Selective Estrogen Receptor Modulators. Antonio Cano

Chapter 6. Pure Antiestrogens

• Carlos Hermenegildo



An antiestrogen is a compound that blocks the action of estrogens. According to McGregor and Jordan (1998), antiestrogens can be classified into two major groups:


Type I

This group is made up of compounds exhibiting mixed estrogenic/antiestroge- nic actions in the laboratory. These compounds are also known as SERMs (selective estrogen receptor modulators) and include both triphenylethylene derivatives (such as tamoxifen, toremifene, idoxifene, or droloxifene) and benzothiophenes (raloxifene) (Bryant and Dere 1998). The agonist-antagonist profile for a given compound is tissue and species specific. Tamoxifen, for instance, inhibits estrogen-stimulated growth of breast cancer cells (antagonistic activity) but stimulates endometrial proliferation (agonistic activity). Tamoxifen is a pure estrogen antagonist in the chick but partial estrogen agonist in the mouse, rat, and human (Baker and Jaffe 1996).


Type II

Type II antiestrogens, pure antiestrogens, or selective estrogen receptor downregulators (SERDs) (Howell et al. 2004b) have no estrogen-like properties in laboratory assays.

To illustrate the different actions of both groups of antiestrogens, Table 6.1 presents the tissue-specific effects obtained with the administration of type I (tamoxifen and raloxifene) and type II (ICI 164384 and fulvestrant) antiestrogens in preclinical studies.

The pure antiestrogens were discovered about 20 years ago by Wakeling and collaborators (Wakeling and Bowler 1987). To date, a few distinct compounds of this group have been discovered. All of them are able to bind to the estrogen receptor (ER) without any estrogenic activity, either in vitro or in vivo, in any studied species or tissues, including all estrogen target tissues such as uterus, mammary gland, ovary, or bone.

Table 6.1. Tissue-associated estrogen activities of various estrogen receptor ligands based on preclinical studies. We present the effects of two estrogen receptor agonists (17β-estradiol and 17a-ethynyl estradiol), two selective estrogen receptor modulators (SERMs, tamoxifen, and raloxifene), and two pure antiestrogens (fulvestrant and ICI 164384) (from Bryant and Dere 1998)

















Partial agonist








ICI 164384 Fulvestrant





The main potential utility of antiestrogens would be the treatment of advanced breast cancer after failure of long-term tamoxifen therapy. Nevertheless, pure antiestrogens could also find application in gynecology and in other nonmalignant conditions (Gradishar and Jordan 1997). In April 2002, fulvestrant was the first pure antiestrogen approved by the Food and Drug Administration for clinical practice (Bross et al. 2003).

The aim of the present review is to update the recent discoveries on the mechanisms of action, biological effects, clinical trials, and potential clinical utility of pure antiestrogens.


Chemical Structure and Classification

The main compounds that have demonstrated a pure antiestrogenic activity in the laboratory are the following (Fig. 6.1):

1. ICI 164384. This is the first pure antiestrogen discovered (Wakeling and Bowler 1987). This compound is a 7a-alkylamine derivative of 17β- estradiol, with a 16-atom carbon chain in the 7a position.

2. Fulvestrant. Also called ICI 182780 and Faslodex, this compound is also a 7a-alkylamine derivative of 17β-estradiol, developed from ICI 164384 to improve the bioavailability and the biological profile of its activity: the amide moiety was replaced by other polar groups and the terminal alkyl function was fluorinated. Those molecular changes made fulvestrant more potent, and its affinity for ERs approximately is 4-5 times higher than that of ICI 164384 (Wakeling et al. 1991). Since both compounds are poorly soluble and have low oral activity, they are being used as depot injections in clinical studies. Fulvestrant is the only pure antiestrogen approved for the treatment of hormone-sensitive breast cancer in postmenopausal women with disease progression following antiestrogen therapy (Bross et al. 2002, 2003).

Fig. 6.1. Chemical structures of pure antiestrogens. Chemical structures of ICI 164384, fulvestrant, RU 58668, and EM-139 are presented (MacGregor and Jordan 1998)

3. RU 58668. This is a 17β-estradiol derivative compound, substituted in the Opposition with a long hydrophobic side chain, producing a spatial arrangement similar to the 7a-substituted compounds in relation to the plane of steroid nucleus (Van de Velde et al. 1994, 1996).

