Michael J. Reed

DIFFERENCES IN CLINICAL AND ENDOCRINE FEATURES BETWEEN OBESE AND NON‐OBESE SUBJECTS WITH POLYCYSTIC OVARY SYNDROME: AN ANALYSIS OF 263 CONSECUTIVE CASES

Two hundred and sixty‐three women with ultrasound‐diagnosed polycystic ovary syndrome were studied of whom 91 (35%) were obese (BMI > 25 kg/m2‐). Obese women with PCOS had a greater prevalence of hirsutism (73% compared with 56%) and menstrual disorders than non‐obese subjects. Total testosterone and androstenedione concentrations in serum were similar in the two subgroups but SHBG concentrations were significantly lower, and free testosterone levels higher, in obese compared with lean subjects. In addition, concentrations of androsterone glucuronide, a marker of peripheral 5α‐reductase activity, were higher in obese than in non‐obese women with PCOS. There were no significant correlations of either SHBG or free testosterone with androsterone glucuronide suggesting that obesity has independent effects on transport and on metabolism of androgen. There were no significant differences between the subgroups in either baseline gonadotrophin concentrations or the pulsatile pattern of LH and FSH secretion studied over an 8‐h period. There was, however, an inverse correlation of FSH with BMI, but only in the obese subgroup. In conclusion, the increased frequency of hirsutism in obese compared with lean women with PCOS is associated with increased bio‐availability of androgens to peripheral tissues and enhanced activity of 5α‐reductase in obese subjects. The mechanism underlying the higher prevalence of anovulation in obese women remains unexplained.

Aromatase activity in normal breast and breast tumor tissues: In vivo and in vitro studies

Infusions of isotopically labeled [3H] androstenedione with measurement of [3H] estrone in normal breast and breast tumor tissue have been carried out in an attempt to determine the contribution that aromatization makes to the estrogen content of breast tissues. After infusion of [3H] androstenedione for 12h there was significant uptake of this steroid by normal breast and breast tumors. [3H] Estrone was detected in all samples of normal breast tissue examined so far but not in all tumors. Aromatase activity when measured in vitro was found to be higher in breast tumors than in fat next to the tumor or normal breast fat. Studies in which we have examined the effect of epidermal growth factor on aromatase activity in cultured breast adipose tissue suggests that the response may be influenced by a subjects menopausal status. Results from these preliminary studies suggest that the aromatization of androgens may make a significant contribution towards the estrogen content of some breast tumors and that growth factors may also be involved in regulating aromatase activity.

The role of cytokines in regulating estrogen synthesis: implications for the etiology of breast cancer

Cytokines, such as IL-6 and tumor necrosis factor (TNF)-α, have an important role in regulating estrogen synthesis in peripheral tissues, including normal and malignant breast tissues. The activities of the aromatase, estradiol 17β-hydroxysteroid dehydrogenase and estrone sulfatase are all increased by IL-6 and TNF-α. Prostaglandin E2 may also be an important regulator of aromatase activity in breast tumors. Macrophages and lymphocytes, which invade many breast tumors, are thought to be an important source of factors that can stimulate estrogen synthesis in malignant breast tissues. The co-ordinated stimulation of the activities of the enzymes that are involved in estrogen synthesis offers an explanation for the high concentrations of estrogens that are present in breast tumors.

Phase I Study of STX 64 (667 Coumate) in Breast Cancer Patients: The First Study of a Steroid Sulfatase Inhibitor

Purpose: Inhibition of steroid sulfatase (STS), the enzyme responsible for the hydrolysis of steroid sulfates, represents a potential novel treatment for postmenopausal women with hormone-dependent breast cancer. Estrone and DHEA are formed by this sulfatase pathway and can be converted to steroids (estradiol and androstenediol, respectively), which have potent estrogenic properties. Experimental Design: STX64 (667 Coumate), a tricylic coumarin-based sulfamate that irreversibly inhibits STS activity, was selected for entry into the first phase I trial of a STS inhibitor in postmenopausal women with breast cancer. STX64 was administered orally (nine patients at 5 mg and five patients at 20 mg) as an initial dose followed 1 week later by 3 × 2 weekly cycles, with each cycle comprising daily dosing for 5 days followed by 9 days off treatment. Blood and tumor tissue samples were collected for the assessment of STS activity and serum was obtained for steroid hormone measurements before and after treatment. Results: The median inhibition of STS activity by STX64 was 98% in peripheral blood lymphocytes (PBL) and 99% in breast tumor tissue at the end of the 5-day dosing period. As expected, serum concentrations of estrone, estradiol, androstenediol, and DHEA all decreased significantly from pretreatment levels. Unexpectedly, androstenedione and testosterone concentrations also decreased. Four patients, all of whom had previously progressed on aromatase inhibitors, showed evidence of stable disease for 2.75 to 7 months. The drug was well tolerated with only minor drug-related adverse events recorded. Conclusion: STX64 is a potent, well-tolerated STS inhibitor. It inhibits STS activity in PBLs and tumor tissues and causes significant decreases in serum concentrations of steroids with estrogenic properties.

