Recent progress in development of aromatase inhibitors

In postmenopausal women with breast cancer, aromatase, which is the enzyme converting androstenedione to estrone and testosterone to estradiol, is the rate-limiting step in estrogen biosynthesis. The currently available aromatase inhibitor, aminogluethimide, effectively blocks estrogen production and produces tumor regressions in patients previously treated with tamoxifen. This drug, however, produces frequent side effects and blocks steroidogenic steps other than the aromatase enzyme. Thus, newer aromatase inhibitors with greater potency and specificity are under intense study. More than 20 such compounds have recently been developed. In several clinical trials, 4-hydroxyandrostenedione, given parenterally, has been highly active and specific for aromatase inhibition in patients with breast cancer. In two large recent studies, one-third of heavily pretreated women experienced objective tumor regression with this therapy. CGS 16949A, a newer agent, is also Phase II clinical trials. This compound is an imidazole derivative with nearly 1000-fold greater potency than aminoglutethimide. An initial Phase I study compared the potency of 0.6–16 mg daily in 12 postmenopausal women and found maximal suppression of urinary and plasma estrogens with 2 mg daily. The degree of inhibition was similar to that induced by aminoglutethimide or by surgical adrenalectomy. No CNS, hematologic or biochemical toxicity was observed. A larger Phase II study in 54 patients confirmed this high degree of potency of CGS since a plateau effect was observed at the 1.8, 2 and 4 mg daily doses. The endocrine effects were not absolutely specific as a blunting of ACTH-stimulated but not basal aldosterone levels were observed. This and other emerging aromatase inhibitors offer promise as pharmacologic methods to inhibit estrogen production specifically and without side effects.     

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

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

The efficacy of CGS 20267 in suppressing estrogen biosynthesis in patients with advanced stage breast cancer

The pharmacologic inhibition of aromatase activity has been the focus of clinical trials in patients with advanced stage breast cancer. Recent developments with imidazole compounds that inhibit aromatase activity suggest their clinical use as potent inhibitors of estrogen biosynthesis in postmenopausal breast cancer patients. In this Phase I, open-label, dose-range finding study, we examined the inhibitory potency of CGS 20267 on blood and urine levels of estradiol, estrone and estrone sulfate in 8 patients with metastatic breast cancer. Studies included evaluation of adrenal and thyroid function to look for evidence of general hydroxylase inhibition at dose levels effective for aromatase blockade. Patients were administered CGS 20267 at doses of 0.1 and 0.25 mg, once a day in ascending doses over a 12-week period. Preliminary data reveal that CGS 20267 elicits a striking suppression in plasma estradiol, estrone and estrone sulphate which was observed in some patients as quickly as within 24 h of the first dose. Estrogen suppression of over 90% was achieved within 2 weeks of therapy. No alterations in either baseline or ACTH (cortrosyn) stimulated cortisol and aldosterone levels were observed through the 12 weeks of therapy. In addition, 24 h urine sodium and potassium values were not appreciably altered during therapy. We conclude that CGS 20267 is a potent, specific inhibitor of estrogen biosynthesis in postmenopausal patients with metastatic breast cancer and effectively reduces blood and urine estrogens to undetectable levels.     

Effect of postmenopausal hormone replacement therapy on lipoprotein and homocysteine levels in Chinese women.

Epidemiological studies have revealed that postmenopausal estrogen replacement therapy results in a marked reduction in the risk for cardiovascular diseases. In the present study, we evaluated plasma lipoprotein profile as well as homocysteine levels in 145 postmenopausal and premenopausal Chinese women living in Hong Kong. We also investigated the effect of hormone-replacement therapy (HRT) with estrogen or estrogen combined with progestin on plasma lipoprotein profile and homocysteine concentrations in those individuals. Postmenopausal women displayed significantly higher plasma levels of total cholesterol, LDL-cholesterol and apoB as well as higher plasma homocysteine levels than that of premenopausal women. HRT with either estrogen (17beta-estradiol or conjugated equine estrogen) alone or estrogen combined with progestin for 3.5-4.5 years significantly improved the lipoprotein profile in postmenopausal women by decreasing the levels of total cholesterol (12-20% reduction), LDL-cholesterol (26-29% reduction) and apoB (21-25% reduction). In women treated with 17beta-estradiol or conjugated equine estrogens their plasma levels of apoAl were significantly elevated (18% elevation) as compared to non-users. HRT also reduced plasma concentrations of homocysteine (13-15% reduction). In conclusion, we found that long-term HRT was associated with improvement in plasma lipoprotein profile and a reduction in homocysteine concentration in postmenopausal women. These results support the notion that the improvement of lipoprotein profile and a reduction in homocysteine concentration may contribute to the beneficial effect of HRT on cardiovascular risk.     

