally, the question of whether the inhibitors are oxidized at the C-19 position in a manner similar to the substrate androstenedione before irreversible binding and inactivation remains to be answered.

    B. Nonsteroidal inhibitors

    1. First- and second-generation inhibitors.

    Nonsteroidal aromatase inhibitors possess a heteroatom as a common chemical feature and interfere with steroid hydroxylations by the binding of this heteroatom with the heme iron of the cytochrome P450s (Fig. 9). Initial nonsteroidal inhibitors were less enzyme specific and inhibited, to varying degrees, other cytochrome P450-mediated hydroxylations of steroidogenesis. Aminoglutethimide was the prototype for nonsteroidal aromatase inhibitors (95). Aminoglutethimide was originally an antiepileptic agent that was removed from the market due to serious side effects. Aminoglutethimide inhibited cytochrome P450SCC and other enzyme pathways but was more selective for cytochrome P450arom. The racemic mixture (dl-aminoglutethimide) inhibits aromatase with an apparent Ki of 700 nM. The d-aminoglutethimide stereoisomer is 20-fold more potent than the l-aminoglutethimide stereoisomer.

    Because aminoglutethimide was the first aromatase inhibitor to be studied in patients, it is referred to as the first-generation aromatase inhibitor. Aminoglutethimide has been used in the clinics with some success to treat patients with advanced breast cancer, but it must be administered with corticosteroid due to the inhibitory effects on cortisol and aldosterone biosynthesis (95, 96). Although effective at decreasing aromatization, it also inhibited a number of other steroidogenic CYP-450 enzymes, which resulted in significant toxicity. The imidazole compound fadrazole is a potent, competitive inhibitor of aromatase both in vitro and in vivo (97). Fadrazole, referred to as a "second-generation inhibitor," is more selective than aminoglutethimide, and its inhibitory activity is 700 times more potent. Clinical studies using fadrazole proved this nonsteroidal inhibitor is effective in the treatment of some postmenopausal women with advanced breast cancer. Both partial and complete responses were observed upon treatment with fadrazole. However, this compound still has some nonselective inhibitory activity with respect to aldosterone, progesterone, and corticosterone biosynthesis. The apparent Ki for the racemate of fadrazole in human placental microsomes is 1.6 nM, and the (–)-S isomer is equipotent to the racemate whereas the (+)-R isomer has a lower inhibitory activity with an apparent Ki of 39 nM (98).

    2. Third-generation triazole inhibitors.

    Several nonsteroidal aromatase inhibitors containing a triazole ring have been successfully developed. Vorozole potently inhibits aromatase in numerous in vitro systems, with an apparent Ki of 1.3 nM in human placental microsomes (99). The racemate has been tested thoroughly, but it is known that the (+)-S isomer is 36 times more potent than the (–)-R isomer (100). Regression of tumors in a hormone-dependent rat tumor model has been demonstrated with various doses of vorozole. Another triazole analog is anastrozole, which is an achiral triazole derivative (101). Anastrozole is a potent aromatase inhibitor with an IC50 of 15 nM in human placental microsomes. In vitro, anastrozole has no effect on numerous other P450 enzymes such as, P450SCC, 11?-hydroxylase, 18-hydroxylase, 17-hydroxylase, and lanosterol-14-demethylase. In vivo studies in monkeys showed that anastrozole, at a dose of less than 0.2 mg/kg·d, reduced peripheral aromatase activity by 50–60% (101). The third triazole derivative, letrozole, is a potent inhibitor of aromatase with an IC50 of 11.5 nM in human placental microsomes (102). In in vitro studies, letrozole has no effect on the biosynthesis of other steroids such as aldosterone, progesterone, or corticosterone. In vivo, letrozole was determined to be orally active and to cause regression of tumors in the 7,12-dimethylbenz(a)anthracene hormone-dependent rat tumor model, and it demonstrated aromatase inhibition in patients (103). A higher degree of specificity has been reached with the new generation of triazole derivatives (letrozole and anastrozole). These newer agents are 100-3000 times more active than aminoglutethimide, and all inhibit whole-body aromatization by greater than 96%.

    3. Flavonoid derivatives as inhibitors.

    Flavonoids are plant natural products present in many food sources, including fruits, vegetables, legumes, and whole grains. The class of flavonoids encompasses flavones, isoflavones, flavanones, and flavonols, each possessing the benzopyranone ring system as the common chemical scaffold. Considerable interest in flavonoids in breast cancer has been stimulated by the hypothesis that these natural products, present in soy and in rye flour, are dietary factors that may be responsible for the lower incidence of breast cancer in women from certain regions of the world (104, 105). Several flavonoids demonstrate inhibitory activities of the aromatase enzyme, thus lowering estrogen biosynthesis and circulating estrogen levels (Fig. 10) (104, 106, 107, 108, 109, 110, 111). However, these natural products demonstrate numerous biological activities and interact with various enzymes and receptor systems of pharmacological significance, thus limiting their therapeutic usefulness.

    Strong evidence for the binding of flavones to the active site of aromatase was obtained by difference spectral absorption studies (106), with 7,8-benzoflavone displacing androstenedione from the aromatase active site and inducing a spectrum consistent with the low-spin state of iron. Reduction of the flavone 4-keto group was detrimental to aromatase inhibition by these compounds (107). Based on data obtained from site-directed mutagenesis studies and ligand docking into a homology model of the aromatase protein, a binding orientation was predicted in which the A and C rings of the flavone mimic the C and D rings of the steroid substrate, respectively. The 2-phenyl substituent is oriented in a region similar to that occupied by the A ring of the steroid. This analysis places the flavone 4-keto functionality in the same position as the steroid 19-angular methyl group with respect to the heme iron (110).

    Medicinal chemistry approaches to develop synthetic flavonoids, chromone, or xanthone analogs with enhanced aromatase inhibitory activity have identified more selective and/or more potent agents for future development (112, 113, 114). Generally, flavones and flavanones have higher aromatase inhibitory activity than isoflavones (Fig. 10). The flavone, chrysin, has an IC50 value of 0.50 μM; apigenin, flavone, flavanone, and quercetin were less efficacious inhibitors with IC50 values of 1.2, 8, 8, and 12 μM, respectively. Isoflavones are significantly less potent as aromatase inhibitors. The most effective isoflavone inhibitor is biochanin A with an IC50 value of 113 μM, approximately 20-fold less potent than chrysin in terms of IC50 values (110, 115). This large difference in potency is the likely reason why there has been little effort to develop aromatase inhibitors on an isoflavone scaffold. On the other hand, we envisioned introduction of the proper functional groups on the isoflavone core could result in the desired aromatase activity. As a proof of principle, a 2-(4-pyridylmethyl)thio functionality was introduced onto the isoflavone nucleus (Fig. 10), and this isoflavone modification afforded a 160-fold enhancement in potency compared with the natural product, biochanin A (116). In human placental microsomal assays, the synthetic pyridyl isoflavone analog, 3-phenyl-7-(phenylmethoxy)-2-[(4'-pyridylmethyl)thio]-4H-1-benzopyran-4-one, exhibited an IC50 value of approximately 210 nM and an apparent Ki value of 220 nM.

    IV. Aromatase Inhibitors in Breast Cancer

    A. First- and second-generation aromatase inhibitors

    1. Aminoglutethimide.

    Aminoglutethimide was the first of these inhibitors evaluated in clinical studies for treatment of hormone-dependent breast cancer (117). Santen et al. (118) extensively evaluated the kinetic and hormonal effects of aminoglutethimide in breast cancer and first proposed aromatase inhibition as the primary mechanism of action of aminoglutethimide therapy. Numerous clinical trials performed since the early 1980s have demonstrated the clinical efficacy of combination therapy of aminoglutethimide with corticosteroid replacement (96, 119) in treatment of hormone-dependent breast cancer. However, side effects of lethargy, ataxia, and morbilliform skin rash and the development of more potent aromatase inhibitors resulted in cessation of further development of aminoglutethimide for breast cancer.

