Selective Estrogen Receptor Modulators (SERMs): Effects on ... - UAH

Current Medicinal Chemistry, 2000, 7, 577-584

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Selective Estrogen Receptor Modulators (SERMs): Effects on Multiple Organ Systems D. Agnusdei* and N. Iori† European Medical Coordinator, Skeletal Diseases, Eli Lilly & Co., and †Clinical Research Physician, Eli Lilly Italy, Florence, Italy Abstract: SERMs represent a structurally diverse group of compounds which interact with the estrogen receptor but which elicit agonist or antagonist activity depending on the organ system and physiological context. Evaluation of the actions of these compounds has led researchers to a fuller understanding not only of the antiestrogens but also of steroid signaling in general. Based on their evolving clinical profiles, SERMs have the potential to address long-term health maintenance needs of women in their non-reproductive years.

Introduction The advent of specific techniques to locate the estrogen receptor has displayed estrogens to have not one but four great central areas of physiological activity. We now know that estrogen, in addition to its reproductive role, restrains and modulates bone turnover, protects the vasculature from the key pathological process of atherogenesis, and may also inhibit the process of amyloid protein deposition in the brain which manifests itself as the Alzheimer’s-related dementia. At the turn of the century, a woman’s menopause signaled the last remaining years of her life. With the average life expectancy of women close to 80 years of age, women can now expect to live up to one-third of their lives in the postmenopausal state. While this natural transformation is welcomed by many, as it frees women from the burden of monthly menstrual cycles, for most, it also accelerates several health problems. With the menopause, a number of hormonal changes occur, the most notable of which is a reduction in estrogen levels. Not only does this result in the well known symptoms of hot flashes, mood swings, etc., but a low estrogen state has been demonstrated to be a major risk factor for the development of osteoporosis and cardiovascular disease [1,2]. Osteoporosis is a systemic skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture

*Address correspondence to this author at the Via Gramsci 731, I50019 Sesto Fiorentino (Florence), Italy; Phone: +39 0554257324; Fax: +39 0554257397; e-mail: [email protected]

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[3]. Studies have demonstrated that menopause accelerates the age related loss of bone common to both genders, so that postmenopausal women can lose up to 5% of their bone mass per year during the rapid phase of bone loss that occurs shortly after menopause [4]. Osteoporotic fractures can result in significant morbidity and mortality to those affected, as well as place a tremendous burden on the health care system. In the European Union member states the total number of hip fractures is estimated to rise from 414.000 at the turn of the century to 972.000 fifty years later, representing an increase of 135%. Osteoporosis is a common health problem which results in approximately one in eight European citizens over the age of 50 having a fracture of the spine. Additionally one in three women and one in nine men over the age of 80 will have a hip fracture. Furthermore, hip fractures are associated with a 20% mortality during the first year and 40% during the three years after the fracture, and more than half of those with hip fractures never regain full mobility [5]. Given the magnitude of the effects of estrogen deficiency, it is no surprise that estrogen has been the mainstay of treatment for postmenopausal women. Estrogen is currently the treatment of choice for preventing postmenopausal osteoporosis [2,6]. Estrogen replacement has also been shown to exert beneficial effects on several risk factors for cardiovascular disease, contributing to an estimated 50% reduction in cardiovascular disease [7,8]. On the other hand, the availability of the results of the Heart and Estrogen/Progestin Replacement Study (HERS) shed new light on this particular aspect of the estrogen effects. The HERS study was a randomized, placebocontrolled trial of conjugated estrogen and medroxyprogesterone acetate in 2763 postmenopausal © 2000 Bentham Science Publishers B.V.