Second line endocrine treatment of postmenopausal advanced breast cancer. Preliminary endocrine results of a 5-ARM randomized phase II trial of medium vs low dose aminoglutethimide, with or without hydrocortisone vs hydrocortisone alone (EORTC 10861)

The supposed mechanism of action of aminoglutethimide (AG), medical adrenalectomy, has been challenged. AG is now considered to act as an inhibitor of the aromatization of mainly adrenal androgens to estrogens in peripheral tissues and/or breast cancer itself. To further establish the AG dose required to sufficiently reduce estrogen levels in plasma and the possible role of hydrocortisone (HC) in combination with AG or by itself, postmenopausal advanced breast cancer patients received AG low (125 mg bid) or medium (250 mg bid) dose alone or combined with HC (20 mg bid) or HC alone (20 mg bid). Preliminary hormonal data show a similar reduction of serum estrone and estrone sulphate by at least some 50% at 8 wk in all treatment groups. At 6 months these effects persist except for patients treated with HC alone. In the latter a normalization of estrone levels is observed with effective suppression of adrenal androgen precursors, suggesting increased aromatase activity with prolonged glucocorticoid treatment.     

Successful Estrogen Therapy for Post-Adrenalectomy Relapses of Breast Cancer

Four postmenopausal women are described in whom breast cancer responded both to bilateral adrenalectomy and bilateral oophorectomy, and subsequently, after relapse, to estrogen therapy. This paradoxical finding demonstrates the complexity of the response of breast carcinoma to hormone manipulations. Simple estrogen dependence and estrogen suppression of pituitary mammotrophins are seen to be inadequate explanations of this phenomenon. Seven fundamental observations are listed that have to be accounted for by hypotheses concerning the endocrinology of breast cancer. It is suggested that in the past we have perhaps overlooked (1) the difficulty of extrapolating observations on experimental animal tumours to spontaneous human neoplasms, (2) the fact that there may be more than one type of breast cancer, and (3) the important role that must be played by the different tissues bearing the metastases.     

Total body aromatization in postmenopausal breast cancer patients is strongly correlated to plasma leptin levels

The adipocytokine leptin has recently been shown to enhance the expression of aromatase via promoter II and I.3 using an AP-1 motif. Thus, we evaluated the correlation between plasma leptin concentrations and total body aromatization (TBA) as well as plasma levels of estrone (E(1)), estradiol (E(2)) and estrone sulfate (E(1)S) in postmenopausal breast cancer patients. Twenty-two postmenopausal women with metastatic breast cancer, participating in tracer studies for the measurement of total body aromatization (TBA) in vivo, were available. In addition, blood samples for plasma estrogens and leptin measurements were available from another 22 breast cancer patients and 114 healthy postmenopausal women participating in the mammography-screening program. Values for TBA varied from 1.46 to 4.72% while plasma leptin levels ranged from 1.83 to 95.51 ng/ml in the same group of patients. All plasma estrogen levels were in the normal range expected for postmenopausal women. We found a significant correlation between pretreatment leptin levels and TBA (r(s) 0.452, P=0.01). In contrast, basal levels of TBA did not correlate to body mass index (BMI) in the same group of patients. Plasma leptin levels correlated to plasma levels of estradiol (r(s) 0.659, P=0.007), and estrone sulfate (r(s) 0.562, P=0.01) in the group of breast cancer patients (n=44) as well as in the group of healthy postmenopausal women (estradiol, r(s) 0.363, P< or =0.001, estrone sulfate r(s) 0.353, P< or =0.001). In conclusion, we found plasma leptin levels to correlate to TBA in breast cancer patients and to plasma levels of estradiol and estrone sulfate in breast cancer patients as well as in healthy postmenopausal females. These findings suggest that leptin may influence on aromatase activity in vivo, providing a possible link between body weight and plasma estrogen levels as well as breast cancer risk.     