    2. 4-Hydroxyandrostenedione.

    The steroidal inhibitor 4-hydroxyandrostenedione, generic name of formestane, was extensively evaluated in clinical trials and was the first aromatase inhibitor approved for general use in Europe. In a series of clinical studies (120), 4-hydroxyandrostenedione treatment in unselected postmenopausal breast cancer patients with either weekly or biweekly im injections resulted in 26% of patients experiencing complete or partial responses and another 25% exhibiting disease stabilization. Decreased serum estrogen levels have been observed in postmenopausal breast cancer patients. The drug is extremely well tolerated, and only a low percentage (13%) of patients experienced pain and/or inflammation at the injection site. Because 4-hydroxyandrostenedione (formestane) was the second aromatase inhibitor to be studied in patients, it is referred to as a second-generation aromatase inhibitor.

    B. Third-generation aromatase inhibitors

    The third-generation aromatase inhibitors include the nonsteroidal inhibitors anastrozole and letrozole and the steroidal inhibitor exemestane. These third-generation aromatase inhibitors have received extensive clinical evaluations (121, 122, 123, 124, 125, 126).

    1. Anastrozole.

    Anastrozole (Arimidex; Astra-Zeneca, London, UK) is a potent nonsteroidal aromatase inhibitor in patients, decreasing plasma estradiol levels in a dose-dependent manner and producing approximately 97% inhibition of estrogen biosynthesis at the dose of 1 mg/d (127). Anastrozole was well tolerated in two large, Phase III trials of anastrozole vs. megestrol acetate in patients who progressed on tamoxifen, and the combined analysis demonstrated a clinically significant advantage over megestrol acetate (128, 129, 130). Two randomized, double-blind studies demonstrated that anastrozole (1 mg daily) was more effective than tamoxifen (20 mg daily) as first-line therapy in postmenopausal women with advanced breast cancer (131, 132, 133). Anastrozole at the recommended therapeutic dose of 1 mg once daily effectively suppressed total-body aromatization, with a mean percentage inhibition of 97.3%, and suppressed plasma estrone and estradiol levels by 81–85% in postmenopausal women with metastatic breast cancer (134). In clinical trials with postmenopausal women, no changes were detected in levels of androgens, an increase in the gonadotropins LH and FSH was observed over time, and a decrease in SHBG was also detected (135).

    2. Letrozole.

    Letrozole (Femara; Novartis, Basel, Switzerland) is a potent nonsteroidal aromatase inhibitor that produces approximately 99% inhibition of estrogen biosynthesis at the dose of 2.5 mg/d in patients (136). Clinical studies, involving postmenopausal women with advanced breast cancer who have had numerous previous endocrine treatments, showed that letrozole produced either a partial response or stabilization of disease in about 40% of the women (137, 138). Letrozole is also well tolerated, causes a marked decrease in serum and urine estrogen levels, and has little effect on other endocrine factors. In clinical trials with postmenopausal women, plasma levels of androgens were unchanged with letrozole treatment, and increases in LH, FSH, and SHBG were detected over time (139). A multicenter, randomized, double-blind study in advanced breast cancer reported letrozole more effective than tamoxifen in response rate, clinical benefit, time to progression, and time to treatment failure (140). Letrozole at the recommended therapeutic dose of 2.5 mg once daily effectively suppressed total-body aromatization, with a mean percentage inhibition of greater than 99.1%, and suppressed plasma estrone and estradiol levels by 84–88% in postmenopausal women with metastatic breast cancer (141).

    3. Exemestane.

    Exemestane (Aromasin; Pfizer, New York, NY) is a potent steroidal inhibitor of human placental aromatase, and a single oral dose of 25 mg exemestane was found to cause a long-lasting reduction in plasma and urinary estrogen levels. Maximal suppression of circulating estrogens occurred 2–3 d after dosing and persisted for 4–5 d (142). The lengthy duration of estrogen suppression is thought to be related to the irreversible nature of the drug-enzyme interaction rather than pharmacokinetic properties of the compound. Exemestane is also well tolerated, causes a marked decrease in serum and urine estrogen levels, and has no effect on other endocrine factors (142, 143, 144, 145, 146). Increased doses of exemestane can lead to suppression of SHBG (147, 148). Exemestane was shown to inhibit peripheral aromatase by 97–98% (144, 149).

    Table 1 compares the inhibitory activities in vitro and in vivo of aromatase inhibitors that have been evaluated clinically. As described earlier, anastrozole and letrozole are both nonsteroidal competitive inhibitors of aromatase, whereas exemestane is a mechanism-based inhibitor. Investigations of these therapeutic agents in cell lines expressing high levels of aromatase, such as JEG-3 and JAr choriocarcinoma cells, have examined the impact of these inhibition characteristics on the residual in vitro aromatase activity and protein levels. Several mechanism-based aromatase inhibitors produce prolonged suppression of aromatase catalytic activity for up to 48 h in cells following short drug exposure (72, 94, 150). On the other hand, the nonsteroidal inhibitor aminoglutethimide resulted in elevated levels of aromatase enzyme activity under similar conditions, possibly due to enzyme stabilization (150). In a study comparing letrozole (3 nM), exemestane (20 nM), anastrozole (40 nM), and aminoglutethimide (10 nM), aromatase activity returned to control levels immediately after letrozole exposure and increased by about 60, 30, and 20% after 4, 24, and 48 h, respectively. After preincubation with anastrozole or aminoglutethimide, cellular aromatase also increased. On the other hand, suppression of aromatase activity was maintained for approximately 24–48 h after the removal of exemestane (151). There was no increase in the aromatase protein level in either experiment. In a study with cultured fibroblasts from mammary adipose tissue preincubated with an antiaromatase agent for 18 h, removal of aminoglutethimide or anastrozole elicited up to 40% increases in aromatase activity, whereas preincubation with exemestane resulted in a marked suppression (152).

    C. Clinical studies

    The third-generation aromatase inhibitors are approved in the United States for the treatment of postmenopausal women with metastatic estrogen-dependent breast cancer. Both anastrozole and letrozole were more effective than tamoxifen as first-line therapy in postmenopausal women with advanced breast cancer (131, 132, 133, 153). Exemestane has also shown enhanced efficacy over tamoxifen as first-line therapy in postmenopausal women with advanced breast cancer (154). In these clinical studies, the aromatase inhibitors demonstrated improved clinical efficacy in primary endpoints of objective response rates (complete response, partial response, or disease stabilization), time to progression, and time to treatment failure. Patients positive for estrogen receptor (estrogen receptor positive) and/or progesterone receptor (progesterone receptor positive) had better response rates when treated with aromatase inhibitors than the patients treated with tamoxifen. Overall, the third-generation aromatase inhibitors are well tolerated in these clinical trials of postmenopausal women with hormone-dependent metastatic breast cancer.