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women who had documented coronary heart disease and an intact uterus at baseline [9]. The primary outcome was fatal and non-fatal coronary heart disease. The results from this study provide no evidence that HRT is useful for secondary prevention in women with coronary heart disease, in fact the therapy did not reduce the risk of fatal or non-fatal coronary heart disease or of all-cause mortality [10]. Furthermore, sustained unopposed estrogen is associated with an increased risk of endometrial hyperplasia and endometrial cancer, necessitating the concomitant use of progesterone in those with an intact uterus [11]. As well, recent large cohort studies suggest that estrogen and estrogen/progesterone therapy may also be associated with an increased risk of breast cancer [12,13], thus diminishing the beneficial impact of estrogen on overall mortality [14]. As a result of these and other problems associated with estrogen therapy, including the perceived risks, undesirable side effects, inconvenience, and the resumption of menses, the compliance rate among women currently using estrogen replacement therapy (ERT) or estrogen/progesterone therapy (HRT) varies from only 12-32% [15-17]. Thus, the benefits of estrogen demonstrated in clinical studies may not be attained in clinical practice if the medication is not taken, resulting in costs incurred by government insurance plans and individuals without the long term benefits realized.

Selective Estrogen Receptor Modulators (SERMs) Because of the multiplicity of estrogen effects on bone, cardiovascular system, uterus, breast, and other tissues, researchers have searched for compounds with selective estrogen agonist activity in bone and cardiovascular system but with estrogen antagonistic activity or no activity in the reproductive tissues. Several synthetic compounds with this spectrum of activities have been described as Selective Estrogen Receptor Modulators (SERMs) and represent several widely varied structural families, including tripheniletylene derivatives such as tamoxifen, dihydronaphtalene derivatives such as nafoxidine, benzopyrans derivatives such as levormeloxifene, and benzotiophene derivatives such as raloxifene. The concept of SERMs is derived from the observation that tamoxifen, an effective adjuvant therapy of breast cancer that has an anti-estrogenic effect on breast tissue, has estrogen-like effects on the skeleton and on the lipid profile [18]. Therefore, an estrogen-like compound that binds with high affinity to the estrogen receptor (ER) could have either estrogen agonist or antagonist activity according to the type of estrogenresponsive tissue. Although the molecular basis for

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tissue specific actions of SERMs is not fully understood, recent advances in the field of ER biology has led to new insights in their mechanism of action. After years of unsuccessful attempts at identifying a variant ER, the recent identification and cloning by Kuiper et al. [19] and Mosselman et al. [20] of a novel, tissue-restricted estrogen receptor (ERβ) in both the rat and human, provided the first evidence that variant, non-mutated forms of the ER exist and are capable of activation by estradiol. It remains to be demonstrated that tissue-restricted ERs, such as ERß will be able to be exploited therapeutically, though this will surely be the goal of future research. Endogenous estradiol exerts its physiological effects by binding to the ER which then dimerizes and binds to the estrogen receptor response element (ERE), a specific promoter sequence on target genes, to activate transcription[21]. This transcriptional activation pathway may be modulated by the presence of other hormones or growth factors, by proteins which bind the ligand, its receptor, the ligand-receptor complex, or the target gene, by estrogen receptor subtypes, or by DNA sequences on the target gene[21,22]. SERMs exert a continuum of estrogen agonist/antagonist effects, due to their preferential binding to ER, as well as the endogenous cellular milieu. Triphenyethylenes such as tamoxifen, and nonsteroid benzothiophenes, such as raloxifene, are SERMs with mixed estrogen agonist/antagonist actions[22-24]. In contrast, 17β-estradiol and ICI 164384 have pure estrogen agonist and antagonist activities, respectively [25].

SERMs: Mechanism of Action The mechanism by which SERMs interact with the ER but display a gene activation profile differing from the prototypic estrogen, estradiol, is incompletely understood. While the estrogen antagonist properties of antiestrogens were originally ascribed to simple competition for ER binding, it is now believed that estrogen antagonism is an active process, derived from manifold ER and, perhaps, non-ER mediated events [26,27]. Importantly, the relationship between the agonist and antagonist properties of antiestrogens appears to depend on the gene or cellular context in which the observation is made, giving rise to an apparent tissue-specific pattern of action unique to each compound. The molecular basis of these interactions is the subject of recent comprehensive reviews and intensive investigation [21,28]. The ER is a ligand-activated transcription factor with six structural domains of overlapping function (labeled A through F). The two transcription activation functions (AF-1 and AF-2) are located in domains A/B and E,