THE RELATIONSHIP BETWEEN 17β‐HYDROXYSTEROID DEHYDROGENASE ACTIVITY AND OESTROGEN CONCENTRATIONS IN HUMAN BREAST TUMOURS AND IN NORMAL BREAST TISSUE

The activity of 17β‐hydroxysteroid dehydrogenase (17βHSD) was measured in human breast tumours and in normal breast tissue from premenopausal, perimenopausal and postmenopausal women. Enzyme activity was higher in tumour tissue than in normal tissue from the same breast and under the conditions of the assay the oxidation of oestradiol was higher than the reduction of oestrone. The physiological status of the women in the study did not relate to the activity of the enzyme in either normal or tumour tissue although fibroadenomas had less activity than adenocarcinomas.

Diet-induced changes in sex hormone binding globulin and free testosterone in women with normal or polycystic ovaries: correlation with serum insulin and insulin-like growth factor-I.

The purpose of this study was to investigate the effect of calorie restriction on serum concentrations of sex hormone binding globulin (SHBG) in women with normal or polycystic ovaries (PCO) and to examine the possible role of insulin and insulin‐like growth factor‐I (IGF‐I) in mediating changes in SHBG levels. Six normal subjects with mean (SD) body mass index (BMI) 25.5 (2.2) and five subjects with PCO (BMI 36.1 (3.7)) were studied before and after 2 or (PCO only) 4 weeks of a very low calorie diet (330 kcal/day; Cambridge Diet). In both normal women and patients with PCO there was a twofold increase in SHBG concentrations after 2 weeks and this was sustained in the PCO subjects for a further 2 weeks. The rise in SHBG was accompanied by a fall in free testosterone concentrations. There were parallel changes in serum insulin and IGF‐I concentrations which decreased during the diet and there were significant negative correlations of SHBG with insulin in both normal subjects (r =−0.62) and women with PCO (r =−0.60). In addition, serum concentrations of an insulin‐dependent small molecular weight (34 kDa) binding protein for IGF‐I (IGF‐BPI) increased significantly during dieting in both groups and were negatively correlated with serum insulin (controls, r =−0.56; PCO, r =−0.68) and positively correlated with serum SHBG levels (controls, r = 0.69; PCO, r = 0.63).

Potent active site-directed inhibition of steroid sulphatase by tricyclic coumarin-based sulphamates

BACKGROUND There is now abundant evidence that inhibition of steroid sulphatase alone or in conjunction with inhibition of aromatase may enhance the response of postmenopausal patients with hormone-dependent breast cancer to this type of endocrine therapy. Additionally, sulphatase inhibition has been proposed to be of potential therapeutic benefit in the immune system and for neuro-degenerative diseases. After the finding that our first highly potent active site-directed steroid sulphatase inhibitor, oestrone-3-O-sulphamate (EMATE), was highly oestrogenic, we proposed non-steroidal coumarin sulphamates such as 4-methylcoumarin-7-O-sulphamate (COUMATE) as alternative non-steroidal steroid sulphatase inhibitors. In this work, we describe how tricyclic coumarin-based sulphamates have been developed which are even more potent than COUMATE, are non-oestrogenic and orally active. We also discuss potential mechanisms of action. RESULTS 4-Ethyl- (4), 4-(n-propyl)- (6), 3-ethyl-4-methyl- (8), 4-methyl-3-(n-propyl)coumarin-7-O-sulphamate (11); the tricyclic derivatives 665COUMATE (13), 666COUMATE (15), 667COUMATE (17), 668COUMATE (20) and the tricyclic oxepin sulphamate (22) were synthesised. In a placental microsome preparation, all of these analogues were found to be more active than COUMATE in the inhibition of oestrone sulphatase, with the most potent inhibitor being 667COUMATE which has an IC(50) of 8 nM, some 3-fold lower than that for EMATE (25 nM). In addition, 667COUMATE was also found to inhibit DHEA-sulphatase some 25-fold more potently than EMATE in a placental microsome preparation. Like EMATE, 667COUMATE acts in a time- and concentration-dependent manner, suggesting that it is an active site-directed inhibitor. However, in contrast to EMATE, 667COUMATE has the important advantage of not being oestrogenic. In addition, we propose several diverse mechanisms of action for this active site-directed steroid sulphatase inhibitor in the light of recent publications on the crystal structures of human arylsulphatases A and B and the catalytic site topology for the hydrolysis of a sulphate ester. CONCLUSIONS A highly potent non-steroidal, non-oestrogenic and irreversible steroid sulphatase inhibitor has been developed. Several mechanisms of action for an active site-directed steroid sulphatase inhibitor are proposed. With 667COUMATE now in pre-clinical development for clinical trial, this should allow the biological and/or clinical significance of steroid sulphatase inhibitors in the treatment of postmenopausal women with hormone-dependent breast cancer and other therapeutic indications to be fully evaluated.