    Current clinical studies of aromatase inhibitors are focusing on the use of the agents in the adjuvant setting for the treatment of early breast cancer (121, 125, 155, 156). These studies assess the effectiveness of aromatase inhibitors following tamoxifen, of aromatase inhibitors alone, and/or of the combination of aromatase inhibitors and tamoxifen in adjuvant therapy. Table 2 summarizes the key adjuvant trials for the third-generation aromatase inhibitors. Early results from three randomized, adjuvant Phase III clinical trials have recently been published. The largest of these trials, Anastrozole, Tamoxifen Alone, or in Combination, recruited 9366 postmenopausal women with early breast cancer, and the patients were randomized into one of the three treatment arms for 5 yr. After a median follow-up of 47 months, the anastrozole arm of the study resulted in statistically significant reduction in breast cancer events and improvement of disease-free survival (157, 158). No differences in disease-free survival were observed between the tamoxifen-alone arm and the combination arm. The MA-17 trial recruited 5187 postmenopausal women who had taken tamoxifen for 5 yr, and these patients were randomized into two treatment arms of letrozole or placebo for an additional 5 yr. After a median follow-up of 2.4 yr, the letrozole-treated patients had a significant reduction of breast cancer events (159). At this analysis, the data safety and monitoring committee for the MA-17 study recommended that the trial be halted early and the participants informed of the positive results. The Intergroup Exemestane Study enrolled 4742 postmenopausal women who had taken tamoxifen for 2 or 3 yr, and these patients were randomized into two treatment arms of tamoxifen for a total of 5 yr or exemestane to complete the 5-yr hormonal therapy. Exemestane therapy after 2–3 yr of tamoxifen therapy significantly reduced breast cancer recurrence and contralateral breast cancer as compared with the standard 5 yr of tamoxifen treatment (160). Based on the results of these multiple, large randomized trials, the American Society of Clinical Oncology technology assessment panel (161) recommends that the "optimal adjuvant hormonal therapy for a postmenopausal woman with receptor-positive breast cancer includes an aromatase inhibitor as initial therapy or after treatment with tamoxifen."

    These multiple, large randomized trials also enabled a more thorough analysis of the tolerability and adverse events of the aromatase inhibitors. In general, patients receiving aromatase inhibitors experienced less gynecological symptoms such as endometrial cancer, vaginal bleeding, and vaginal discharges. Fewer cerebrovascular events and venous thromboembolic events were also observed with patients receiving aromatase inhibitors. No information is yet available on the effects of aromatase inhibitors on serum lipid levels, cardiovascular disease, and coronary heart disease risk. On the other hand, musculoskeletal effects and bone toxicity are associated with aromatase inhibitors. The percentages of musculoskeletal effects, which include increased arthritis, arthralgia, and/or myalgia, were small but showed statistically significant increases with aromatase inhibitors compared with tamoxifen. All three aromatase inhibitors were associated with increased fractures when compared with tamoxifen or placebo. The Anastrozole, Tamoxifen Alone, or in Combination trial reported a 7.1% fracture incidence in the anastrozole arm vs. tamoxifen at 4.4% (157, 158). In the MA-17 study, fractures in the letrozole-treated patients were 3.6% compared with 2.9% in placebo, after 2 or 3 yr of prior tamoxifen treatment (159). Exemestane was associated with osteoporosis and/or increased fractures (7.41%) when compared with tamoxifen (5.7%) in the Intergroup Exemestane Study trial. Baseline bone mineral density evaluations and potential bisphosphonate therapy are recommended (161).

    Other ongoing clinical studies are designed to compare the various aromatase inhibitors and/or combination therapies in early-stage breast cancer or in the chemoprevention setting. For example, MA-27 is a Phase III adjuvant trial in postmenopausal women with primary breast cancer comparing exemestane with anastrozole, with or without celecoxib, a COX-2 inhibitor. The potential for aromatase inhibitors in the chemoprevention setting in women with increased risk for the development of breast cancer is also being considered. In preclinical models, aromatase inhibitors reduce tumor formation in the carcinogen-induced rat mammary tumor studies (162, 163, 164, 165). The International Breast Cancer Intervention Study II will compare anastrozole vs. placebo in a prevention study, and the accompanying Ductal Carcinoma in Situ Study will compare tamoxifen vs. anastrozole in women with locally excised ductal carcinoma in situ (166). A Canadian breast cancer prevention study, NCIC MAP3, is a three-arm study of placebo vs. exemestane vs. exemestane and celecoxib (166). Important endpoints in such trials may include not only reduction in tumor incidence but also may examine effects of aromatase inhibitors on bone mineral density and serum lipid levels.

    V. Conclusions

    Aromatase inhibitors, both steroidal and nonsteroidal agents, have been shown to be useful for the treatment of breast cancer. These compounds work by preventing the synthesis of estrogens in the body. Aromatase inhibitors appear to be more effective in postmenopausal women than in premenopausal women due to the fact that the major source of estrogen biosynthesis in postmenopausal women is adipose tissue. Over the years, both steroidal and nonsteroidal inhibitors have developed into very potent compounds that are highly selective for aromatase vs. other steroidogenic cytochrome P450 enzymes. The potent, selective and orally-active third-generation aromatase inhibitors, anastrozole, letrozole, and exemestane, were initially approved for clinical use as endocrine therapy in postmenopausal patients failing antiestrogen therapy alone or multiple hormonal therapies. More recent clinical studies have shown that these aromatase inhibitors are more effective than tamoxifen in postmenopausal patients with metastatic breast cancer, and these agents are the approved therapy for the treatment of postmenopausal women with metastatic estrogen-dependent breast cancer. Furthermore, the third-generation aromatase inhibitors are now being studied in the adjuvant setting either alone or in combination with other agents. Based on the results of these multiple, large randomized trials, the American Society of Clinical Oncology panel recommends adjuvant hormonal therapy for a postmenopausal woman with receptor-positive breast cancer with an aromatase inhibitor as initial therapy or after treatment with tamoxifen.

    Acknowledgments

    We thank the Ohio Supercomputer Center (OSC) for computational resources.

    Footnotes

    This work was supported by NIH Grant R01 CA73698 (to R.W.B.), the Chemistry & Biology Interface Training Program, NIH Grant T32 GM08512 (to E.S.D.-C.), U.S. Army Medical Research and Material Command (USAMRMC) Breast Cancer Program Idea Grant DMAD-17–00-1–0388 (to R.W.B.), and USAMRMC Breast Cancer Program Predoctoral Fellowship DMAD17–02-1–0529 (to J.C.H.).

    First Published Online April 6, 2005

    Abbreviations: COX, Cyclooxygenase; NADPH, reduced nicotinamide adenine dinucleotide phosphate; PGE2, prostaglandin E2.

    References

    American Cancer Society 2004 Facts and figures. Atlanta: American Cancer Society

    Weigel NL, Rowan BG 2001 Estrogen and progesterone action. In: DeGroot LJ, Jameson JL, Burger HG, et al, eds. Endocrinology. Philadelphia: WB Saunders; 2053–2060

    Dickson RB, Lippman ME 1995 Molecular basis of breast cancer. In: Mendelsohn J, Howley PH, Israel MA, Liotta LA, eds. The molecular basis of cancer. Philadelphia: WB Saunders; 358–386

    Simpson ER, Mahendroo MS, Means GD, Kilgore MW, Hinshelwood MM, Graham-Lorence S, Amarneh B, Ito Y, Fisher CR, Michael MD 1994 Aromatase cytochrome P450, the enzyme responsible for estrogen biosynthesis. Endocr Rev 15:342–355

    James VH, McNeill JM, Lai LC, Newton CJ, Ghilchik MW, Reed MJ 1987 Aromatase activity in normal breast and breast tumor tissues: in vivo and in vitro studies. Steroids 50:269–279

    Miller WR, O’Neill J 1987 The importance of local synthesis of estrogen within the breast. Steroids 50:537–548

    Reed MJ, Owen AM, Lai LC, Coldham NG, Ghilchik MW, Shaikh NA, James VH 1989 In situ oestrone synthesis in normal breast and breast tumour tissues: effect of treatment with 4-hydroxyandrostenedione. Int J Cancer 44:233–237