Selective Estrogen Receptor Modulators (SERMs)

respectively [29,30]. Binding of ligand to the latent ER induces conformational changes which favors dissociation of heat shock proteins followed by ER dimerization and, later, binding of cell-specific adapter proteins to the active, dimerized complex [26]. It is believed that the strength of the ligand-ER induced activating function is modulated by these cell-specific adapter proteins and perhaps by other signaling pathway transcription factors, e.g. AP-1 [31]. Extending this model of ER-mediated gene activation to several classes of antiestrogens, McDonnell et al. demonstrated that while ICI 164,384 (a primarily “pure” antagonist), 4-hydroxytamoxifen (a potent metabolite of tamoxifen), and raloxifene all bound ER, each affected different transcriptional profiles depending on the cell and gene promoter context in vitro [32]. By examining susceptibility of the ER-antiestrogen complexes to protease degradation, structural (conformational) distinctions between the complexes were deduced which could account for the observed divergent genomic actions. They postulated that these three antiestrogens form distinct ER-ligand complexes representing different points along a continuum of potential ER activation (ie, intrinsic estrogenicity). Consistent with this model, tamoxifen binding to the ER distinctly affects the two transcription activation functions of the ER compared with estradiol, such that AF-2 (the hormone-dependent transcription activation function) is usually enhanced by estradiol binding, while it may be suppressed by tamoxifen in a doseand cell specific manner [29,30]. Therefore, the estrogen agonist/antagonist profile of tamoxifen in a given tissue results from the relative strength of AF-2 inhibition versus constitutive (hormone-independent) AF-1 activation, both of which may be modulated by cell-specific adapter proteins and transcription factors [28]. The conformation of the ER-ligand complex could impose structural specificity on these putative proteinprotein and protein-DNA interactions [32]. Another cellular mechanism likely to play a role in cell- and promoter-specific actions of the antiestrogens has been elucidated by work from Yang et al. [33] who compared the ability of estrogens and antiestrogens to initiate transcription of the TGF-β3 gene in an in vitro system. While it is activated by both estrogen and antiestrogens via the ER, TGFβ3 was studied because it is unusually responsive to activation by antiestrogens [33] and it is believed to be important in bone remodeling. They demonstrated that raloxifene and some estradiol metabolites, but not estradiol itself, interacted with ER and activated the TGF-β3 gene, even when the ER region containing the DNA-binding


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domain (Domain C) was mutated. These findings suggested the presence of a novel pathway for ERmediated gene activation not requiring direct ER-DNA contact. They were able to identify a region of the TGFβ3 gene promoter, distinct from the canonical estrogen response element (ERE), which recognized the raloxifene-bound ER. They termed this region the raloxifene response element (RRE) and concluded that tissue specificity of ER-ligand could be imparted via interactions with gene-specific response elements. Further evidence of novel, non-ERE promoter sequences able to mediate antiestrogen genomic actions has recently been reported for the human retinoic acid receptor-α1 gene [34]. The recent advances in the molecular pharmacology of antiestrogens, as outlined above, have already paid off in new ways to screen for molecules with potential therapeutic utility. As the complexity of the steroid/anti-steroid signaling pathway is further unraveled, we will likely witness a wide array of therapies based on selective modulation of steroid receptor activity. Rationale for the SERM classification emerged from preclinical studies of raloxifene--the first compound to be labeled as a SERM--demonstrating that, in rodents, raloxifene prevented estrogen-deficiency bone loss and lowered serum cholesterol following ovariectomy without stimulating proliferation of the endometrium [36]. Raloxifene thus exhibited apparent estrogenagonist effects in some tissues (e.g., bone, liver) but, at the same doses, was neutral with respect to estrogenic activity in others (endometrium). Based on its pattern of tissue specificity, raloxifene was deemed representative of a second generation SERM, the first generation represented by tamoxifen and its derivatives, which demonstrated estrogen-agonist properties in the endometrium [35]. For many of the SERMs in development, few or no clinical studies examining their SERM properties have been published. What clinical data are available come primarily from two compounds, tamoxifen and raloxifene.