Estradiol and progesterone receptor levels in human breast cancer in relation to cytosol and plasma estrogen level

Estradiol receptor (ER) and progesterone receptor (PR) content along with the cytosol and plasma estrone and estradiol levels in 15 premenopausal and 26 postmenopausal women with breast cancer in different clinical stages (T₁₂₃, N₀₁, M₀) were measured. ER-positive tumor frequency and the ER content tended to be higher in postmenopausal than in premenopausal patients. There was no evidence for a relationship between high cytosol estrogen levels and low receptor measurements. The estrogen concentration was higher in ER-positive tumor cytosols than in those of ER-negative tumors; the differences were significant in postmenopausal women, only, with P < 0.05 for estrone and P < 0.01 for estradiol values. Twelve pairs of tumor and normal tissue from the same breast, were also studied: seven of which contained ER-positive and five ER-negative tumors. The ER-positive tumors showed a clear trend to higher estradiol content as compared to the corresponding normal tissues. The circulating level of estradiol in postmenopausal women, was higher (P < 0.05) in ER-positive tumors than that in ER-negative tumors. Our results indicate that: (a) false negative ER assays are not likely to be due to the presence of endogenous estrogens, (b) higher amounts of estrone and estradiol are contained in ER-positive tumors than in negative ones.     

Estrogens in tissues: uptake from the peripheral circulation or local production

Because intratissue levels of estrogens may be more important for the understanding of the endocrine status of an organ than are peripheral plasma levels, the concentrations of estrone and estradiol have been measured in normal and pathological breast and uterine specimens. Some breast tumors were collected in countries with differences in incidence and natural history of the disease. In other samples the subcellular distribution of the steroids was studied. Estrone levels did show much less variation than estradiol levels. Not related to estrogen receptors, estradiol levels were higher in uterine than in breast tissues. Also, the subcellular distribution observed could not be explained by changes in receptors. Malignant breast tumors of premenopausal and postmenopausal women contained similar amounts of estradiol. Unexplained large differences were found in the intratissue estradiol levels obtained in different countries.     

Plasma estrogen suppression with aromatase inhibitors evaluated by a novel, sensitive assay for estrone sulphate

Aromatase inhibition is a well-defined treatment option for postmenopausal breast cancer. Although several aromatase inhibitors such as aminoglutethimide, formestane, fadrozole have been found to inhibit in vivo aromatization by>85%, previous studies reported plasma estrogen levels to be sustained at approximately 20–50% of their control level during treatment with these drugs. The discrepancy could be due to lack of sensitivity or non-specific crossreactions in the radioimmunoassay (RIA) methods. Mean plasma levels of estrone (E₁) and estradiol (E₂) in postmenopausal women are approximately 80 and 20 pmol/l, respectively; on the contrary, mean plasma levels of the estrogen conjugate estrone sulphate (E₁S) are approximately 4–500 pmol/l. Most RIA methods for plasma E₂ and E₁ measurements have sensitivity limits in the range of 2–3 and 7–10 pmol/l, respectively; accordingly, the suppression of plasma estrogens by more than 80–90% will produce hormone values below the sensitivity limit of the method in many patients. Recently, we developed a new method to determine plasma E₁S. This assay has a sensitivity limit of 2.7 pmol/l. In theory, this method may allow the determination of plasma E₁S levels suppressed to less than 2% of control values in the majority of patients. Using this method, we found different aromatase inhibitors such as formestane, aminoglutethimide, formestane and aminoglutethimide administered in concert or anastrozole to suppress plasma E₁S levels down to 24, 13, 7 and 4%, respectively. The suppression of plasma E₁S evaluated with this method thus approaches the percentage aromatase inhibition measured with tracer studies.     