    Bulun SE, Price TM, Aitken J, Mahendroo MS, Simpson ER 1993 A link between breast cancer and local estrogen biosynthesis suggested by quantification of breast adipose tissue aromatase cytochrome P450 transcripts using competitive polymerase chain reaction after reverse transcription. J Clin Endocrinol Metab 77:1622–1628

    Simpson ER, Mahendroo MS, Means GD, Kilgore MW, Corbin CJ, Mendelson CR 1993 Tissue-specific promoters regulate aromatase cytochrome P450 expression. J Steroid Biochem Mol Biol 44:321–330

    Kellis JT, Vickery LE 1987 Purification and characterization of human placental aromatase cytochrome P-450. J Biol Chem 262:4413–4420

    Akhtar M, Calder MR, Corina DL, Wright JN 1982 Mechanistic studies on C-19 demethylation in oestrogen biosynthesis. Biochem J 201:569–580

    Graham-Lorence S, Amarneh B, White RE, Peterson JA, Simpson ER 1995 A three-dimensional model of aromatase cytochrome P450. Protein Sci 4:1065–1080

    Hackett JC, Brueggemeier RW, Haddad CM 2005 The final catalytic step of cytochrome P450 aromatase: a density functional theory study. J Am Chem Soc 10.1021/ja044716w

    Meunier B, de Visser SP, Shaik S 2004 Mechanism of oxidation reactions catalyzed by cytochrome p450 enzymes. Chem Rev 104:3947–3980

    Bulun SE, Takayama K, Suzuki T, Sasano H, Yilmaz B, Sebastian S 2004 Organization of the human aromatase p450 (CYP19) gene. Semin Reprod Med 22:5–9

    Gartner CA, Thompson SJ, Rettie AE, Nelson SD 2001 Human aromatase in high yield and purity by perfusion chromatography and its characterization by difference spectroscopy and mass spectrometry. Protein Expr Purif 22:443–454

    Zhao Y, Agarwal VR, Mendelson CR, Simpson ER 1997 Transcriptional regulation of CYP19 gene (aromatase) expression in adipose stromal cells in primary culture. J Steroid Biochem Mol Biol 61:203–210

    Simpson ER, Clyne C, Rubin G, Boon WC, Robertson K, Britt K, Speed C, Jones M 2002 Aromatase—a brief overview. Annu Rev Physiol 64:93–127

    Dowsett M, Macaulay V, Gledhill J, Ryde C, Nicholls J, Ashworth A, McKinna JA, Smith IE 1993 Control of aromatase in breast cancer cells and its importance for tumor growth. J Steroid Biochem Mol Biol 44:605–609

    Miller WR, Mullen P, Sourdaine P, Watson C, Dixon JM, Telford J 1997 Regulation of aromatase activity within the breast. J Steroid Biochem Mol Biol 61:193–202

    Reed MJ, Topping L, Coldham NG, Purohit A, Ghilchik MW, James VH 1993 Control of aromatase activity in breast cancer cells: the role of cytokines and growth factors. J Steroid Biochem Mol Biol 44:589–596

    Quinn AL, Burak WE, Brueggemeier RW 1999 Effects of matrix components on aromatase activity in breast stromal cells in culture. J Steroid Biochem Mol Biol 70:249–256

    Sasano H, Edwards DP, Anderson TJ, Silverberg SG, Evans DB, Santen RJ, Ramage P, Simpson ER, Bhatnagar AS, Miller WR 2003 Validation of new aromatase monoclonal antibodies for immunohistochemistry: progress report. J Steroid Biochem Mol Biol 86:239–244

    Zhao Y, Agarwal VR, Mendelson CR, Simpson ER 1996 Estrogen biosynthesis proximal to a breast tumor is stimulated by PGE2 via cyclic AMP, leading to activation of promoter II of the CYP19 (aromatase) gene. Endocrinology 137:5739–5742

    Hughes R, Timmermans P, Schrey MP 1996 Regulation of arachidonic acid metabolism, aromatase activity and growth in human breast cancer cells by interleukin-1? and phorbol ester: dissociation of a mediatory role for prostaglandin E2 in the autocrine control of cell function. Int J Cancer 67:684–689

    Harris RE, Namboodiri KK, Farrar WB 1996 Nonsteroidal antiinflammatory drugs and breast cancer. Epidemiology 7:203–205

    Harris RE, Robertson FM, Abou-Issa HM, Farrar WB, Brueggemeier R 1999 Genetic induction and upregulation of cyclooxygenase (COX) and aromatase (CYP19): an extension of the dietary fat hypothesis of breast cancer. Med Hypotheses 52:291–292

    Arun B, Goss P 2004 The role of COX-2 inhibition in breast cancer treatment and prevention. Semin Oncol 31:22–29

    Terry MB, Gammon MD, Zhang FF, Tawfik H, Teitelbaum SL, Britton JA, Subbaramaiah K, Dannenberg AJ, Neugut AI 2004 Association of frequency and duration of aspirin use and hormone receptor status with breast cancer risk. JAMA 291:2433–2440

    Brueggemeier RW, Quinn AL, Parrett ML, Joarder FS, Harris RE, Robertson FM 1999 Correlation of aromatase and cyclooxygenase gene expression in human breast cancer specimens. Cancer Lett 140:27–35

    Brodie AM, Lu Q, Long BJ, Fulton A, Chen T, Macpherson N, DeJong PC, Blankenstein MA, Nortier JW, Slee PH, van de Ven J, van Gorp JM, Elbers JR, Schipper ME, Blijham GH, Thijssen JH 2001 Aromatase and COX-2 expression in human breast cancers. J Steroid Biochem Mol Biol 79:41–47

    Richards JA, Brueggemeier RW 2003 Prostaglandin E2 regulates aromatase activity and expression in human adipose stromal cells via two distinct receptor subtypes. J Clin Endocrinol Metab 88:2810–2816

    Brueggemeier RW, Diaz-Cruz ES, Li PK, Sugimoto Y, Lin YC, Shapiro CL, Translational studies on aromatase, cyclooxygenases, and enzyme inhibitors in breast cancer. J Steroid Biochem Mol Biol, in press

    Diaz-Cruz ES, Shapiro CL, Brueggemeier RW 2005 Cyclooxygenase inhibitors suppress aromatase expression and activity in breast cancer cells. J Clin Endocrinol Metab 90:2563–2570

    Carmichael PL 1998 Mechanisms of action of antiestrogens: relevance to clinical benefits and risks. Cancer Invest 16:604–611

    Jordan VC 1995 Tamoxifen: toxicities and drug resistance during the treatment and prevention of breast cancer. Annu Rev Pharmacol Toxicol 35:195–211

    Harvey HA, Lipton A, Santen RJ 1982 Aromatase: new perspectives for breast cancer. Cancer Res 42(Suppl):3261s–3467s

    Santen RJ 1987 Aromatase conference: future perspectives. Steroids 50:1–665

    Brodie AM, Brodie HJ, Callard G, Robinson CH, Roselli C, Santen RJ 1993 Proceedings of the Third International Aromatase Conference. J Steroid Biochem Mol Biol 44:321–696

    Simpson ER, Bhatnagar A, Brodie AM, Brueggemeier RW, Chen S, DeCoster R, Dowsett M, Harada N, Naftolin F, Osawa Y, Plourde P, Reed MJ, Santen RJ 1997 Proceedings of the Fourth International Aromatase Conference. J Steroid Biochem Mol Biol 61:107–425

    Simpson ER 2001 Aromatase 2000: Sixth International Aromatase Conference. J Steroid Biochem Mol Biol 79:1–314