Tamoxifen: A First Generation SERM Tamoxifen was first used in the early 1970’s for treatment of advanced breast cancer in postmenopausal women [37]. It is still widely employed for this use and has since become a standard of adjuvant therapy [38]. Most of the approximately six 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 chemoprevention agent and its use has been recently approved by the FDA.

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Although there was concern that tamoxifen might accelerate post-menopausal bone loss, several human studies of variable quality and size, in women receiving adjuvant tamoxifen for treatment of early stage breast cancer, and later in healthy volunteers demonstrated that tamoxifen has a beneficial effect on bone [18, 3943]. The bone preserving effect of tamoxifen in the lumbar spine was apparent after six months of therapy, plateaued after one year, and remained stable during five years of follow-up [18]. In contrast to the usually observed regional effect of HRT on the skeleton, tamoxifen treatment resulted in similar, modest increases in bone mineral density (BMD) at both the hip and spine [42-44]. As opposed to the favorable effect of tamoxifen on BMD in postmenopausal women, the two studies specifically examining its skeletal effects in premenopausal women have shown decreases in BMD at the radius, spine, and hip [43,45]. Tamoxifen, thus appears to have estrogen-like properties in bone when used in the presence of low circulating estrogen levels, but antagonist effects when circulating estrogen levels are high. These findings suggest that the prevailing estrogen milieu directly affects tamoxifen’s ultimate activity in bone, thus implicating the ER as a direct mediator of tamoxifen’s bone effects. One might expect, therefore, that the combination of tamoxifen and HRT in a postmenopausal woman would result in at least a partial amelioration of estrogen’s benefit. A preliminary report testing this hypothesis, however, demonstrated instead further increases in proximal femur BMD and no effect on spine BMD when HRT was added to tamoxifen therapy [44]. Clearly, more work is needed to better understand how tamoxifen interacts with ER to influence bone metabolism. In addition to its estrogen-like effects in bone, the SERM profile of tamoxifen includes estrogen-like effects on serum lipids, vaginal and endometrial proliferation in postmenopausal women. The proliferative effect in the endometrium might relate to the observed increase in endometrial cancer risk with long-term tamoxifen therapy [46] and thus does not fully circumvent the problems caused by unopposed estrogen therapy. Additionally, although available data are limited, tamoxifen also appears to increase the risk of venous thromboembolism 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 to 5 versus placebo) [47-51].

Raloxifene: A Second Generation SERM Raloxifene was initially developed as a therapy for breast cancer. In preclinical studies raloxifene inhibited

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binding of estradiol to the ER and estradiol-dependent proliferation of MCF-7 breast cancer cells [52]. Consistent with these observations, studies in vivo demonstrated anti-tumor activity in carcinogen-induced tumors in rodents of a magnitude similar to what had been observed previously with tamoxifen [53]. As might be expected based on its putatively similar mechanism of action in breast tissue, raloxifene demonstrated a lack of anti-tumor effect in a small phase II trial in postmenopausal women with advanced breast cancer who were refractory to recent tamoxifen therapy [54]. The distinct SERM profile of raloxifene on the skeleton, serum lipids and uterine endometrium has been demonstrated in a rodent model. In rats subjected to ovariectomy, treatment with raloxifene preserved bone mass in the distal femur and proximal tibia compared to vehicle-treated rats [36]. In these same animals raloxifene treatment resulted in a significant (50-75%) reduction of serum cholesterol levels without significant effect on triglyceride levels. These effects on the skeleton and serum lipids occurred with minimal effects on uterine weight which were not associated with changes in the histologic appearance of the endometrium. The beneficial effects of raloxifene treatment on bone density were maintained during long-term dosing and were associated with favorable effects on bone biomechanical properties [55]. Histomorphometric analysis of bone demonstrated that raloxifene decreased bone resorption similarly to estrogens [56]. As suggested from the preclinical studies, raloxifene appears to have a clinical profile distinct from either estradiol or tamoxifen. In an eight week randomized, double-blind, placebo-controlled, multi-center phase II study, 251 healthy postmenopausal women received Premarin  (conjugated equine estrogens) 0.625 mg/day or raloxifene (200 or 600 mg/day). Administration of raloxifene at either dose decreased bone turnover, as assessed by a panel of biochemical markers of bone metabolism, equivalently to treatment with Premarin. Furthermore, raloxifene treatment significantly lowered serum total and LDL-cholesterol levels but did not increase triglycerides or HDLcholesterol levels as did Premarin. In contrast to the endometrial proliferation observed in Premarin-treated subjects, treatment with raloxifene 600 mg/day suppressed endometrial proliferation, as judged by histological grading of biopsy material obtained before and after treatment. Beneficial effects on bone turnover and serum lipids were also observed in subjects treated with lower doses of raloxifene [57]. The only adverse event possibly related to raloxifene in phase II studies was vasodilatation (hot flushes), which occurred with significantly greater frequency only in the group of women receiving 600 mg/day.

