Aust NZ J Ohstet Gynnecol 1999; 39: 3: 334-340

Occasional Review

Modern Antioestrogens and the Coming Revolution Women’s Health Care

in

Amanda Evans’ MRANZCOG, FRACGP, Beverley Vollenhoven’ PhD, FRANZCOG, CREI and

Monash University Department of Obstetrics and Gynaecology, Monush Medical Centre, Victoria David Healy’ PhD, FRANZCOG, CREI

Summary: This review will focus on antioestrogens and selective oestrogen receptor modulators (SERMS). The more traditional SERMS, clomiphene citrate and tamoxifen, will be reviewed along with such modern drugs as raloxifene and faslodex, with emphasis upon their actions on breast, uterus, bone and lipids. The future potential of these medications, in the management of oestrogen-dependent gynaecological conditions such as endometriosis, dysfunctional uterine bleeding, fibroids and breast cancer will be discussed.

New and recent knowledge of oestrogen receptors (ER) is opening up treatment possibilities for con- ditions such as cancer of the breast, uterine fibroids, endometriosis, dysfunctional uterine bleeding, osteoporosis, and postmenopausal cardiovascular disease. The current modalities of treatment for conditions such as endometriosis and fibroids have included medical treatments, such as danazol, and gonadotrophin hormone-releasing hormone agonists. The treatment of postmenopausal women with hor- mone replacement therapy has side-effects including breast problems and the need for endometrial protection with progestogens which may themselves cause unwanted side-effects. The solution to these imperfect treatments lies in better understanding of the oestrogen receptor and its molecular and cellular biology. This should permit developing for the first time more selective treatments which target the tissue concerned without adversely affecting other oestrogen receptive tissues and organ systems.

This review will discuss the current state of knowledge of ER and the currently available medi- cations such as clomiphene citrate (CC), tamoxifen and raloxifene, as well as new products undergoing phase 2 and 3 clinical research trials, such as faslodex. We will also briefly review phytooestrogens, and their clinical applications.

I . Registrar. 2. Senior Lecturer. 3 . Professor and Chairman Address for correspondence: Professor David Healy, Monash Medical Centre. Department of Obstetrics and Gynaecology. 246 Clayton Road, Clayton 3 168, Victoria.

OESTROGEN RECEPTOR In order to better understand the mechanism of

action of these modern antioestrogens, it is important that we review what is currently known about the ER. Recent discoveries related to the structure of this receptor have shed new light on the way selective oestrogen receptor modulators (SERMS) can have different effects in various tissues and organ systems. A schematic diagram of the (ER) is shown in figure 1 (courtesy of Dr Peter Fuller, Prince Henry’s Institute of Medical Research at the Monash Medical Centre). Upon binding of agonist, in this case oestradiol, (indicated in figure 1 by the dark black square) the ER changes its conformation in the area called the ligand binding domain, enabling recruitment of coactivators (AF-I and AF-2). This allows the ER to interact with the DNA more effectively, and to activate transcription. The mechanism of action of pure antioestrogens such as ICI 164,384 (no generic name, preclinical trials only), appears to be via an increase in receptor turnover or the occupation of the ER ligand binding domain by a nonfunctional ligand, (in this case ICI 164,384 indicated in figure 1 by the dark T) rendering the receptor incapable of dimerization (1). In the case of a mixed agonisUantagonist, such as 4-hydroxytamoxifen (a SERM), activation of oestrogen dependent gene transcription may depend on the relative ratio of coactivators and corepressors within the cell, that deteimines the relative agonistic or antagonist potential of different compounds. These coactivators and corepressors appear to act as accelerators or brakes that modulate transcriptional regulation of hormone responsive target gene expression (2) (figure 2). In figure 1 tamoxifen can be seen to be interacting with DNA but less effectively since the coactivator AF-2 has not been able to bind to the ligand binding domain due to the altered shape of the dimerized ER. Thus, the

AMANDA EVANS ET A L 335

oestradiol

oestradiol

AF-1 TS2 ,/

tamoxifen ICI 164384

DNA

Figure 1 . Aschematic diagram of oestrogen receptors (ER) and its interaction with the ligands oestradiol, tamoxifen, and ICI 164384. (Courtesy of Dr. Peter Fuller, Prince Henry’s Institute of Medical Research.)

recent discovery of coactivators and corepressors expands our knowledge of the mechanisims of steroid receptor action.