    Johnston JO, Metcalf BW 1984 Aromatase: a target enzyme in breast cancer. In: Sunkara P, ed. Novel approaches to cancer chemotherapy. New York: Academic Press; 307–328

    Banting L, Smith HJ, James M, Jones G, Nazareth W, Nicholls PJ, Hewlins MJ, Rowlands MG 1988 Structure-activity relationships for non-steroidal inhibitors of aromatase. J Enzym Inhib 2:215–229

    Banting L, Nicholls PJ, Shaw MA, Smith HJ 1989 Recent developments in aromatase inhibition as a potential treatment for oestrogen-dependent breast cancer. Prog Med Chem 26:253–298

    Covey DF 1988 Aromatase inhibitors: specific inhibitors of oestrogen biosynthesis. In: Berg D, Plempel M, eds. Sterol biosynthesis inhibitors. Chichester, UK: Ellis Horwood; 534–571

    Brueggemeier RW 1990 Biochemical and molecular aspects of aromatase. J Enzym Inhib 4:101–111

    Brueggemeier RW 1994 Aromatase inhibitors—mechanisms of steroidal inhibitors. Breast Cancer Res Treat 30:31–42

    Cole PA, Robinson CH 1990 Mechanism and inhibition of cytochrome P-450 aromatase. J Med Chem 33:2933–2942

    Brodie AM, Njar VC 1996 Aromatase inhibitors and breast cancer. Semin Oncol 23:10–20

    Brodie A, Lu Q, Long B 1999 Aromatase and its inhibitors. J Steroid Biochem Mol Biol 69:205–210

    Santen RJ, Harvey HA 1999 Use of aromatase inhibitors in breast carcinoma. Endocr Relat Cancer 6:75–92

    Schwarzel WC, Kruggel WG, Brodie HJ 1973 Studies on the mechanism of estrogen biosynthesis. 8. The development of inhibitors of the enzyme system in human placenta. Endocrinology 92:866–880

    Siiteri PK, Thompson EA 1975 Studies of human placental aromatase. J Steroid Biochem 6:317–322

    Brueggemeier RW, Floyd EE, Counsell RE 1978 Synthesis and biochemical evaluation of inhibitors of estrogen biosynthesis. J Med Chem 21:1007–1011

    Henderson D, Norbisrath G, Kerb U 1986 1-Methyl-1,4-androstadiene-3,17-dione (SH 489): characterization of an irreversible inhibitor of estrogen biosynthesis. J Steroid Biochem 24:303–306

    Brueggemeier RW, Katlic NE 1987 Effects of the aromatase inhibitor 7 -(4'-amino)phenylthio-4-androstene-3,17-dione in MCF-7 human mammary carcinoma cell culture. Cancer Res 47:4548–4551

    Brueggemeier RW, Katlic NE, Kenreigh CA, Li PK 1992 Aromatase inhibition by 7-substituted steroids in human choriocarcinoma cell culture. J Steroid Biochem Mol Biol 41:85–90

    Brueggemeier RW, Li PK 1988 Effects of the aromatase inhibitor 7 -(4'-amino)phenylthio-4-androstene-3,17-dione on 7,12-dimethylbenz(a)anthracene-induced mammary carcinoma in rats. Cancer Res 48:6808–6810

    Li PK, Brueggemeier RW 1990 Synthesis and biochemical studies of 7-substituted 4,6-androstadiene-3,17-diones as aromatase inhibitors. J Med Chem 33:101–105

    Kellis JT, Childers WE, Robinson CH, Vickery LE 1987 Inhibition of aromatase cytochrome P-450 by 10-oxirane and 10-thiirane substituted androgens. Implications for the structure of the active site. J Biol Chem 262:4421–4426

    Childers WE, Shih MJ, Furth PS, Robinson CH 1987 Stereoselective inhibition of human placental aromatase. Steroids 50:121–134

    Shih MJ, Carrell MH, Carrell HL, Wright CL, Johnston JO, Robinson CH 1987 Stereoselective inhibition of aromatase by novel epoxysteroids. J Chem Soc Chem Comm 213–214

    Bednarski PJ, Porubek DJ, Nelson SD 1985 Thiol-containing androgens as suicide substrates of aromatase. J Med Chem 28:775–779

    Wright JN, Calder MR, Akhtar M 1985 Steroidal C-19 sulfur and nitrogen derivatives designed as aromatase inhibitors. J Chem Soc Chem Comm 1733–1735

    Peet NP, Burkhart JP, Wright CL, Johnston JO 1992 Time-dependent inhibition of human placental aromatase with a 2,19-methyleneoxy-bridged androstenedione. J Med Chem 35:3303–3306

    Peet NP, Johnston JO, Burkhart JP, Wright CL 1993 A-ring bridged steroids as potent inhibitors of aromatase. J Steroid Biochem Mol Biol 44:409–420

    Johnston JO, Wright CL, Burkhart JP, Peet NP 1993 Biological characterization of A-ring steroids. J Steroid Biochem Mol Biol 44:623–631

    Metcalf BW, Wright CL, Burkhart JP, Johnston JO 1981 Substrate-induced inactivation of aromatase by allenic and acetylenic steroids. J Am Chem Soc 103:3221–3222

    Covey DF, Hood WF, Parikh VD 1981 10?-Propynyl-substituted steroids. Mechanism-based enzyme-activated irreversible inhibitors of estrogen biosynthesis. J Biol Chem 256:1076–1079

    Marcotte PA, Robinson CH 1982 Synthesis and evaluation of 10?-substituted 4-estrene-3,17-diones as inhibitors of human placental microsomal aromatase. Steroids 39:325–344

    Johnston JO, Wright CL, Metcalf BW 1984 Biochemical and endocrine properties of a mechanism-based inhibitor of aromatase. Endocrinology 115:776–785

    Johnston JO, Wright CL, Metcalf BW 1984 Time-dependent inhibition of aromatase in trophoblastic tumor cells in tissue culture. J Steroid Biochem 20:1221–1226

    Longcope C, Femino A, Johnston JO 1988 Inhibition of peripheral aromatization in baboons by an enzyme-activated aromatase inhibitor (MDL 18,962). Endocrinology 122:2007–2011

    Johnston JO 1987 Biological characterization of 10-(2-propynyl) estr-4-ene-3,17-dione (MDL 18,962), an enzyme-activated inhibitor of aromatase. Steroids 50:105–120

    Brodie AM, Garrett WM, Hendrickson JR, Tsai-Morris CH, Marcotte PA, Robinson CH 1981 Inactivation of aromatase in vitro by 4-hydroxy-4-androstene-3,17-dione and 4-acetoxy-4-androstene-3,17-dione and sustained effects in vivo. Steroids 38:693–702

    Brodie AM, Schwarzel WC, Shaikh AA, Brodie HJ 1977 The effect of an aromatase inhibitor, 4-hydroxy-4-androstene-3,17-dione, on estrogen-dependent processes in reproduction and breast cancer. Endocrinology 100:1684–1695

    Wing LY, Garrett WM, Brodie AM 1985 Effects of aromatase inhibitors, aminoglutethimide, and 4-hydroxyandrostenedione on cyclic rats and rats with 7,12-dimethylbenz(a)anthracene-induced mammary tumors. Cancer Res 45:2425–2428

    Brodie AM, Garrett WM, Hendrickson JR, Tsai-Morris CH 1982 Effects of aromatase inhibitor 4-hydroxyandrostenedione and other compounds in the 7, 12-dimethylbenz(a)anthracene-induced breast carcinoma model. Cancer Res 42(Suppl):3360s–3364s

    Coombes RC, Goss P, Dowsett M, Gazet JC, Brodie A 1984 4-Hydroxyandrostenedione in treatment of postmenopausal patients with advanced breast cancer. Lancet 2:1237–1239