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By performing studies of calcium kinetics, Heaney and Draper demonstrated that bone remodeling declined significantly and similarly in estrogen/progesteroneand raloxifene-treated subjects. Based on measurement of urinary calcium excretion and determination of absorption efficiency, they concluded that the acute effect of raloxifene on calcium kinetics and bone remodeling were similar to that of HRT in early postmenopausal women [58]. In a multicenter European study, 601 early postmenopausal women were randomly assigned to receive raloxifene 30, 60, or 150 mg/d or placebo. All subjects received a calcium supplement (400-600 mg/day). Bone mineral density, which was measured twice a year over 24 months by DEXA, decreased significantly at all skeletal sites with placebo, and significantly increased with raloxifene at the spine, hip, and total body at the three doses. At 24 months, the mean increase with raloxifene 60 mg compared with placebo was 2.4% at the lumbar spine and at the total hip, and 2% at the total body. Markers of bone formation (serum osteocalcin and bone specific alkaline phosphatase) and of resorption (urinary CrossLaps) decreased significantly to the premenopausal range within 3 to 6 months of treatment with raloxifene [59]. In a large double-blind, placebo-controlled, randomized clinical study, named Multiple Outcomes of Raloxifene Evaluation (MORE), involving 7705 osteoporotic patients (mean age 67 years) with and without vertebral fractures, two-years treatment with raloxifene produced a substantial decrease in the rate of new vertebral fractures. The two year analysis demonstrated that women taking raloxifene who had no spinal fractures upon entry into the study were 52% less likely to have a first spinal fracture and women with a previous spinal fracture were 38% less likely to have new spinal fractures when compared with calcium and vitamin D-supplemented placebo patients [60]. The magnitude of the raloxifene effect in the spine at 2 years is clinically significant, and is comparable to the magnitude of effect of alendronate 5 or 10 mg after 2.9 years (48% risk reduction). Raloxifene has been shown to be as good at reducing fractures in osteoporotic patients as any currently available agent used both for prevention and/or treatment of postmenopausal osteoporosis. Moreover, the results of these studies clearly indicate that raloxifene exerts its beneficial effect both in healthy, or osteopenic postmenopausal women, and in elderly women with osteoporosis and established osteoporosis. Finally, biomechanical and histomorphometric studies performed in animals and postmenopausal osteoporotic women have shown that the bone mass gained after raloxifene treatment is of normal quality, and the structural integrity of the bone tissue is preserved.