4. EM-139. This compound is also a lα derivative of 17β estradiol, with a structure similar to that of ICI 164384 (Doualla-Bell et al. 1995).

Other compounds, such as ZK-703 and ZK-253, are currently under preclinical testing, and preliminary data show a pure antiestrogen activity in xenograft breast cancer models (Hoffmann et al. 2004).


Mechanism of Action

Pure antiestrogens are distinguishable form other SERMs in terms of their mechanism of action, although both classes of agent mediate their effects through the two types of estrogen receptors (ERa and ERβ

Type I antiestrogens are competitive inhibitors of the binding of estrogens to ER. As demonstrated for raloxifene, these compounds seem to form a complex with the ER that retains partial transcription activity as a result of imperfect changes in the tertiary structure of the complex (Brzozowski et al. 1997). Due to this partial agonistic activity, type I antiestrogens show a wide range of biological functions, from complete antagonism to partial agonism, depending upon the species, tissue, or target genes studied (Bryant and Dere 1998).

Pure antiestrogens also act as competitive inhibitors of the estradiol-ER complex. For instance, ICI 164384 is a competitive antagonist of both ERa and ERβ (Barkhem et al. 1998). In MCF-7 cells, similar amounts of estradiol and RU58668 are bound to ER (Jensen and Khan 2004).

Distinct mechanisms of action have been ascribed to pure antiestrogens (Fig. 6.2). According to one proposal, pure antiestrogens impede the dimerization of two ER-ligand complexes, preventing binding to DNA and, as a consequence, gene activation (Fawell et al. 1990). However, it was later reported that the pure antiestrogen-ER complex is able to bind to EREs, as has been demonstrated for ICI 164384 and RU 58668 (Barsalou et al. 1998), though the transcription unit formed is inactive (Pink and Jordan 1996).

Pure antiestrogens also exert a unique mechanism of action: they decrease intracellular levels of ER (Wakeling 2000). Fulvestrant is able to bind to newly synthesized ER in cell cytoplasm, modifying the cytoplasm-nucleus net flux of the ER by diminishing its transport into the nucleus. The paralyzed receptors are then rapidly destroyed (Dauvois et al. 1993) in a process implying an increased turnover of ER by the ubiquitin-proteasome pathway, since the complex formed between ER and fulvestrant causes a high ubiqui- tination and rapid destruction of the receptor (Wijayaratne and McDonnell 2001).

Fig. 6.2. Proposed mechanisms of action of pure antiestrogens (fulvestrant). 1 Fulvestrant (ICI) binds to estrogen receptor (ER). 2 Fulvestrant binding to ER accelerates receptor degradation (“ER down-regulator”). 3 Rate of dimerization and nuclear localization of fulvestrant-ER complex is reduced. 4 Reduced binding of fulvestrant-ER to ERE. 5 No transcription of estrogen-responsive genes; since AF-1 and AF-2 are inactive, no coactivators are recruited and the activity of RNA polymerase II is not activated (or inhibited) (Wakeling 2000)

Also, RU 58668 modifies the subcellular distribution of ER, appearing as clusters in the perinuclear region of cytoplasm, without association to specific cellular structures. This means that after RU 58668 treatment, ER is sequestered in the cytoplasm associated to short half-life proteins (probably induced by RU 58668 treatment) that impede its entry into the nucleus (Devin-Leclerc et al. 1998).

As a consequence of the above-cited studies, it has been suggested that the title of pure antiestrogen should be given to those compounds that are capable of blocking the entry of the ER into the nucleus. This mechanism of action would be the essential difference between pure antiestrogens and SERMs (type I antiestrogens). In this sense, type I antiestrogens induce an increase in the amount of ER in the cell nucleus, while pure antiestrogens diminish it (and therefore they can also be named SERDs) (Devin-Leclerc et al. 1998; Howell et al. 2004b).