    Goss PE, Powles TJ, Dowsett M, Hutchison G, Brodie AM, Gazet JC, Coombes RC 1986 Treatment of advanced postmenopausal breast cancer with an aromatase inhibitor, 4-hydroxyandrostenedione: phase II report. Cancer Res 46:4823–4826

    Dowsett M 1994 Aromatase inhibition: basic concepts, and the pharmacodynamics of formestane. Ann Oncol 5(Suppl 7):S3–S5

    Goss PE, Jarman M, Wilkinson JR, Coombes RC 1986 Metabolism of the aromatase inhibitor 4-hydroxyandrostenedione in vivo. Identification of the glucuronide as a major urinary metabolite in patients and biliary metabolite in the rat. J Steroid Biochem 24:619–622

    Wiseman LR, Goa KL 1996 Formestane. A review of its pharmacological properties and clinical efficacy in the treatment of postmenopausal breast cancer. Drugs Aging 9:292–306

    Covey DF, Hood WF 1981 Enzyme-generated intermediates derived from 4-androstene-3,6,17-trione and 1,4,6-androstatriene-3,17-dione cause a time-dependent decrease in human placental aromatase activity. Endocrinology 108:1597–1599

    Covey DF, Hood WF 1982 Aromatase enzyme catalysis is involved in the potent inhibition of estrogen biosynthesis caused by 4-ace. Mol Pharmacol 21:173–180

    Snider CE, Brueggemeier RW 1987 Potent enzyme-activated inhibition of aromatase by a 7-substituted C19 steroid. J Biol Chem 262:8685–8689

    Li PK, Brueggemeier RW 1990 7-Substituted 1,4,6-androstatriene-3,17-diones as enzyme-activated irreversible inhibitors of aromatase. J Steroid Biochem 36:533–539

    Ebrahimian S, Chen HH, Brueggemeier RW 1993 Synthesis and biochemical studies of 7-substituted androsta-1,4-diene-3,17-diones as enzyme-activated irreversible inhibitors of aromatase. Steroids 58:414–422

    O’Reilly JM, Brueggemeier RW 1996 7-Arylaliphatic androsta-1,4-diene-3,17-diones as enzyme-activated irreversible inhibitors of aromatase. J Steroid Biochem Mol Biol 59:93–102

    Giudici D, Ornati G, Briatico G, Buzzetti F, Lombardi P, Di Salle E 1988 6-Methylenandrosta-1,4-diene-3,17-dione (FCE 24304): a new irreversible aromatase inhibitor. J Steroid Biochem 30:391–394

    Zaccheo T, Giudici D, Lombardi P, Di Salle E 1989 A new irreversible aromatase inhibitor, 6-methylenandrosta-1,4-diene-3,17-dione (FCE 24304): antitumor activity and endocrine effects in rats with DMBA-induced mammary tumors. Cancer Chemother Pharmacol 23:47–50

    Zaccheo T, Giudici D, Di Salle E 1993 Inhibitory effect of combined treatment with the aromatase inhibitor exemestane and tamoxifen on DMBA-induced mammary tumors in rats. J Steroid Biochem Mol Biol 44:677–680

    Di Salle E, Giudici D, Briatico G, Ornati G 1990 Novel irreversible aromatase inhibitors. Ann NY Acad Sci 595:357–367

    Brueggemeier RW, Li PK, Chen HH, Moh PP, Katlic NE 1990 Biochemical and pharmacological development of steroidal inhibitors of aromatase. J Steroid Biochem Mol Biol 37:379–385

    Cocconi G 1994 First generation aromatase inhibitors—aminoglutethimide and testololactone. Breast Cancer Res Treat 30:57–80

    Hoffken K 1993 Experience with aromatase inhibitors in the treatment of advanced breast cancer. Cancer Treat Rev 19(Suppl B):37–44

    Trunet PF, Vreeland F, Royce C, Chaudri HA, Cooper J, Bhatnagar AS 1997 Clinical use of aromatase inhibitors in the treatment of advanced breast cancer. J Steroid Biochem Mol Biol 61:241–245

    Furet P, Batzl C, Bhatnagar A, Francotte E, Rihs G, Lang M 1993 Aromatase inhibitors: synthesis, biological activity, and binding mode of azole-type compounds. J Med Chem 36:1393–1400

    Vanden Bossche H, Willemsens G, Roels I, Bellens D, Moereels H, Coene MC, Le Jeune L, Lauwers W, Janssen PA 1990 R 76713 and enantiomers: selective, nonsteroidal inhibitors of the cytochrome P450-dependent oestrogen synthesis. Biochem Pharmacol 40:1707–1718

    Wouters W, Snoeck E, De Coster R 1994 Vorozole, a specific non-steroidal aromatase inhibitor. Breast Cancer Res Treat 30:89–94

    Plourde PV, Dyroff M, Dukes M 1994 Arimidex: a potent and selective fourth-generation aromatase inhibitor. Breast Cancer Res Treat 30:103–111

    Bhatnagar AS, Hausler A, Schieweck K, Lang M, Bowman R 1990 Highly selective inhibition of estrogen biosynthesis by CGS 20267, a new non-steroidal aromatase inhibitor. J Steroid Biochem Mol Biol 37:1021–1027

    Demers LM 1994 Effects of Fadrozole (CGS 16949A) and Letrozole (CGS 20267) on the inhibition of aromatase activity in breast cancer patients. Breast Cancer Res Treat 30:95–102

    Adlercreutz H 1995 Phytoestrogens: epidemiology and a possible role in cancer protection. Environ Health Perspect 103(Suppl 7):103–112

    Wang C, Makela T, Hase T, Adlercreutz H, Kurzer MS 1994 Lignans and flavonoids inhibit aromatase enzyme in human preadipocytes. J Steroid Biochem Mol Biol 50:205–212

    Kellis Jr JT, Vickery LE 1984 Inhibition of human estrogen synthetase (aromatase) by flavones. Science 225:1032–1034

    Ibrahim AR, Abul-Hajj YJ 1990 Aromatase inhibition by flavonoids. J Steroid Biochem Mol Biol 37:257–260

    Le Bail JC, Laroche T, Marre-Fournier F, Habrioux G 1998 Aromatase and 17?-hydroxysteroid dehydrogenase inhibition by flavonoids. Cancer Lett 133:101–106

    Le Bail JC, Pouget C, Fagnere C, Basly JP, Chulia AJ, Habrioux G 2001 Chalcones are potent inhibitors of aromatase and 17?-hydroxysteroid dehydrogenase activities. Life Sci 68:751–761

    Kao YC, Zhou C, Sherman M, Laughton CA, Chen S 1998 Molecular basis of the inhibition of human aromatase (estrogen synthetase) by flavone and isoflavone phytoestrogens: a site-directed mutagenesis study. Environ Health Perspect 106:85–92

    Pouget C, Fagnere C, Basly JP, Besson AE, Champavier Y, Habrioux G, Chulia AJ 2002 Synthesis and aromatase inhibitory activity of flavanones. Pharm Res 19:286–291

    Pouget C, Fagnere C, Basly JP, Habrioux G, Chulia AJ 2002 Design, synthesis and evaluation of 4-imidazolylflavans as new leads for aromatase inhibition. Bioorg Med Chem Lett 12:2859–2861

    Recanatini M, Bisi A, Cavalli A, Belluti F, Gobbi S, Rampa A, Valenti P, Palzer M, Palusczak A, Hartmann RW 2001 A new class of nonsteroidal aromatase inhibitors: design and synthesis of chromone and xanthone derivatives and inhibition of the P450 enzymes aromatase and 17-hydroxylase/C17,20-lyase. J Med Chem 44:672–680

