An in vitro model showing adaptation to long-term oestrogen deprivation highlights the clinical potential for targeting kinase pathways in combination with aromatase inhibition

Aromatase inhibitors (AI) have improved the treatment of oestrogen receptor positive (ER+) breast cancer. Despite the efficacy of these agents over 40% of patients relapse with endocrine resistant disease. Here we describe an in vitro model of acquired resistance to long-term oestrogen deprivation (LTED). The LTED cells retain expression of the ER and appear hypersensitive to oestrogen as a result of altered kinase activity. Furthermore analysis of temporal changes in gene expression during the acquisition of resistance highlight growth factor receptor pathways as key mediators of this adaptive process.     

Predictors of response to aromatase inhibitors

Aromatase inhibitors are now considered to be part of the endocrine treatment for most hormone receptor-positive breast cancer in post-menopausal women for both early and advanced disease. Despite the impressive efficacy of these agents, up to 50% of treated patients exhibit de novo or intrinsic resistance to aromatase inhibitors and hence identification of response predictors is essential to allow treatment to be directed towards responsive populations and for alternative or additional therapies to be offered to resistant patients. Emerging data seem to suggest a role for the conventional tumour markers of oestrogen receptor and progesterone receptor as possible predictors of response but, particularly in the adjuvant setting, the extent to which these are useful has not been fully elucidated. Data from both the neo-adjuvant and advanced disease settings suggest that response to aromatase inhibitors does not appear to be adversely affected by HER-2 overexpression. Within neo-adjuvant aromatase inhibitor studies, the proliferation marker Ki67 has shown a significant correlation with relapse-free survival, suggesting a role in prediction for measurement of Ki67 and other dynamic markers of response. Analysis of multiple gene expression changes over a short treatment period may also have potential clinical utility for prediction of response.     

Inhibition of aromatase: insights from recent studies

Aromatase is the rate limiting enzyme that catalyzes the conversion of androgens to estrogens. Blockade of this step allows treatment of diseases that are dependent upon estrogen. Over the past two decades, highly potent and specific aromatase inhibitors have been developed which block total body aromatization by over 99%. An important recent question is whether aromatase inhibitors are superior to the antiestrogens for treatment of hormone-dependent breast cancer. The third generation aromatase inhibitors have been compared to tamoxifen for the treatment of breast cancer in the advanced, adjuvant, and neoadjuvant settings. All of these studies suggest the superiority of aromatase inhibitors over tamoxifen. The mechanism responsible for the superiority of the aromatase inhibitors relates to the estrogen agonistic effects of tamoxifen. During exposure to estrogen deprived conditions and to tamoxifen, breast cancer cells adapt and upregulate the MAP kinase and PI-3 kinase pathways. These growth factor signaling pathways potentiate the estrogen agonistic properties of tamoxifen. Data from a large adjuvant therapy trial (ATAC trial) provide evidence that the aromatase inhibitors may also be superior for breast cancer prevention. The mechanism for superiority in this setting probably relates to the genotoxic effects of estradiol metabolites. The aromatase inhibitors may be also useful for the treatment of endometriosis and for ovulation induction as evidenced by preliminary data. The recent advances in development of the aromatase inhibitors clearly demonstrate the utility of these agents for treatment of breast cancer and potentially for other indications.     

Where do selective estrogen receptor modulators (SERMs) and aromatase inhibitors (AIs) now fit into breast cancer treatment algorithms?

The agents used for endocrine therapy in patients with breast cancer have changed markedly over the past decade. Tamoxifen remains the anti-oestrogen of choice, but could be replaced by the oestrogen receptor down-regulator ICI 182780 or by the fixed ring triphenylethylene arzoxifene (previously SERM III) soon. Whilst aminoglutethimide and 4-OH androstenedione were the aromatase inhibitors of choice, they have been replaced by non-steroidal (anastrozole and letrozole) and steroidal (exemestane) inhibitors of high potency and low side effect profile. Previously, often used treatments such as progestogens (megestrol acetate and medroxyprogesterone acetate) and androgens are now rarely used or confined to fourth or fifth line treatments. The LHRH agonist, goserelin, remains the treatment of choice for pre-menopausal patients with advanced breast cancer although recent randomised trials indicate a response, time to progression and survival advantage for the combination of goserelin and tamoxifen compared with goserelin alone.