Further to the above, work by Korach (3) and others has discovered 2 ER subtypes, namely ER-a and ER- b. Mice with their ER genetically eliminated or knocked out have been studied. These mice have been cloned and throughout embryogenesis and fetal life and beyond to sexual maturity, they have never ‘seen’ an ER. The gene for ER-a was eliminated from the first cloned group, and the mice observed as they reached maturity. The female mice appeared to develop a condition that could be compared to polycystic ovary syndrome (PCOS) in women. They had multiple cysts on their ovaries, relative infertility, but could be hyperstimulated to reproduce, and had a hormone profile similar to PCOS ie. raised lute- nizing hormone (LH) and dihydroepiandrosterone sulphate (DHEAS).

Corepressor Mixed AgonisVAntagonist Coactivator

Corepressor t 4

inactive Rc

Coactivator t 4

active Rc

Figure 2. A simplified model of the mechanism of action of corepressorq and coactivators. (Adaptation reference 2.)

A second group of mice are now being studied where the gene for ER-b has been eliminated. Although these mice have not been fully studied as yet it appears that they develop an ovarian dysgenesis- type syndrome. It is becoming apparent that both types of ER are necessary for normal function in both males and females, and that ER-a and ER-b appear to have quite distinct but interrelated functions.

Thus, the discovery of corepressors and coacti- vators, ER subtypes, and a repertoire of accessory DNA-bound proteins, lends further to our under- standing of how the ER is influenced by SERMS. It seems that in different tissues there will be different concentrations of these various components that allow SERMS to have varying agonistic and antagonistic actions. The goal will be to find the ideal SERM that lowers the risk of breast and endometrial cancer, yet has a positive effect on bone, the cardiovascular system, hepatic function and still alleviates the symptoms of menopause such as hot flushes, dry vagina and mood change.

CLOMIPHENE CITRATE Clomiphene citrate is a triphenylethylene derivative

with antioestrogenic effects. The structure of CC is indicated in figure 3, along with the structures of other antioestrogens and SERMS. Clomiphene citrate is an orally active nonsteroidal agent distantly related to diethylstilboestrol. It is a racemic mixture of 2 stereochemical isomers, originally described as cis and trans. This designation is now recognized to have been inaccurate and the isomers have been relabelled as zu and enclomiphene (4). The individual isomers of CC have been reported to have similar effects on bone metabolism, while the uterine effects are primarily induced by zuclomiphene (5).

336 AUST AND NZ JOURNAL OF OBSTETRICS AND GYNAECOLOCY

Clomiphene citrate

C2H5

tamoxifen

raloxifene hydrochloride ICI 182,780

Figure 3. Structures of clorniphene citrate, tamoxifen, raloxifene hydrochloride and faslodex

The similarity of structure to an oestrogenic substance is a clue to its mechanism of action. CC exerts only a very weak biological oestrogenic effect. It is taken up by the ER and occupies it for weeks rather than hours. It modifies hypothalamic activity by lowering the concentration of intracellular ER. Like other oestrogen antagonists, the agonist-antagonist profile of CC varies depending upon the endocrine

One ReceptorlTwo Pathways

/ \ 17-epiestriol 4 Oestrogen or raloxifene

/ \

Oestrogen response Raloxifene response element element

Figure 4. A schematic diagram of the interaction of raloxifene plus adaptor protein with oestrogen receptor, compared with oestradiol. (Adaptation, Science 1996. 273: 1171)

milieu. In typical clinical use for initiation of ovulation in the presence of significant levels of endogenous oestrogen, CC is primarily an oestrogen antagonist (6). But other actions include direct antioestrogen effects upon the endometrium and cervical mucus, as well as antagonizing oestrogen activity in mammary tissue. Caution needs to be exercised in the use of CC in the treatment of anovulatory infertility. Rossing and colleagues (7) identified 11 invasive or borderline ovarian tumours in a cohort of CC-users attending infertility clinics. The adjusted relative tumour risk amongst women who received CC compared with other infertile women was 2.3 (95% CI 0.5-1 1.4). A significant risk was seen only for the 5 women who had taken CC for greater than 12 months, Although no conclusive evidence is available, especially in women with PCOS, women receiving CC need to be carefully monitored, and not merely given a prescription for 6-12 months supply of tablets.