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In a recent study, raloxifene showed to be effective in inhibiting aortic accumulation of cholesterol in ovariectomized cholesterol-fed rabbits. This effect was similar to the effect of HRT, at pharmacological doses [61]. Using surrogate markers of cardiovascular efficacy in long-term controlled studies, raloxifene consistently reduced total cholesterol and LDL-cholesterol (10% and 14%, respectively), with little or no effect on HDLcholesterol and triglycerides. These effects were also studied in a six-month study of 390 healthy postmenopausal women. In this study the decrease in total cholesterol and LDL-cholesterol were confirmed and a significant reduction in fibrinogen and Lp(a) was also observed. Clinical evidence suggests that elevated fibrinogen and Lp(a) serum levels are considered to be independent risk factors for heart disease and atherosclerosis, respectively [62]. Raloxifene is well tolerated. There was no difference between raloxifene and placebo in the overall discontinuation rate which was about 13% in each therapy group. For two adverse events there was consistent statistical evidence that raloxifene increases the incidence over placebo: these were leg cramps, and hot flushes. Leg cramps were reported by 5.5% of women overall compared with 1.9% in the placebo treated women. In women treated with raloxifene the incidence of hot flushes was 24.3% compared to 18.2% with placebo; the effect is mostly early and almost always within the first 6 months. Raloxifene use is not associated with vaginal atrophy or urinary dysfunction. In the reported frequency of serious adverse events, raloxifene is associated with an increased incidence of venous thromboembolic events (VTE). The relative risk of VTE associated with raloxifene therapy (pooling all doses) versus placebo in all placebo-controlled clinical trials is similar to the relative risk of VTE associated with HRT, between 2 and 3 overall. There were no differences between raloxifene and placebo on all-cause mortality at any dose. In healthy postmenopausal women, several doubleblind placebo controlled studies evaluated the effects of raloxifene on the uterus. Uterine safety was assessed from evaluations of transvaginal ultrasound (TVU) measuring uterine volume and endometrial thickening, hysteroscopy or saline infusion ultrasonography with endometrial biopsies in case of endometrial thickening, and the incidence of postmenopausal bleeding. Globally, from 831 women in all dose groups, more than 3000 TVUs examinations were evaluated. Raloxifene treated women consistently had an endometrial thickness which was indistinguishable from placebo. In the HRT-controlled database (6 studies) a statistically significant greater

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incidence of vaginal bleeding was observed among HRT recipients (79%) compared with the raloxifene recipients, (5%, which could not be differentiated from placebo). Endometrial biopsies demonstrated non proliferative endometrium in all patients treated with raloxifene 60 mg/day. Raloxifene did not increase the risk of endometrial cancer and although based on few cases it is possible that raloxifene confers protection [63]. Safety data from osteoporosis studies include ~ 14,800 patient-years cumulative exposure to raloxifene, and ~ 6,750 patient-years cumulative exposure to placebo (median exposure 28 months). Data obtained 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. For all placebo-controlled studies, a total of 58 cases of newly-diagnosed breast cancer were reported. Given the ratio of randomized patients [raloxifene: placebo of approximately 2:1], and based on an intent-to-treat analysis, there was a relative risk (RR) of 0.46 [CI: 0.28, 0.75], corresponding to a 54% reduction in risk, of developing breast cancer; counting only women who received at least 18 months of exposure the RR for combined doses of raloxifene compared with placebo was: 0.23[CI: 0.10, 0.49], corresponding to a 77% reduction in risk [64,65]. The available clinical data suggests that raloxifene has a unique SERM profile which is distinct from both estrogens and tamoxifen. Treatment with raloxifene revealed estrogenic effects on the skeleton and serum lipids without concomitant stimulation of endometrial proliferation.

Other SERMs Recent clinical and preclinical studies suggest that several compounds may demonstrate a SERM profile. In addition to tamoxifen, other triphenylethylene derivatives (e.g., toremifene, droloxifene, idoxifene) have been evaluated clinically for the treatment of breast cancer [66-68]. The triphenyethylene derivatives generally have favorable effects on cholesterol metabolism [67], although there may be differences in their effects on serum lipoproteins when compared to tamoxifen [69]. In breast cancer patients, for example, while both toremifene and tamoxifen decreased total and LDL cholesterol levels, toremifene increased serum HDL cholesterol levels by a statistically significant 14% while treatment with tamoxifen decreased HDL cholesterol by 4% [69]. There is limited information about the effect of these compounds on the uterus and skeleton. One study reported that toremifene increased endometrial thickness and

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caused estrogenic changes in Pap smears in breast cancer patients in a manner and degree similar to tamoxifen [70].

Conclusions SERMs represent a structurally diverse group of compounds which interact with the estrogen receptor but which elicit agonist or antagonist activity depending on the organ system and physiological context. Evaluation of the actions of these compounds has led researchers to a fuller understanding not only of the antiestrogens but also of steroid signaling in general. Based on their evolving clinical profiles, SERMs have the potential to address long-term health maintenance needs of women in their non-reproductive years [71].

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