The newer treatments have led to questions concerning the optimum sequence of agents to use in advanced breast cancer and as neo-adjuvant and adjuvant therapy in relation to surgery. Two trials of anastrozole compared with tamoxifen and one trial of letrozole compared with tamoxifen indicate that the new triazole aromatase inhibitors have a significant advantage over the anti-oestrogen with respect to time to progression and survival. Similarly, triazole aromatase inhibitors give faster and more complete responses compared with tamoxifen when used in post-menopausal women before surgery.

Major research questions remain with respect to the aromatase inhibitors used as adjuvant therapy. Anastrozole is being tested alone or in combination with tamoxifen compared with tamoxifen in the ‘so-called’ ATAC trial. Over 9000 patients have been randomised to this important study: the results will be available late-2001. A similar study comparing letrozole and tamoxifen started recently under the auspices of the Breast International Group. Importantly, this trial is also comparing the sequence of tamoxifen followed by letrozole (or vice versa). A similar trial of exemestane given after 2–3 years of tamoxifen compared with 5 years of tamoxifen is recruiting well as is a study comparing letrozole (or placebo) for 5 years after 5 years of adjuvant tamoxifen. These studies may show that aromatase inhibitors are superior to tamoxifen or that a sequence is preferable.

ICI 182780 causes complete oestrogen receptor down-regulation leading to a the lack of agonist activity of the drug. Two trials of ICI 182780 compared with anastrozole for advanced disease will report later this year and a comparison with tamoxifen next year. Arzoxifene (SERM III) is being tested against tamoxifen. These studies are likely to result in new anti-oestrogens being introduced into the clinic.

Most of our endocrine treatments deprived the tumour cell of oestradiol. In vitro experiments with MCF-7 cells indicate that tumour cells can adapt and then grow in response to low oestrogen concentrations in the tissue—culture medium. Importantly, the cells were shown to apoptose in response to high oestrogen concentrations. A recent clinical trial has demonstrated a high response rate to stilboestrol given after a median of four previous oestrogen depriving endocrine therapies. These data and the newer treatments available indicate a need to re-think our general approach to endocrine therapy and endocrine prevention.     

An integrated view of aromatase and its inhibition

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

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

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

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.     

Aromatase, aromatase inhibitors, and breast cancer

Estrogens are known to be important in the growth of breast cancers in both pre and postmenopausal women. As the number of breast cancer patients increases with age, the majority of breast cancer patients are postmenopausal women. Although estrogens are no longer made in the ovaries after menopause, peripheral tissues produce sufficient concentrations to stimulate tumor growth. As aromatase catalyzes the final and rate-limiting step in the biosynthesis of estrogen, inhibitors of this enzyme are effective targeted therapy for breast cancer. Three aromatase inhibitors (AIs) are now FDA approved and have been shown to be more effective than the antiestrogen tamoxifen and are well tolerated. AIs are now a standard treatment for postmenopausal patients. AIs are effective in adjuvant and first-line metastatic setting. This review describes the development of AIs and their current use in breast cancer. Recent research focuses on elucidating mechanisms of acquired resistance that may develop in some patients with long term AI treatment and also in innate resistance. Preclinical data in resistance models demonstrated that the crosstalk between ER and other signaling pathways particularly MAPK and PI3K/Akt is an important resistant mechanism. Blockade of these other signaling pathways is an attractive strategy to circumvent the resistance to AI therapy in breast cancer. Several clinical trials are ongoing to evaluate the role of these novel targeted therapies to reverse resistance to AIs. Article from the special issue on 'Targeted Inhibitors'.     

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.     

Contribution of aromatase to the deoxyribonucleic acid synthesis of MCF-7 human breast cancer cells and its suppression by aromatase inhibitors.

We have studied the effects of various steroids on DNA synthesis in MCF-7 human breast carcinoma cells, which have aromatase activity and which exert an oestrogen receptor-mediated growth, to assess the significance of intracellular aromatase on growth stimulation as well as inhibition by aromatase inhibitors. The cells were cultured for 96 h in phenol red-free medium containing 10% charcoal-treated fetal bovine serum and test reagents and pulse-labelled with [3H]thymidine. Physiological concentrations of oestradiol, oestrone, testosterone (T) and androstenedione (AD) stimulated thymidine incorporation. However, oestrone-sulphate and dihydrotestosterone (DHT) only stimulated at concentrations greater than the physiological levels. T and DHT stimulation was blocked by tamoxifen, but not by cyproterone acetate, suggesting that the stimulation was mediated via the oestrogen receptor but not by the androgen receptor. Stimulation by T and AD was reduced by aminoglutethimide and 14 alpha-hydroxy-4-androstene-3,6,17-trione, both of which inhibit aromatase activity, however, stimulation by nonaromatizable DHT was not reduced by the inhibitors, suggesting that androgens were converted by the intracellular aromatase to oestrogens which stimulated the thymidine incorporation. It is suggested that intracellular aromatase significantly contributes to the stimulation of DNA synthesis and that aromatase inhibitors suppress the stimulation.     