    Brueggemeier RW, Richards JA, Joomprabutra S, Bhat AS, Whetstone JL 2001 Molecular pharmacology of aromatase and its regulation by endogenous and exogenous agents. J Steroid Biochem Mol Biol 79:75–84

    Hodek P, Trefil P, Stiborova M 2002 Flavonoids-potent and versatile biologically active compounds interacting with cytochromes P450. Chem Biol Interact 139:1–21

    Kim YW, Hackett JC, Brueggemeier RW 2004 Synthesis and aromatase inhibitory activity of novel pyridine-containing isoflavones. J Med Chem 47:4032–4040

    Lipton A, Santen RJ 1974 Proceedings: medical adrenalectomy using aminoglutethimide and dexamethasone in advanced breast cancer. Cancer 33:503–512

    Santen RJ, Samojlik E, Lipton A, Harvey H, Ruby EB, Wells SA, Kendall J 1977 Kinetic, hormonal and clinical studies with aminoglutethimide in breast cancer. Cancer 39:2948–2958

    Santen RJ, Manni A, Harvey H, Redmond C 1990 Endocrine treatment of breast cancer in women. Endocr Rev 11:221–265

    Brodie AM 1994 Aromatase inhibitors in the treatment of breast cancer. J Steroid Biochem Mol Biol 49:281–287

    Goss PE, Strasser K 2001 Aromatase inhibitors in the treatment and prevention of breast cancer. J Clin Oncol 19:881–894

    Ingle JN 2001 Current status of adjuvant endocrine therapy for breast cancer. Clin Cancer Res 7:4392s–4396s

    Buzdar A, Howell A 2001 Advances in aromatase inhibition: clinical efficacy and tolerability in the treatment of breast cancer. Clin Cancer Res 7:2620–2635

    Murray R 2001 Role of anti-aromatase agents in postmenopausal advanced breast cancer. Cancer Chemother Pharmacol 48:259–265

    Hamilton A, Volm M 2001 Nonsteroidal and steroidal aromatase inhibitors in breast cancer. Oncology (Huntingt) 15:965–972

    Buzdar AU 2001 Endocrine therapy in the treatment of metastatic breast cancer. Semin Oncol 28:291–304

    Geisler J, King N, Dowsett M, Ottestad L, Lundgren S, Walton P, Kormeset PO, Lonning PE 1996 Influence of anastrozole (Arimidex), a selective, non-steroidal aromatase inhibitor, on in vivo aromatization and plasma oestrogen levels in postmenopausal women with breast cancer. Br J Cancer 74:1286–1291

    Jonat W, Howell A, Blomqvist C, Eiermann W, Winblad G, Tyrrell C, Mauriac L, Roche H, Lundgren S, Hellmund R, Azab M 1996 A randomized trial comparing two doses of the new selective aromatase inhibitor anastrozole (Arimidex) with megestrol acetate in postmenopausal patients with advanced breast cancer. Eur J Cancer 32A:404–412

    Buzdar AU, Jones SE, Vogel CL, Wolter J, Plourde P, Webster A 1997 A phase III trial comparing anastrozole (1 and 10 milligrams), a potent and selective aromatase inhibitor, with megestrol acetate in postmenopausal women with advanced breast carcinoma. Arimidex Study Group. Cancer 79:730–739

    Buzdar A, Jonat W, Howell A, Jones SE, Blomqvist C, Vogel CL, Eiermann W, Wolter JM, Azab M, Webster A, Plourde PV 1996 Anastrozole, a potent and selective aromatase inhibitor, versus megestrol acetate in postmenopausal women with advanced breast cancer: results of overview analysis of two phase III trials. Arimidex Study Group. J Clin Oncol 14:2000–2011

    Bonneterre J, Thurlimann B, Robertson JF, Krzakowski M, Mauriac L, Koralewski P, Vergote I, Webster A, Steinberg M, von Euler M 2000 Anastrozole versus tamoxifen as first-line therapy for advanced breast cancer in 668 postmenopausal women: results of the Tamoxifen or Arimidex Randomized Group Efficacy and Tolerability Study. J Clin Oncol 18:3748–3757

    Nabholtz JM, Buzdar A, Pollak M, Harwin W, Burton G, Mangalik A, Steinberg M, Webster A, von Euler M 2000 Anastrozole is superior to tamoxifen as first-line therapy for advanced breast cancer in postmenopausal women: results of a North American multicenter randomized trial. Arimidex Study Group. J Clin Oncol 18:3758–3767

    Bonneterre J, Buzdar A, Nabholtz JM, Robertson JF, Thurlimann B, von Euler M, Sahmoud T, Webster A, Steinberg M 2001 Anastrozole is superior to tamoxifen as first-line therapy in hormone receptor positive advanced breast carcinoma. Cancer 92:2247–2258

    Geisler J, King N, Dowsett M, Ottestad L, Lundgren S, Walton P, Kormeset PO, Lonning PE1996 Influence of anastrozole (Aromidex), a selective, non-steroidal aromatase inhibitor, on in vivo aromatization and plasma oestrogen levels in postmenopausal women with breast cancer. Br J Cancer 74:1286–1291

    Bajetta E, Martinetti A, Zilembo N, Pozzi P, La Torre I, Ferrari L, Seregni E, Longarini R, Salvucci G, Bombardieri E 2002 Biological activity of anastrozole in postmenopausal patients with advanced breast cancer: effects on estrogens and bone metabolism. Ann Oncol 13:1059–1066

    Dowsett M, Jones A, Johnston SR, Jacobs S, Trunet P, Smith IE 1995 In vivo measurement of aromatase inhibition by letrozole (CGS 20267) in postmenopausal patients with breast cancer. Clin Cancer Res 1:1511–1515

    Dombernowsky P, Smith I, Falkson G, Leonard R, Panasci L, Bellmunt J, Bezwoda W, Gardin G, Gudgeon A, Morgan M, Fornasiero A, Hoffmann W, Michel J, Hatschek T, Tjabbes T, Chaudri HA, Hornberger U, Trunet PF 1998 Letrozole, a new oral aromatase inhibitor for advanced breast cancer: double-blind randomized trial showing a dose effect and improved efficacy and tolerability compared with megestrol acetate. J Clin Oncol 16:453–461

    Buzdar A, Douma J, Davidson N, Elledge R, Morgan M, Smith R, Porter L, Nabholtz J, Xiang X, Brady C 2001 Phase III, multicenter, double-blind, randomized study of letrozole, an aromatase inhibitor, for advanced breast cancer versus megestrol acetate. J Clin Oncol 19:3357–3366

    Bajetta E, Zilembo N, Dowsett M, Guillevin L, Di Leo A, Celio L, Martinetti A, Marchiano A, Pozzi P, Stani S, Bichisao E 1999 Double-blind, randomised, multicentre endocrine trial comparing two letrozole doses, in postmenopausal breast cancer patients. Eur J Cancer 35:208–213

    Mouridsen H, Gershanovich M, Sun Y, Perez-Carrion R, Boni C, Monnier A, Apffelstaedt J, Smith R, Sleeboom HP, Janicke F, Pluzanska A, Dank M, Becquart D, Bapsy PP, Salminen E, Snyder R, Lassus M, Verbeek JA, Staffler B, Chaudri-Ross HA, Dugan M 2001 Superior efficacy of letrozole versus tamoxifen as first-line therapy for postmenopausal women with advanced breast cancer: results of a phase III study of the International Letrozole Breast Cancer Group. J Clin Oncol 19:2596–2606