TAMOXIFEN Tamoxifen and its more potent metabolite 4-

hydroxytamoxifen, a triphenylethyene derivitive (figure 3 for structure) was first used in the early 1970’s for the treatment of advanced breast cancer in postmenopausal women. It is still widely employed for this purpose and has since become the standard adjuvant treatment. Most of the 6 million patient- years of exposure with tamoxifen is a result of its use to treat breast cancer. Recently however there has been interest in using tamoxifen as a breast cancer preventitive agent and perhaps as an HRT substi- tute in women with a family history of breast cancer. These potential uses are envisioned as a direct result of tamoxifen’s emerging SERM profile.

AMANDA EVANS ET AL 337

Tamoxifen has also been used for ovulation induction since 1971. It produces a 60% ovulation rate and conception rates of 35-40%. It may have added benefits over CC for women who have PCOS, or corpus luteal defect, and abnormal cervical mucus, due to its oestrogen-like action on the endometrium and cervical mucus. The dose is 10-20 mg twice daily on cycle days 3 to 7 and doses are increased in 10-20 mg increments to a maximum dose of 80 mg per day (personal communication, Dr James Evans, FRANZCOG, Royal Womens Hospital).

Tamoxifen exhibits an antioestrogen activity on the breast in postmenopausal women, but enhances endometrial cell proliferation, In menstruating women, tamoxifen exhibits an antioestrogen effect on the neuroendocrine axis, which leads to elevated oestradiol concentrations ( 1 ). Tamoxifen has a bone-preserving effect in postmenopausal women (6) but additional studies are required to determine if tamoxifen will protect against the rapid bone loss that occurs just after the time of the menopause. In contrast to this action in post-menopausal women, tamoxifen seems to cause a decrease in bone mineral density (BMD) in premenopausal women (8). Tamoxifen thus appears to have oestrogen like properties in bone when used in the presence of very low circulating oestradiol concentrations, but antagonistic effects when circulating oestradiol concentrations are high. Tamoxifen also has oestrogen like effects on serum lipids and hence a potential for cardioprotection.

Although available data are limited, tamoxifen appears to increase the risk of venous thrombo- embolism (VTE) in postmenopausal women to an extent at least as great as has been recently documented for HRT (relative risk of idiopathic venous thromboembolism approximately 3-5 versus placebo) (8). The mechanism may be by decreasing antithrombin -3 concentrations (9). Thus the action of tamoxifen on the ER appears dependent on the prevai 1 ing oestrogen milieu.

Tamoxifen was initially used in the palliative treatment of advanced breast cancer in postmenopausal women. Numerous randomized trials (9) now show that adjuvant treatment with tamoxifen improves relapse-free and overall survival for early node negative and positive breast cancer. These trials also demonstrate a decrease in contralateral breast cancer among women receiving tamoxifen compared with controls, and this has led to clinical trials evaluating tamoxifen as a preventitive agent (9). The limiting factors to the use of tamoxifen are increased VTE, endometrial hyperplasia and promotion of preexisting endometrial carcinoma. More importantly is the development of drug resistance. Although theory dictates that recurrence results from the development of ER negative metastases, there are currently alternative explanations for the failure of tamoxifen.

The oestrogen-like properties of tamoxifen, appear to encourage the growth of ER-positive tumours that will grow with either tamoxifen or oestradiol. The mechanism may be an ER mutation. This tamoxifen- stimulated growth was predicted to occur after laboratory experiments, and may occur after 5 years of clinical use (9).