Plasma estrone sulfate levels in postmenopausal women

Estrone sulfate levels were measured in the plasma of 63 postmenopausal women. The assay method involved prior extraction of the free estrogens, enzyme hydrolysis of the estrone sulfate with sulfatase and radioimmunoassay of the estrone liberated. The plasma levels ranged from 37 to 320 pg/ml (expressed as free estrone) with a mean value of 178 ± 79 pg/ml. As observed in premenopause, estrone sulfate is quantitatively the most important circulating estrogen in postmenopausal women.     

Development of (p-O-sulfamoyl)-N-alkanoyl-phenylalkyl amines as non-steroidal estrone sulfatase inhibitors

Estrogen levels in breast tumors of postmenopausal women are as much as 10 times higher than estrogen levels in plasma, presumably due to in situ formation of estrogen. The major source of estrogen in breast cancer cells may be conversion of estrone sulfate to estrone by the enzyme estrone sulfatase. Thus, inhibitors of estrone sulfatase are potential agents for treatment of estrogen-dependent breast cancer. Several steroidal compounds have been developed that are potent estrone sulfatase inhibitors, most notably estrone-3-O-sulfamate. However, these compounds and their metabolites may have undesired effects, including estrogenicity. To avoid the problems associated with a potentially active steroid nucleus, we designed and synthesized a series of nonsteroidal estrone sulfatase inhibitors, the (p-O-sulfamoyl)-N-alkanoyl phenylalkyl amines. The compounds synthesized vary in the length of their alkanoyl chain and in the number of carbons separating the phenyl ring and the carbonyl carbon. The ability of these compounds to inhibit estrone sulfatase activity was tested using human placental microsomes and intact cultured human breast cancer cells. Estrogenicity was also evaluated, using growth of estrogen-dependent human breast cancer cells. All of the test compounds inhibited estrone sulfatase activity of human placental microsomes to some extent, with the most effective compound having an IC50 value of 72 nM. In general, compounds with longer alkanoyl chains (12-14 carbons) were more effective than those with shorter chains. The test compounds also inhibited estrone sulfatase activity in intact cultures of MDA-MB-231 human breast cancer cells. Again, the longer chain compounds were more effective. In both the placental and breast cancer cell sulfatase assays, the optimal distance between the phenyl ring and the carbonyl carbon was 1-2 carbons. The MCF-7 cell proliferation assay revealed that estrone and estrone-3-O-sulfamate were both estrogenic, but the (p-O-sulfamoyl)-N-alkanoyl phenylalkyl amines were not. Our data indicate the utility of (p-O-sulfamoyl)-N-alkanoyl phenyl alkylamines for inhibition of estrone sulfatase activity. Furthermore, our data support the concept that nonsteroidal estrone sulfatase inhibitors may be useful as therapeutic agents for estrogen-dependent breast cancers.     

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

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

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

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

Postmenopausal estrogen synthesis and metabolism: alterations caused by aromatase inhibitors used for the treatment of breast cancer

Inhibition of postmenopausal estrogen production by aromatase inhibitors is an established drug treatment modality for postmenopausal breast cancer. In this article postmenopausal estrogen disposition and the alterations caused by treatment with aromatase inhibitors are reviewed. Recent investigations have challenged the hypothesis that aromatization of androstenedione into estrone is the sole production pathway for estrogens in postmenopausal women. The finding that estrogens persist in the plasma of patients receiving aminoglutethimide treatment despite a near total inhibition of the aromatase enzyme suggests that alternative pathways for estrogen synthesis exist. While nonspecific actions of aromatase inhibitors may be disadvantageous, certain effects may also be beneficial. Recent findings that aminoglutethimide may induce estrone sulfate metabolism questions whether this "prototype" aromatase inhibitor might have a dual mechanism of action. The importance of investigating the possible influence of different aromatase inhibitors on all components of estrogen disposition is considered.     