    Geisler J, Haynes B, Anker G, Dowsett M, Lonning PE 2002 Influence of letrozole and anastrozole on total body aromatization and plasma estrogen levels in postmenopausal breast cancer patients evaluated in a randomized, cross-over study. J Clin Oncol 20:751–757

    Evans TR, Di Salle E, Ornati G, Lassus M, Benedetti MS, Pianezzola E, Coombes RC 1992 Phase I and endocrine study of exemestane (FCE 24304), a new aromatase inhibitor, in postmenopausal women. Cancer Res 52:5933–5939

    Bajetta E, Zilembo N, Noberasco C, Martinetti A, Mariani L, Ferrari L, Buzzoni R, Greco M, Bartoli C, Spagnoli I, Danesini GM, Artale S, Paolini J 1997 The minimal effective exemestane dose for endocrine activity in advanced breast cancer. Eur J Cancer 33:587–591

    Geisler J, King N, Anker G, Ornati G, Di Salle E, Lonning PE, Dowsett M 1998 In vivo inhibition of aromatization by exemestane, a novel irreversible aromatase inhibitor, in postmenopausal breast cancer patients. Clin Cancer Res 4:2089–2093

    Lonning PE, Bajetta E, Murray R, Tubiana-Hulin M, Eisenberg PD, Mickiewicz E, Celio L, Pitt P, Mita M, Aaronson NK, Fowst C, Arkhipov A, Di Salle E, Polli A, Massimini G 2000 Activity of exemestane in metastatic breast cancer after failure of nonsteroidal aromatase inhibitors: a phase II trial. J Clin Oncol 18:2234–2244

    Lonning PE 2000 Pharmacology and clinical experience with exemestane. Expert Opin Investig Drugs 9:1897–1905

    Johannessen DC, Engan T, Di Salle E, Zurlo MG, Paolini J, Ornati G, Piscitelli G, Kvinnsland S, Lonning PE 1997 Endocrine and clinical effects of exemestane (PNU 155971), a novel steroidal aromatase inhibitor, in postmenopausal breast cancer patients: a phase I study. Clin Cancer Res 3:1101–1108

    Boeddinghaus IM, Dowsett M 2001 Comparative clinical pharmacology and pharmacokinetic interactions of aromatase inhibitors. J Steroid Biochem Mol Biol 79:85–91

    Miller WR, Dixon JM 2002 Endocrine and clinical endpoints of exemestane as neoadjuvant therapy. Cancer Control 9:9–15

    Yue W, Brodie AM 1997 Mechanisms of the actions of aromatase inhibitors 4-hydroxyandrostenedione, fadrozole, and aminoglutethimide on aromatase in JEG-3 cell culture. J Steroid Biochem Mol Biol 63:317–328

    Soudon J 2000 Comparison of in vitro exemestane activity versus other antiaromatase agents. Clin Breast Cancer 1(Suppl 1):S68–S73

    Miller WR, Dixon JM 2000 Antiaromatase agents: preclinical data and neoadjuvant therapy. Clin Breast Cancer 1(Suppl 1):S9–S14

    Brodie AH, Mouridsen HT 2003 Applicability of the intratumor aromatase preclinical model to predict clinical trial results with endocrine therapy. Am J Clin Oncol 26:S17–S26

    Paridaens R, Dirix L, Lohrisch C, Beex L, Nooij M, Cameron D, Biganzoli L, Cufer T, Duchateau L, Hamilton A, Lobelle JP, Piccart M 2003 Mature results of a randomized phase II multicenter study of exemestane versus tamoxifen as first-line hormone therapy for postmenopausal women with metastatic breast cancer. Ann Oncol 14:1391–1398

    Ingle JN 2001 Aromatase inhibition and antiestrogen therapy in early breast cancer treatment and chemoprevention. Oncology (Huntingt) 15:28–34

    Buzdar AU 2003 Advances in endocrine treatments for postmenopausal women with metastatic and early breast cancer. Oncologist 8:335–341

    Baum M, Budzar AU, Cuzick J, Forbes J, Houghton JH, Klijn JG, Sahmoud T 2002 Anastrozole alone or in combination with tamoxifen versus tamoxifen alone for adjuvant treatment of postmenopausal women with early breast cancer: first results of the ATAC randomised trial. Lancet 359:2131–2139

    Baum M, Buzdar A, Cuzick J, Forbes J, Houghton J, Howell A, Sahmoud T 2003 Anastrozole alone or in combination with tamoxifen versus tamoxifen alone for adjuvant treatment of postmenopausal women with early-stage breast cancer: results of the ATAC (Arimidex, Tamoxifen Alone or in Combination) trial efficacy and safety update analyses. Cancer 98:1802–1810

    Goss PE, Ingle JN, Martino S, Robert NJ, Muss HB, Piccart MJ, Castiglione M, Tu D, Shepherd LE, Pritchard KI, Livingston RB, Davidson NE, Norton L, Perez EA, Abrams JS, Therasse P, Palmer MJ, Pater JL 2003 A randomized trial of letrozole in postmenopausal women after five years of tamoxifen therapy for early-stage breast cancer. N Engl J Med 349:1793–1802

    Coombes RC, Hall E, Gibson LJ, Paridaens R, Jassem J, Delozier T, Jones SE, Alvarez I, Bertelli G, Ortmann O, Coates AS, Bajetta E, Dodwell D, Coleman RE, Fallowfield LJ, Mickiewicz E, Andersen J, Lonning PE, Cocconi G, Stewart A, Stuart N, Snowdon CF, Carpentieri M, Massimini G, Bliss JM 2004 A randomized trial of exemestane after two to three years of tamoxifen therapy in postmenopausal women with primary breast cancer. N Engl J Med 350:1081–1092

    Winer EP, Hudis C, Burstein HJ, Wolff AC, Pritchard KI, Ingle JN, Chlebowski RT, Gelber R, Edge SB, Gralow J, Cobleigh MA, Mamounas EP, Goldstein LJ, Whelan TJ, Powles TJ, Bryant J, Perkins C, Perotti J, Braun S, Langer AS, Browman GP, Somerfield MR 2005 American Society of Clinical Oncology technology assessment on the use of aromatase inhibitors as adjuvant therapy for postmenopausal women with hormone receptor-positive breast cancer: status report 2004. J Clin Oncol 23:619–629

    Moon RC, Steele VE, Kelloff GJ, Thomas CF, Detrisac CJ, Mehta RG, Lubet RA 1994 Chemoprevention of MNU-induced mammary tumorigenesis by hormone response modifiers: toremifene, RU 16117, tamoxifen, aminoglutethimide and progesterone. Anticancer Res 14:889–893

    De Coster R, Van Ginckel RF, Callens MJ, Goeminne NK, Janssens BL 1992 Antitumoral and endocrine effects of (+)-vorozole in rats bearing dimethylbenzanthracene-induced mammary tumors. Cancer Res 52:1240–1244

    Lubet RA, Steele VE, Casebolt TL, Eto I, Kelloff GJ, Grubbs CJ 1994 Chemopreventive effects of the aromatase inhibitors vorozole (R-83842) and 4-hydroxyandrostenedione in the methylnitrosourea (MNU)-induced mammary tumor model in Sprague-Dawley rats. Carcinogenesis 15:2775–2780

    Schieweck K, Bhatnagar AS, Batzl C, Lang M 1993 Anti-tumor and endocrine effects of non-steroidal aromatase inhibitors on estrogen-dependent rat mammary tumors. J Steroid Biochem Mol Biol 44:633–636

    Goss PE 2003 Breast cancer prevention—clinical trials strategies involving aromatase inhibitors. J Steroid Biochem Mol Biol 86:487–493(Robert W. Brueggemeier, J)

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