An analysis of the meta-analysis carried out by McLennan, 1998 (10) indicates that treatment with tamoxifen for women with breast cancer leads to a 10% reduction in recurrence in ER-negative tumours, and a 23% reduction in recurrence in ER-negative and progesterone receptor-positive tumours. There is no benefit for tumours which are both ER and progesterone receptor negative. For ER-positive tumours there is a 50% reduction in recurrence and 28% decreased mortality. The maximum benefit is seen after 5 years of treatment. The addition of tamoxifen to chemotherapy results in a significant improvement in recurrence risk and mortality risk. Of greater interest is the possibility of tamoxifen as a preventative agent in breast cancer. There has been some data on this recently, but the literature is mixed in its results. After 5 years of tamoxifen treatment there is a 47% reduction in risk for cancer of the contralateral breast. The potential of tamoxifen as an HRT alternative in women with contraindications to oestrogen therapy needs to be further studied for its cardiovascular and bone protective effects.

The lessons learnt with tamoxifen have led to further investigations on other versions of SERMS. Chemical manipulation of tamoxifen resulted in more triphenylethyenes eg; droloxifene and idoxifene. However, it is the benzothiopene raloxifene that has undergone the most research. Raloxifene has been submitted to the relevant Australian authorities for release.

RALOXIFENE The structure of raloxifene can be seen in figure 3. It

was originally developed as a treatment for metastatic breast cancer, but in phase 2 clinical trials it proved to be ineffective against these metastatic tumour cells. The increased risk of breast cancer shown with oestrogen replacement therapy in some studies (1 1) has led to the need for alternative therapies for post- menopausal pathologies such as coronary artery disease and osteoporosis. Hence raloxifene was developed further because of its oestrogen-like action on bone and lipid metabolism which was not associated with endometrial proliferation (1 1).

The mechanism of action of raloxifene is not completely understood and remains controversial. One theory of its action involves the raloxifene-response element (figure 4) (12). Raloxifene is thought to produce its effects by binding to the ER and activating a unique DNA response element termed the raloxifene-

338 AUST A N D NZ JOURNAL OF OBSTETRICS AND GYNAECOLOCY

response element. The presence of an adaptor protein, as yet unidentified, is necessary for raloxifene’s action. The observation that individual oestrogens modulate multiple DNA response elements may explain the tissue selective oestrogen agonist or antagonistic activity of compounds such as raloxifene. Raloxifene is currently marketed under the name of Evista, is supplied in 60 mg tablet form, and is absorbed rapidly after oral administration. The recommended daily dose is 60 mg at any time of day without regard to meals. Raloxifene reduces resorption of bone and decreases overall bone turnover, hence it is indicated for postmenopausal osteoporotic women ( 1 3).

Delmas and colleagues (14) studied 601 post- menopausal women randomly assigned to receive 30, 60, and 150 mg of raloxifene or placebo for 24 months. They studied bone mineral density of the lumbar spine and hip, serum lipid concentrations and endometrial thickness. Endometrial thickness was similar in the raloxifene and placebo groups, as was reporting of hot flushes and vaginal bleeding. Serum concentrations of cholesterol and low density lipoproteins decreased in the raloxifene group, however high density lipoproteins and triglycerides did not change. In those receiving raloxifene there was an increase in bone density of 2.4%, those on placebo decreased their bone density by 0.8% and this was statistically significant.

Interim results of the MORE study (15), a double blind placebo controlled clinical trial to evaluate the effect of raloxifene on the risk of vertebral fracture, in which 7,705 osteoporotic women have been studied for 24 months, showed a reduction in the rate of vertebral fracture in the raloxifene group by one half. The trial continues to assess the effects at 36 months. The MORE study indicates that raloxifene markedly reduced the risk of newly diagnosed breast cancer and might decrease the risk of newly diagnosed endometrial cancer during the 2 years of use by osteoporotic women with no history of breast or endometrial cancer. Breast and endometrial cancer cases were reported from a safety database. After a median of 28.9 months follow-up, 32 cases of breast cancer had been confirmed: 11 (0.2 I %) of the women assigned to raloxifene and 21 (0.82%) of the women assigned to placebo, this result remains significant when adjusted for interim monitoring. Compared with the rate in the placebo group, the overall relative risk of endometrial cancer is 0.38 (16).