Local endocrine effects of aromatase inhibitors within the breast

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

Future uses for aromatase inhibitors in breast cancer

Over recent years highly potent, well-tolerated aromatase inhibitors have been developed, which essentially obliterate peripheral aromatase activity in postmenopausal women. Their role as the optimal second-line agents (post-tamoxifen) for the treatment of advanced breast cancer has recently been established in large comparative clinical trials. Their testing as adjuvant therapy is warranted, but their eventual application in this (or the prophylactic) setting will be dependent on the currently unknown effects of profound oestrogen deprivation on the physiology of postmenopausal women as well as on its efficacy. It is also possible that these new compounds could suppress oestrogen synthesis in premenopausal women, but the consequences on ovarian folliculogenesis might prevent their widespread use in this group of patients.     

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 and COX-2 expression in human breast cancers

We have investigated aromatase and the inducible cyclooxygenase COX-2 expression using immunocytochemistry in tumors of a series of patients with advanced breast cancer treated with aromatase inhibitors. Aromatase was expressed in 58/102 breast cancers. This is similar to the percentage previously reported for aromatase activity. Interestingly, aromatase was expressed in a variety of cell types, including tumor, stromal, adipose, and endothelial cells. Since prostaglandin E₂ is known to regulate aromatase gene expression and is the product of COX-2, an enzyme frequently overexpressed in tumors, immunocytochemistry was performed on the tissue sections using a polyclonal antibody to COX-2. Aromatase was strongly correlated (P<0.001) with COX-2 expression. These results suggest that PGE₂ produced by COX-2 in the tumor may be important in stimulating estrogen synthesis in the tumor and surrounding tissue. No correlation was observed between aromatase or COX-2 expression and the response of the patients to aromatase inhibitor treatment. However, only 13 patients responded. Nine of these patients were aromatase positive. Although similar to responses in other studies, this low response rate to second line treatment suggests that tumors of most patients were no longer sensitive to the effects of estrogen. Recent clinical studies suggest that greater responses occur when aromatase inhibitors are used as first line treatment. In the intratumoral aromatase mouse model, expression of aromatase in tumors is highly correlated with increased tumor growth. First line treatment with letrozole was effective in all animals treated and was more effective than tamoxifen in suppressing tumor growth. Letrozole was also effective in tumors failing to respond to tamoxifen, consistent with clinical findings. In addition, the duration of response was significantly longer with the aromatase inhibitor than with tamoxifen, suggesting that aromatase inhibitors may offer better control of tumor growth than this antiestrogen.     

What do we know about the mechanisms of aromatase inhibitor resistance?

Clinical trials have demonstrated the importance of aromatase inhibitor (AI) therapy in the effective treatment of hormone-dependent breast cancers. Yet, as with all prolonged drug therapy, resistance to aromatase inhibitors does develop. To date, the precise mechanism responsible for resistance to aromatase inhibitors is not completely understood. In this paper, several mechanisms of de novo/intrinsic resistance and acquired resistance to AIs are discussed. These mechanisms are hypothesized based on important findings from a number of laboratories.

To better understand this question, our lab has generated, in vitro, breast cancer cell lines that are resistant to aromatase inhibitors. Resistant cell lines were generated over a prolonged period of time using the MCF-7aro (aromatase overexpressed) breast cancer line. These cell lines are resistant to the aromatase inhibitors letrozole, anastrozole and exemestane and the anti-estrogen tamoxifen, for comparison. Two types of resistant cell lines have been generated, those that grow in the presence of testosterone (T) which is needed for cell growth, and resistant lines that are cultured in the presence of inhibitor only (no T). In addition to functional characterization of aromatase and ER in these resistant cell lines, microarray analysis has been employed in order to determine differential gene expression within the aromatase inhibitor resistant cell lines versus tamoxifen, in order to better understand the mechanism responsible for AI resistance on a genome-wide scale. We anticipate that our studies will generate important information on the mechanisms of AI resistance. Such information can be valuable for the development of treatment strategies against AI-resistant breast cancers.     