In explaining these observed differences between both classes of antiestrogens, it has been proposed that the large side chains in the pure antiestrogen molecules are responsible for the mechanism of differences observed in the action. The binding of estradiol, raloxifene, and ICI 164384 to ER has been studied by crystallography. Raloxifene and ICI 164384 bind to the same aminoacids as estradiol, but the side chain of both compounds interacts differently with several amino acids of the binding domain. Such interaction modifies the tertiary structure of the complex, which may explain the differences in their actions (Brzozowski et al. 1997; Schafer et al. 1999; Pike et al. 2001; Lonard and Smith 2002).

In addition to theirinterferencewithERphysiology, alternativemechanisms of action have been reported that help to explain the antiestrogenic potential of pure antiestrogens. Among other things, pure antiestrogens seem to inhibit some enzymatic activities involved in estrogen synthesis. EM-139 was the first pure antiestrogen reported to inhibit an enzyme, 17β-hydroxysteroid dehydrogenase, thus reducing the peripheral conversion of estrone into estradiol (Li et al. 1995). Additionally, fulvestrant has been reported to inhibit aromatase activity in vitro. This inhibition is not due to down-regulation of the aromatase transcript; on the contrary, its activity remains inhibited even after the pure antiestrogen is removed from the cells, suggesting that fulvestrant remains bound to the enzyme (Long et al. 1998).

Finally, it has been suggested that fulvestrant, in addition to its antiestrogenic activity, has also significant antiprogestin activity, comparable to the activity of the antiprogestin RU-486 (Nawaz et al. 1999).


Effects of Pure Antiestrogens

The majority of the actions of pure antiestrogens have been described in studies designed in cell cultures (effects in vitro) or in experiments performed in animals. During the last few years, only a few clinical studies have been completed. The main objective of the majority of the studies has been to demonstrate the pure antiestrogenic action of these compounds.


In Vitro Studies

These studies have been focused on the effects of pure antiestrogens on gene expression, on cell growth and proliferation, and on the effects on different growth factors.

Effects on Gene Expression

In a study of global gene expression in MCF-7 cells, fulvestrant antagonized estradiol action on > 95% of estradiol-regulated genes. Moreover, the antagonism of fulvestrant was not accompanied by partial agonism, in comparison with the other tested compounds (raloxifene and hydroxytamoxifen), supporting the full pure antiestrogen activity of this compound. There were also genes specifically down-regulated by fulvestrant, and the majority of these genes appear to be regulators of the cell cycle, cell proliferation, and DNA synthesis. Therefore, by down-regulating the expression of these genes, fulvestrant has an additional beneficial effect against the proliferation of breast cancer cells (Frasor et al. 2004).

Effects on Cell Growth and Proliferation

In the initial studies with pure antiestrogens, both ICI 164384 and fulvestrant inhibited cell growth and arrested the cell cycle in the G1 phase. These effects were two orders of magnitude more potent than those achieved with tamoxifen in the same experimental conditions (Wakeling and Bowler 1987).

When used in tumor cells, fulvestrant was initially described as a potent, competitive growth inhibitor of ER-positive, human breast cancer MCF-7 cells, whose growth is stimulated by estradiol. The compound was ineffective in tumor cell lines without ER, such as MDA-MB-231. The inhibitory effects were more pronounced with fulvestrant than with tamoxifen in the same cell line (Wakeling et al. 1991).

Effects on Growth Factors

Pure antiestrogens have been demonstrated to block some of the effects of estrogens on growth factors. Estrogens increase the transforming growth factor a (TGFa) production, which in turn stimulates cell growth and, in a process that implies the epidermal growth factor (EGF), increases cell replication. ICI 164384 and fulvestrant block estradiol-stimulated TGFa production (Wakeling et al. 1989; MacGregor and Jordan 1998; Tong et al. 2002).

Fulvestrant has been reported to decrease both insulin-like growth factor I (IGF-I) stimulated cell growth and IGF-I receptor mRNA (Huynh et al. 1996). Moreover, in the human fetal osteoblast cell line (hFOB/ER9 cells), both ICI 164384 and fulvestrant blocked the estradiol-induced increase in IGF-I mRNA levels (Kassem et al. 1998).


Experiments in Animals

To date, all the experiments done in animals with pure antiestrogens have disregarded any estrogenic actions of these compounds. Some of the described effects are presented here, arranged by the tissue or organ where they have been described (Table 6.2).