The pharmacology of letrozole

Recent clinical trials indicate that the third-generation aromatase inhibitors may be more effective than tamoxifen as first line endocrine therapy in ER+ metastatic breast cancer in postmenopausal women. This review will focus exclusively on the pharmacology of the non-steroidal inhibitor letrozole. Aromatase derived from a variety of sources is inhibited at low nM concentrations of the drug. In non-cellular systems, letrozole is 2-5 times more potent than anastrozole and exemestane in its inhibition of aromatase, whilst in cellular systems it is 10-20 times more potent. Anti-tumour effects of letrozole have been demonstrated in several animal models. In postmenopausal women, letrozole commonly suppresses circulating concentrations of estrone and estradiol to below the sensitivity limit of the assays used to measure them. In a recent randomized cross-over study, letrozole (2.5mg daily) achieved a significantly greater suppression of the plasma concentrations of both estrone and estrone sulphate than anastrozole (1mg daily) and a greater inhibition of in vivo aromatization. Letrozole appears to have a small effect on adrenal steroidogenesis such that a small number of patients exhibit an abnormal response to synthetic ACTH during letrozole therapy. This is unlikely to have any clinical significance. In short-term studies letrozole has been shown to increase markers of bone resorption indicating the need to monitor bone integrity when the drug is used for extended periods of time. A consistent effect of letrozole on serum lipids has not been demonstrated.     

Sister-chromatid exchange frequencies in postmenopausal hormone replacement patients

Our objective was to evaluate the frequency of sister-chromatid exchange (SCE) during hormone replacement therapy in postmenopausal women. Thirty-four asymptomatic postmenopausal women with a minimum 12 months since last menstrual period and surgical menopausal women were included in the study. Seventeen patients who were in spontaneous menopause were administered conjugated estrogen and medroxyprogesterone acetate (group A), and the others who were in surgical menopause were given 17beta-estradiol only (group B). Peripheral lymphocytes were obtained at the beginning and at the end of the third month of therapy. The mean age of the patients was 50. 67+/-4.79. There were statistically significant differences in terms of SCE frequencies between pre- and posttreatment levels of both groups (p<0.001 and p=0.003, respectively). It is likely that estrogens with or without progesterone have an effect in increased SCE frequency and this issue may be an evidence for the increased potential for malignancies.     

Recent data on intratumor estrogens in breast cancer

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

Radioimmunoassay of 16 alpha-hydroxyestrone in human urine

A radioimmunoassay for the quantitation of the sum of free, glucuronidated and urine is described. The method is reliable and accurate. Using this method, urinary excretion of 16 alpha-hydroxyestrone was determined in normal men, premenopausal women, and postmenopausal women. The values were compared to the urinary excretion of estrone and estradiol. In two women, the urinary excretion of the three estrogens was measured in daily samples throughout a normal menstrual cycle. We conclude that 16 alpha-hydroxyestrone is a quantitatively important urinary estrogen. Inclusion of the measurement of 16 alpha-hydroxyestrone should yield a more accurate assessment of estrogen metabolism.     

Steroidal oxathiazine inhibitors of estrone sulfatase

The presence of estrone sulfatase in breast tumors and the high levels of circulating estrone sulfate may contribute the major portion of estrogen synthesized locally in breast tissues through conversion of estrone sulfate to estrone by the enzyme. Using inhibitors of estrone sulfatase for the treatment of estrogen-dependent (estrogen receptor positive, ER(+)) breast cancer could be a very effective therapeutic strategy for the treatment of estrogen-dependent breast tumors in postmenopausal women. Therefore, we designed and synthesized several steroidal 2',3'-oxathiazines that inhibit estrone sulfatase and have greatly reduced estrogenic side effects. Our in vitro studies indicate that the oxathiazine compounds have inhibitory activity on estrone sulfatase in MCF-7 human breast cancer cells. These estrone sulfatase inhibitors (ESIs) also inhibit the growth of MCF-7 cells induced by estrone sulfate. In addition, our in vivo experiments demonstrate that our ESIs have moderate antitumor activity against MCF-7 breast cancer xenografts in Balb/c athymic nude mice. The synthesis and biological activity of a number of these unique steroidal ESIs are described.