Aromatase inhibitors: cellular and molecular effects

Marked cellular and molecular changes may occur in breast cancers following treatment of postmenopausal breast cancer patients with aromatase inhibitors. Neoadjuvant protocols, in which treatment is given with the primary tumour still within the breast, are particularly illuminating. In Edinburgh, we have shown that 3 months treatment with either anastrozole, exemestane or letrozole produces pathological responses in the majority of oestrogen receptor (ER)-rich tumours (39/59) as manifested by reduced cellularity/increased fibrosis. Changes in histological grading may also take place, most notably a reduction in mitotic figures. This probably reflects an influence on proliferation as most tumours (82%) show a marked decrease in the proliferation marker, Ki67. These effects are generally more dramatic than seen with tamoxifen given in the same setting. Differences between aromatase inhibitors and tamoxifen are also apparent in changes in steroid hormone expression. Thus, immuno-staining for progesterone receptor (PgR) is reduced in almost all cases by aromatase inhibitors, becoming undetectable in many. This contrasts with effects of tamoxifen in which the most common change on PgR is to increase expression. Changes in proliferation occur rapidly following the onset of exposure to aromatase inhibitors. Thus, neoadjuvant studies with letrozole in which tumour was sampled before and after 14 days and 3 months treatment show that decreased expression of Ki67 occur at 14 days and, in many cases, the effect is greater at 14 days than 3 months. These early changes precede evidence of clinical response but do not predict for it. However, this study design has allowed RNA analysis of sequential biopsies taken during the neoadjuvant therapy. Based on clustering techniques, it has been possible to subdivide tumours into groups showing distinct patterns of molecular changes. These changes in tumour gene expression may allow definition of tumour cohorts with differing sensitivity to aromatase inhibitors and permit early recognition of response and resistance.     

DCIS and aromatase inhibitors

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

Aromatase inhibitors: future directions

Estrogen and its cognate estrogen receptor are key players in the etiology and progression of breast cancer. Aromatase inhibitors, suppressing tumor and plasma estrogen levels by blocking testosterone conversion to estrogen, have been proven to provide the most effective endocrine therapy for postmenopausal breast cancer patients. Aromatase inhibitors are now the first choice endocrine therapy in the metastatic setting for postmenopausal women. These endocrine agents also seem likely to soon become the standard adjuvant therapy, either alone or in sequence with tamoxifen, though their long-term toxicity and the optimum duration of therapy still remain to be defined. Advanced experimental studies and some clinical observations reveal the importance of blocking both the genomic and non-genomic activities of the estrogen receptor, as well as its crosstalk with growth factor and other cellular signaling, for greatest effectiveness of endocrine therapy. Consequently, these studies provide a mechanistic explanation for the superb performance of aromatase inhibitors, and also suggest how inhibiting selected growth factor receptors might delay or prevent the onset of resistance to aromatase inhibitors and other endocrine therapies.     

Clinical development of aromatase inhibitors for the treatment of breast and prostate cancer

Numerous aromatase inhibitors are under development for breast cancer treatment. The major aims are to obtain a drug which at its dose of maximum efficacy has no effect on other endocrine systems, has no clinical side-effects and its convenient to administer. During the early clinical stages of development detailed endocrine and pharmacokinetic analyses are a valuable aid in the establishment of a drug's selectivity and its optimum dose, route and frequency of administration. The optimal dose may be defined as the minimum that will achieve maximal and sustained suppression of aromatase activity. This has generally been measured indirectly by comparing the suppression of plasma oestrogen levels at a selection of dosages. This approach has major advantages in speeding dose selection for therapeutic clinical trials. However, it also has some disadvantages including the unproven assumption that clinical response has a direct relationship with the degree of oestrogen suppression. In addition there are technical difficulties of analysis, of wide variability in endocrine response between patients and of demonstrating oestrogen suppression to be equivalent between doses (necessary to show maximal suppression). The direct measurement of aromatase inhibition in vivo by isotopic infusion analysis provides support to these indirect estimates. Its value is shown by our recent results with CGS16949A. The additional value of collating pharmacokinetic and endocrine measurements is apparent from our investigations of 4-hydroxyandrostenedione (4-OHA) and pyridoglutethimide. A consideration of our experience with these inhibitors may be helpful in directing the development of future agents.

Whilst the value of aromatase inhibition in breast cancer is established its value in prostatic cancer is in doubt: we have found that 4-OHA is only poorly efficacious in advanced prostatic cancer.