The main potential utility of the pure antiestrogens is in the treatment of breast cancer. Several studies on their effects on the breast demonstrate both the pure antiestrogenic action of the tested compounds and their beneficial effects on breast cancer treatment. In experiments conducted in nude mice xenotrans- planted with two different human estradiol-dependent breast tumors, a single injection of fulvestrant provided an antitumor efficacy equivalent to that of daily tamoxifen treatment for at least 4 weeks (Wakeling et al. 1991). Additionally, RU 58668 was able to induce up to 30% disappearance of MCF-7 breast cancer tumors implanted in nude mice (Van de Velde et al. 1995). Moreover, a 3-week treatment with fulvestrant in control rats induced a great mammary atrophy, as a consequence of an increased epithelial cell apoptosis (Lim et al. 2001).

Table 6.2. Effects of pure antiestrogens on experimental animals







Mammary atrophy



(Lim et al.




Nude mice


(Wakeling et al.



Development block

Immature rat


(Wakeling et al.




Mature rat


(Wakeling et al.






(Dukes et al.

1992, 1993)


Block of endometrial tumor progression

Athymic mice

ICI 164384

(Gottardis et al.



Decreased trabecular bone density


ICI 164384

(van Bezooijen et al. 1998)


Reduced bone volume



(Gallagher et al. 1993)

Cardiovascular effects

Block of cholesterol- lowering activity of estradiol



(Lundeen et al.



Block of vascular smooth muscle cell proliferation


ICI 164384

(Cathapermal et al. 1998)


Block of estradiol- induced increase in blood flow in aorta



(Hegele- Hartung et al. 1997)


The effects of pure antiestrogens in the uterus have also been extensively studied, since it is an estrogen-dependent organ and the target of the main side effects of tamoxifen therapy, such as endometrial hyperplasia, hypertrophy of glandular epithelium, or even focal cellular atypia (Sourla et al. 1997).

Fulvestrant has demonstrated high antiuterotrophic potency in several animal models. This compound has been reported to block the uterus development in immature rats (Wakeling et al. 1991) and to promote the involution of uterus in adult normal (Dukes et al. 1993) and ovariectomized monkeys (Dukes et al. 1992). In vivo, RU 58668 displayed a total antiuterotrophic activity in mice and rats without exhibiting any agonistic effect (Van de Velde et al. 1994).

When studied in a model of human endometrial carcinoma, such as EnCa101 tumors in athymic mice, ICI 164384 not only showed no stimulatory activity on tumor progression but also blocked the tamoxifen-stimulated growth of the tumor (Gottardis et al. 1990).

The overall uterine effects obtained in animals treated with the different compoundsmakeitpossibletoassumethatpureantiestrogens couldbeusedin the treatment of endometrial disorders and endometrial carcinoma (Gradishar and Jordan 1997). Skeleton

The effects of the pure antiestrogens on the skeleton are controversial, although it seems ICI 164384 and fulvestrant decrease bone density. It has been demonstrated that treatment of rats with the pure antiestrogen ICI 164384 induced a significant decrease in trabecular bone mineral density, comparable to that observed after ovariectomy (van Bezooijen et al. 1998). Administration of fulvestrant to adult female rats reduced bone volume at the proximal tibial metaphysis and increased the osteoclast surface. When administered to ovariectomized rats, fulvestrant inhibited the estradiol-stimulated cancellous bone formation, while affecting neither longitudinal nor periosteal tibial growth (Gallagher et al. 1993).

Cardiovascular Effects

Estrogens are thought to exert their cardiovascular effects by acting on blood lipoproteins or by direct effects on blood vessels. In studies performed in rats, fulvestrant had no effect on plasma cholesterol levels. When administered along with estradiol, however, it blocked the cholesterol-lowering activity of estradiol (Lundeen et al. 1997).

By acting on the vessel wall, estradiol significantly inhibited superoxide anion-induced vascular smooth muscle cell proliferation, whereas the pure antiestrogen ICI 164384 reversed the inhibitory effect of estradiol (Cathapermal et al. 1998). Fulvestrant also reversed the estradiol-induced increase in blood flow in rabbit aorta (Hegele-Hartung et al. 1997).


Clinical Studies

There is very little information concerning the performance of pure antiestrogens in clinical conditions, and all studies have been done with fulvestrant.