In addition, raloxifene significantly reduced the incidence of both newly diagnosed ER-positive breast tumours and progesterone receptor-positive tumours, without affecting the incidence rate of receptor-negative tumours, consistent with raloxifene’s established activity in hormone responsive breast cancer. Further analyses of combined placebo controlled studies in this cohort of postmenopausal women with no

history of breast cancer, provide overwhelming evidence that for patients assigned to raloxifene, there is a profound reduction in the risk of developing carcinoma of the breast (17).

Walsh and colleagues (18) studied the effects of raloxifene on markers of cardiovascular risk in post- menopausal women, and compared them with those induced by hormone replacement therapy. They studied 390 women for 3 and 6 months, randomized to receive raloxifene 60 or 120 mg, or HRT (conjugated equine oestrogens 0.625 mg, medroxyprogesterone acetate 2.5 mg) or placebo. Raloxifene lowered low density lipids (LDL) by 12% compared with HRT 14%; raloxifene raised HDL by 17% compared with HRT 33%. Raloxifene favourably alters biochemical markers of cardiovascular risk by decreasing LDL-c, fibrinogen and lipoprotein-a, and by increasing high density lipids (HDL)2-C without raising triglycerides. In contrast to HRT, raloxifene had no effect on HDL- C and PAIL1 (plasminogen activator inhibitor-1), and had a lesser effect on HDL2-C and lipoprotein a. Further clinical studies are needed to determine whether these favourable biochemical effects seen with raloxifene are associated with protection against cardiovascular disease.

FASLODEX Faslodex is an example of a pure antioestrogen

(figure 3). Oestrogen deprivation is fundamental to the treatment of many benign and malignant diseases of the breast and reproductive tract. In premenopausal women this is achieved by the ablation of ovarian function through surgical, radiotherapeutic or medical means, and in postmenopausal women by the use of aromatase inhibitors. An alternative approach to oestrogen withdrawal is antioestrogens, and these are being investigated for the treatment of breast cancer and other benign oestrogen-dependent gynaecological disease such as endometriosis, uterine fibroids and dysfunctional uterine bleeding. The potential of faslodex (ICI 182,780) as a useful agent to the gynaecologist is heightened by the evidence that, unlike other oestrogen manipulating drugs, the antioestrogen effects of faslodex are achieved at doses that do not adversely affect bone mineral density. An agent that is bone sparing while also inducing profound hypo- oestrogenism would therefore represent a major advance in this treatment area.

Currently a long acting intramuscular formulation of faslodex is available for investigational purposes. A randomized, multicentre placebo-controlled dose ranging trial comparing faslodex with Zoladex in patients with uterine fibroids awaiting hysterectomy was recently completed. The objectives were to determine which of the 3 intramuscular doses 50, 125, or 250 mg, when given over a 3-month period was the most effective at inhibiting endometrial growth and

AMANDA EVANS E I AL 339

shrinking fibroids, when compared with placebo and which caused less bone resorption measured using biochemical markers, when compared with Zoladex. The results of this trial are awaited.

Fuchs-Young and colleagues (19) used a panel of cell lines derived from spontaneous Eker rat leiomyomas and examined the oestrogen-responsive phenotype of these tumour cells. Leiomyoma-derived ELT cell lines proliferated in response to oestrogen, and oestrogen-induced cell proliferation could be inhibited by faslodex as well as the SERMS raloxifene and tamoxifen. In addition to inhibiting cell growth these antagonists also inhibited oestrogen-induced increases in progesterone-receptor expression. These data indicate that these compounds may be effective for the treatment of fibroids.

In 1991 DeFriend et a1 (20) reported the results from 56 postmenopausal patients with primary breast cancer, who were randomized into control or faslodex groups. After 7 days the tumours were resected and tissue samples evaluated. Tumours treated with faslodex had decreased expression of ER and progesterone receptors. Cellular proliferation was also reduced. The drug was well tolerated and there were no untoward side-effects over this short period of time. This study demonstrates that faslodex has the ability to inhibit tumour growth in vivo. Similar studies in tamoxifen-resistant breast cancer showed a 69% reduction in tumour size over 25 months with the use of faslodex (21). The duration of remission was also longer for cancers treated with the ICl compound compared with megestrol acetate, 26 months compared to 14 months (22). These results demon- strate the usefulness of antioestrogens as second line treatment for tamoxifen-resistant breast cancer. Other antioestrogens such as RU 58,668 and EM- 139 are currently being developed.