Control of aromatase activity in breast cancer cells: The role of cytokines and growth factors

The aromatase complex has a key role in regulating oestrogen formation in normal and malignant breast tissues. Using dexamethasone-treated fibroblasts, derived from breast tumours, breast tumour cytosol and breast tumour-derived conditioned medium (CM) markedly stimulate aromatase activity. The cytokine, interleukin-6 (IL-6) has been identified as a factor present in CM which is capable of stimulating aromatase activity. To examine whether IL-6 may have a role in vivo in regulating breast tissue aromatase activity, IL-6 production and aromatase activity in breast tumour and adipose tissue from breast quadrants were examined. In 5/6 breasts examined so far, aromatase activity was highest in adipose tissue in the breast quadrant containing the tumour or on which the tumour impinged. There was a significant correlation (P < 0.05, Kendalls rank correlation) between IL-6 production and aromatase activity in these breast tissues. It is concluded that IL-6 may have an important role in regulating aromatase activity in breast tissues.

Stimulation of aromatase activity in breast fibroblasts by tumor necrosis factor

The conversion of androstenedione to estrone, the reaction mediated by the aromatase enzyme complex, may make an important contribution to the synthesis of estrogens in breast tissues. In the present study, the effect of the cytokine. TNF alpha, on aromatase activity was examined in breast fibroblasts derived from normal and malignant breast tissue. TNF alpha (2.5-10.0 ng/ml), in the presence of stripped fetal calf serum and dexamethasone, significantly stimulated fibroblast aromatase activity in a dose-dependent manner. IL-1 and IL-6 also stimulated fibroblast aromatase activity, but no marked synergism between TNF alpha and IL-1 or IL-6 was detected. Using a specific radioimmunoassay, significant concentrations of TNF alpha were detected in samples of breast cyst fluid and breast tumor cytosol, which had previously been shown to stimulate aromatase activity, but not in conditioned medium from breast tumor-derived fibroblasts. As TNF alpha may be preferentially expressed and produced in the adipose tissue component of the breast, this cytokine may have an important role in regulating estrogen synthesis in normal and malignant breast tissues.

Regulation of estrogen synthesis in postmenopausal women

The decrease in ovarian estrogen production that occurs at the menopause may lead to an increase in peripheral aromatase activity. While estrogens can have beneficial effects on some body tissues, such as bone and the cardiovascular system, they also have a crucial role in supporting the growth and development of breast tumors. A number of factors, including interleukin-6 (IL-6), tumor necrosis factor alpha (TNFalpha), and prostaglandin E(2) (PGE(2)), which can stimulate aromatase activity, have now been identified. As plasma concentrations of some cytokines increase at the menopause, this may account for the increased peripheral aromatase activity that is detected in older women. Macrophages and lymphocytes which infiltrate breast tissue are now thought to be an important source of cytokines that can stimulate aromatase activity in this tissue. Studies, we have recently carried out, have suggested that the endogenous estrogen metabolite, 2-methoxy-estradiol, may be able to modulate the ability of cytokines and PGE(2) to stimulate aromatase activity. Understanding the role of endogenous estrogen metabolites in regulating estrogen synthesis may give rise to new strategies for the prevention or treatment of breast cancer.