Pure antiestrogenic activity must be sustained over time to achieve its effects, mainly an effective inhibition of estrogen-controlled proliferation. Therefore, exposure to fulvestrant via chronic administration is required. Since oral delivery is not an appropriate route of administration, fulvestrant is administered by a long-acting, intramuscular formulation (Robertson and Harrison 2004). It is given as a 250 mg dose in a prolonged-release intramuscular formulation. Plasma concentrations of fulvestrant are measurable up to 28 d after dosing, with peak plasma concentrations occurring 1 -11 d after dosing, reaching Cmavalues of about 6 ng/ml (Robertson and Harrison 2003). Tolerability

In general, fulvestrant is well tolerated in studies conducted in healthy volunteers (Addo et al. 2002) and in clinical trials (Howell et al. 2002,2004a; Osborne et al. 2002). The most common adverse effects are nausea, asthenia, pain, vasodilatation, and headache (Robertson et al. 2003; Howell et al. 2004a). In trials in which fulvestrant was compared to anastrazole (an aromatase inhibitor), the incidence of adverse events relevant to endocrine therapy (gastrointestinal disturbances, hot flushes, vaginitis, weight gain, thromboembolic disease, urinary tract infection, and joint disorders) were similar for both groups, with the exception of joint disorder incidence, which was lower with fulvestrant (Robertson et al. 2003). In the trial comparing fulvestrant with tamoxifen, the incidence of hot flushes was lower in patients treated with fulvestrant, without any difference in the other above-mentioned adverse events relevant to endocrine therapy (Howell et al. 2004a).

Clinical Efficacy Studies

The first clinical trial of fulvestrant was conducted to assess its tolerance, pharmacokinetics, and short-term biological effects in women with primary breast cancer. Control group patients received no treatment. The treated patients received daily intramuscular injections of fulvestrant at doses of 6 or 18 mg for 7 d prior to primary breast surgery. There were no effects on serum gonadotropin or sex hormone binding globulin levels, suggesting a lack of agonist activity of the compound at the pituitary or hepatic level. Fulvestrant significantly reduced the tumor expression of ER, progesterone receptor, and Ki67, a nuclear antigen whose expression is closely related to cell proliferation (DeFriend et al. 1994). Similar, comparative studies performed with tamoxifen and fulvestrant showed no effect of tamoxifen on tumor expression of ER (McClelland et al. 1996).

In tumor samples derived from the study of DeFriend (DeFriend et al. 1994), ER protein content was suppressed by fulvestrant, whereas the levels of EGF receptor (EGFR) and its ligand TGFa were unaltered by treatment. Since a loss of endocrine sensitivity has been attributed to tumors with elevated levels of EGFR and TGFa, treatment with fulvestrant preserves the hormone response of tumor cells (McClelland et al. 1996).

Fulvestrant has also been administered to premenopausal women. The administration of 12 mg/d for 7 d in the follicular phase prior to hysterectomy produced no changes in luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels or in ovarian function. As expected, fulvestrant caused a potent antiestrogenic action in the endometrium, blocking the physiological increase of the endometrial thickness (Thomas et al. 1994). In another study of similar design, fulvestrant administration prior to hysterectomy reduced endometrial ER and Ki67 expression (Dowsett et al. 1995).

The effect of long-term treatment (up to 33 months) with fulvestrant in patients with advanced tamoxifen-resistant breast cancer was first studied in a small group of patients (n = 19) in phase I/II clinical trials (Howell et al. 1995, 1996). A clinical benefit (complete response + partial response + stable disease ≥ 24 weeks) rate of 69% was obtained in patients treated with fulvestrant, without serious side effects from the treatment. Moreover, the high level of response suggested that fulvestrant was not cross-resistant with tamoxifen. The LH and FSH levels rose after suspension of tamoxifen and then remained stable thereafter, suggesting no effect of fulvestrant on the pituitary-hypothalamic axis. There were no significant changes in serum levels of prolactin, the sex-hormone-binding globulin (SHBG). Compared to the effects of tamoxifen, which reduces serum levels of LH and FSH (Willis et al. 1977) and reduces LDL and cholesterol levels and increases SHBG, HDL, and triglycerides levels (Sakai et al. 1978; Love et al. 1990), those of long-term treatment with fulvestrant did not modify levels of total cholesterol, LDL- cholesterol, HDL-cholesterol, or tryglycerides (Howell et al. 1996).