PHYTOOESTROGENS Phytooestrogens are naturally occurring com-

pounds which have varying agonistic and antago- nistic effects on oestrogen responsive tissues. They therefore can be classified as SERMS. There has been limited research on these compounds, and the numbers of subjects in the trials have been small. The most supportive data are related to effects of soy protein supplements on lipids and lipoproteins and on vascular function (24). Cancer data is still in its infancy. Japanese women have a low rate of breast cancer, and Hirayama (23) reported a significant graded inverse association between risk of breast cancer and consumption of miso (soybean paste soup). It may be that phytooestrogen ingestion needs to be lifelong and combined with other low-risk dietary constituents and behaviours for the protective effects to be significantly manifest (24).

There are 3 main classes of phytooestrogens: isoflavones (soy and beans), lignans (fruits, vegetables and seeds), and coumestans (bean sprouts). The majority of these compounds are nonsteroidal in structure and vastly less potent than the synthetic oestrogens (by a factor of 10 to the negative 5). The relative potencies (compared with oestradiol, given an arbitrary value of IOO), are: coumestrol 0.202, genistein (an isoflavone) 0.084. Genistein has been shown to exhibit antiproliferative effects in human ER- positive MCF-7 breast cancer cell lines in vitro. Extrapolation of cell culture to humans is questionable as minimum cell concentrations of genistein need to be 10-100 mmol/L, whereas high soy diet results in an approximate plasma level of 0.5-4 mmol/L. The other observed effects of phytooestrogens include, reduction in cholesterol and cardioprotective effects in rhesus monkey (25), and reduced bone loss in ovariectomized rats (26). Work with phytooestrogens includes the broader aspects of modifying diet and lifestyle, and a closer look at the mechanism of action of these compounds to investigate their clinical uses.

CONCLUSION New understanding of the ER has led to the

development of many compounds which will benefit women in reproductive and postreproductive years. The potential use of compounds such as tamoxifen for breast cancer prevention is new and exciting and is already showing positive benefits. Compounds such as raloxifene may prove to be a vast improvement in treatment of osteoporosis compared with currently available drugs. Women with endometriosis, fibroids, endometrial hyperplasia and dysfunctional uterine bleeding may benefit greatly from antioestrogens such as faslodex and SERMS. As our understanding of the ER and mechanism of action of these compounds progresses, the potential for better, more effective and more selective drugs is unlimited. The investigation into naturally-occurring SERMS such as phyto- oestrogens requires a medical profession open- minded towards alternative therapies, for therein may be a wealth of discoveries for the benefit of women.

References I . Editorial J Clin Endocrinol Metah 1998; 83: 3-S. 2. Hirotaka S, O’Malley B. Role of Coactivators and corepressors

in the Mechanism of Steroid/Thyroid receptor action. Rec Prog Horm Res 1997; 52: I4 I - 16.5.

3. Couse JF, Korach KS, Lindzey J , Grandien K , Gustafssen JA. Tissue distribution and quantitative analysis of oeatrogen Receptor-a and Estrogen receptor-b Messenger Ribonucleic Acid in the wild type and Er-Knockout mouse. Endocrinol 1997; 138: 46 13-462 I.

4. Speroff L, Glass RH, Kase NG. Clinical Gynecologic Endocrinology and Infertility. Fifth edition, Baltimore Maryland. Williams and Wilkins, 1994; Chapter 30, p. 898.

5. Grese TA, Dodge JA. Selective Estrogen Receptor Modulators. Curr Pharmaceut Des 1998; 4: 7 1-92,

340 AUST A N D NZ JOURNAL OF OBSTETRICS AND GYNAECOLOC~

6. Baker V, Jaffe RB. Clinical Uses of Antioestrogens. Obstet Gynecol Surv 1996; 5 1 : 45-59.

7. Rossing MA, Moore DE. Daling J, Weiss N, Self S. Ovarian tumours in a cohort of infertile women. N Engl J Med 1994.33 I : 77 1 -776.