Three randomized phase III trials have evaluated the efficacy of fulves- trant. The first two trials were designed to compare the efficacy of fulvestrant (250 mg) with anastrazole (1 mg), an inhibitor of aromatase, for the treatment of postmenopausal women with advanced disease previously treated with antiestrogenic therapy (mainly tamoxifen) (Howell et al. 2002; Osborne et al. 2002). Trial 0021, conducted in North America, and trial 0020, conducted in Europe, Australia, and South Africa, were designed to allow the combination of their results (Morris and Wakeling 2002). In both trials, fulvestrant (total n = 851 patients) was at least as effective as anastrazole, with time to disease progression of disease slightly higher (Howell et al. 2002; Osborne et al. 2002). The combined analysis of both trials revealed that time to disease progression of disease was significantly higher (30%) in the fulvestrant-treated group (Morris and Wakeling 2002).

In the third phase III trial, fulvestrant was compared with tamoxifen in 587 postmenopausal patients with metastatic/locally advanced breast cancer previously untreated for advanced disease. At a median followup of 14.5 months, there was no significant difference between fulvestrant and tamoxifen for time to progression. Nevertheless, fulvestrant showed only noninferiority to tamoxifen in the receptor-positive group, and the time to treatment failure was significantly worse for fulvestrant when all patients were considered (Howell et al. 2004a). Data analysis reflects a higher rate of early progressions in the fulvestrant group. Moreover, pharmacokinetic studies demonstrate that ful- vestrant takes 3-6 months to reach steady-state plasma levels, suggesting that either a loading dose or doses of fulvestrant may be required (Howell et al. 2004b).


Clinical Utility

The main potential clinical utility of the pure antiestrogens is their use as a second-line treatment in patients with tamoxifen-resistant breast cancer. Tamoxifen is, for the moment, the first option in breast cancer expressing ER, but in a number of patients, tumors develop resistance to tamoxifen (Jordan 1993). Different hypotheses have been proposed to explain this resistance: alterations in tamoxifen metabolism (Osborne et al. 1991), specific mutations on the genes encoding RE protein (which could explain the fact that an antiestrogen transmits an estrogenic signal) (Jiang et al. 1992), alterations induced by tamoxifen in the gene regulation mechanisms (Johnston et al. 1997), and others, such as alterations in ER phosphorylation and direct effects on genes (MacGregor and Jordan 1998).

Pure antiestrogens, which act by different mechanisms of action, probably are not affected by the mechanisms of tamoxifen resistance. As a result, those compounds might be a good choice as second-line hormonotherapy of breast cancer after failure of tamoxifen treatment, as has been reported in clinical trials (DeFriend et al. 1994; Howell et al. 1995,1996,2004a; Morris and Wakeling 2002).

It seems reasonable that pure antiestrogens might be used as a good alternative to tamoxifen in the treatment of breast cancer, due to their beneficial effects, without increasing the risk of endometrium cancer (Simard et al. 1997).

Nevertheless, their potential use in large treatments will depend on systemic actions, since the beneficial effects may be counterbalanced by deleterious consequences on the cardiovascular (Hegele-Hartung et al. 1997) and skeletal systems (Gallagher et al. 1993). Moreover, most pure antiestrogens have a poor oral bioavailability. Therefore, the use of other routes of administration, such as intravenously, is mandatory. In some cases, to circumvent such problems, the production of nanospheres loaded with the pure antiestrogen RU 58668 has been tested (Ameller et al. 2004).

Finally, therapeutic sequencing of different hormonal agents is fast becoming a common clinical practice, and fulvestrant is a good treatment choice to extend the opportunity for using endocrine therapies before reliance upon cytotoxic chemotherapy is necessary. Further research is required in order to evaluate the optimal sequence, both in clinical practice as well as in the laboratory, to choose the correct treatment of breast cancer in each person after the appearance of tamoxifen-induced drug resistance (Robertson 2004; Osipo et al. 2004; Johnston 2004; Robertson et al. 2005).


The preparation of this review was supported by Grant 03/0831 from Fondo de Investigation Sanitaria, Madrid, Spain.


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