8. Mitlak BH, Cohen FJ. In search of optimal long term female hormone replacement: The potential of Selective Estrogen Receptor Modulators. Horm Res 1997: 48: 155-163.

9. England GM, Jordan VC. Pure antioestrogens as a new therapy for breast cancer. Oncol Res 1997; 9: 397-402.

10.McLennan R. Tamoxifen for early breast cancer: an overview of the randomised trials. Early breast cancer trialists collaborative group. An analysis of the metaanalysis. Lancet 1998; 35 I : 91 14. Victorian cooperative oncology group newsletter 1998; 42: 7- 10.

1 1 . Kearney CE, Purdie DW. Selective oestrogen receptor modulators. Climacteric 1998; 1: 143-147.

12.Young NA, Venugopalen M, Hardikar S, Glasebrook A. Identification of an oestrogen response element activated by metabolites of 17b-estradiol and raloxifene. Science 1996; 273: 30.

13. Evista product information, Eli Lilly and Co. Indianapolis. IN 46285 USA.

14.Delmas PD, Bjarrason NH, Mitlak BH. Effects of raloxifene on bone mineral density, serum cholesterol concentrations, and uterine endometrium in postmenopausal women. N Engl J Med 1997; 337: 1641-1647.

15.Ettinger B, Black D, Cummings S et al. Raloxifene reduces the risk of incident vertebral fractures: 24-month interim analyses. European Congress of Osteoporosis, Berlin Sept 1998.

16.Cummings SR, Norton L, Eckertsd S et al. Raloxifene reduces the risk of breast cancer and may decrease the risk of endometrial cancer in post menopausal women. The two year findings of the MORE trial. American Society of Clinical Oncologists, Los Angeles May, 1998.

17.Jordon VC, Glusman JE, Eckerts S et a]. Incident primary breast cancers are reduced by raloxifene: integrated data from multicenter, double blind randomised trials in 12,000 post- menopausal women. American Society of Clinical Oncologists, Los Angeles, May, 1998.

18. Walsh BW, Kuller LH, Wild RA. Effects of raloxifene on serum lipids and coagulation factors in healthy postmenopausal women. JAMA 1998; 279: 1445-145 I .

19.Fuchs-Young R, Howe S, Hale L et al. Inhibition of oestrogen stimulated growth of uterine leiomyomas by selective oestrogen receptor modulators. Mol Carc 1996; 17: 151-159.

20.DeFriend DJ, Howell A, Nicholson R et al. Investigation of a new pure antioestrogen (ICI 182,780) in women with primary breast cancer. Cancer Res 1994; 54: 408-41 4.

2 I . Howell A, DeFreind D, Robertson J et al. Response to a specific antioestrogen (ICI 182,780) in tamoxifen resistant breast cancer. Lancet 1995; 345: 29-30.

22. Robertson JFR, Howell A, De Friend DJ, Blarney RW, Walton P. Duration of remission to ICI 182-780 compared to megestrol acetate in tamoxifen resistant breast cancer. Breast 1997; 6:

23. Hirayama T. A large scale cohort study on cancer risks by diet with special reference to the risks of reducing effects of green yellow vegetable consumption. Diet Nutr Canc. Tokyo: Japanese scientific Society Press, 1986; 41 -53.

24.Murkies A, Wilcox G, Davis SR. Clinical review Phyto- estrogens. J Clin Endocrinol Metab 1998: 83: 297-303.

25.Anthony MS, Clarkson TB, Weddle DL. Effects of soy protein phytoestrogens on cardiovascular risk factors in rhesus monkeys. J Nutr 1995; 125: 803-804.

26.Arjmandi BH, Alekel L, Hollis BW. Dietary soybean protein prevents bone loss in an ovariectomised rat model of osteoporosis. J Nutr 1996; 126: 16 1 - 167.

186- 189.