Cancer Chemotherapy & Biotherapy: Principles & Practices, 4th Edition

Hormonal Therapy for Breast Cancer

Peter F. Lebowitz

Sandra M. Swain


The hormonal treatment of breast cancer was first used over 100 years ago and now plays a critical role in the prevention and treatment of the disease at all stages.1 The correct application and further development of this therapy requires an appreciation of (a) estrogen synthesis and metabolism, (b) the endocrine regulation of these processes, (c) the molecular basis of estrogen signaling through the estrogen receptors, (d) the response of cancer cells to the modulation of estrogen receptor signaling, and (e) the pharmacology of the growing number of hormonal therapeutics. This chapter aims to provide a reference source on this basis, focusing on hormonal therapies that affect the estrogen and progesterone nuclear hormone receptors to achieve control of tumor growth.


Early Clinical Observations

In 1896, Dr. George Beatson reported on the first successful use of estrogen deprivation to treat breast cancer.2 Based on observations in farm animals that the ovaries have effects on lactation, he hypothesized that the removal of the ovaries might lead to decreased growth of breast tumors. His report describes the treatment of a premenopausal patient with advanced breast cancer who underwent a bilateral oophorectomy and experienced marked regression of her tumor. While ovarian ablation was used over the next few decades to treat breast cancer, the scientific underpinnings of these early clinical observations were just beginning to be understood.

Discovery of Estrogen and the Estrogen Receptor (ER)

The demonstration by Knauer in 1900 that ovarian transplants prevented uterine atrophy and loss of sexual function accompanying ovariectomy established the hormonal nature of ovarian function in regulating reproductive function.3 Over 20 years later, in 1923, Allen and Doisy developed a rat bioassay for assessing changes in the vaginal smear induced by ovarian extracts.4 The identification of a female sex hormone in the blood of various species was demonstrated in 1925, and in 1926 Loewe and Lange discovered a hormone in the urine of menstruating women that varied in concentration with the phase of the menstrual cycle.5, 6 This hormone was also found in large amounts in the urine of pregnant women.7 This finding led to the isolation and crystallization of an active substance, later identified and synthesized as estradiol.8

While the identification of estradiol was critical in the further study of this hormonal system, it took another few decades before the second major component of this system was identified, the estrogen receptor (ER). In 1963, Jensen and Jacobsen used radioactively labeled estradiol to determine the target tissues of the “hormone of estrus.”9 This, eventually, lead to the discovery and characterization of the ER by Toft and Gorski.10, 11 In 1971, Jensen built on this discovery with the development of an ER assay that could predict clinical response to hormonal manipulation.12

The First Antiestrogens

Around the time of the seminal work resulting in the identification of the ER, scientists at ICI Pharmaceuticals were developing the first antiestrogens. The trans-isomer of triphenylethylene, initially named “ICI 46,474,” was discovered in an attempt to use antiestrogens for postcoital contraception. Ironically, this compound, eventually named “tamoxifen,” was later marketed as a fertility treatment. In the early 1970s, tamoxifen was shown to induce breast cancer regression.13 Since then, it has become a prototype drug for the hormonal treatment of cancer, with use in prevention and treatment of all stages of breast cancer. Tamoxifen is not a steroid and is associated with a better side effect profile than high-potency estrogen treatment, the contemporary treatment for advanced breast cancer at the time when tamoxifen was introduced.14 Because long-term tamoxifen administration is safe and well tolerated, clinical use in the adjuvant and prevention settings is possible.15 Indeed, the introduction of tamoxifen likely contributed to the recent decline in breast cancer mortality observed in high-incidence Western countries.16

Figure 37.1 The structure and numbering of the cyclopentane-perhydrophenanthrene nucleus.

Since the 1970s, the armamentarium of medical hormonal therapies has expanded considerably with aromatase inhibitors, luteinizing hormone-releasing hormone (LHRH) agonists, progestins, and selective estrogen down-regulators. A growing number of clinical trials are ongoing to improve the safety and efficacy of endocrine therapy for breast cancer.


Structure of Estrogen

Estrogens, like all steroids, have a hydrated four-ring structure (cyclopentane-perhydro-phenanthrene), in which a five-sided cyclopentane ring (designated the D ring) is attached to three six-sided phenanthrene rings (designated the A, B, and C rings) (Figures 37.1 and 37.2). The carbon atoms that form these rings are numbered 1 to 17. Additional side chain carbons in sex steroids are attached to either carbon atom 10 or 13 and carbon atom 18 for estrogens. Corticoids and progestins are characterized by a two-carbon side chain (carbons 21 and 22) attached to carbon atom 17.

Figure 37.2 The structure and names of the five major steroid hormones. (Trivial name is followed by systematic.) Cortisol, 4-pregnen-11β,17α, 21-triol-3,20-dione; aldosterone, 4-pregnen-11β,21-diol-18-al-3,20-dione; progesterone, 4-pregnen-3,20-dione; testosterone, 4-androsten-17β-ol-3-one; estradiol, 1,3,5(10)-estratrien-3,17β-diol.

The three endogenous estrogens are estradiol, estrone, and estriol. Estradiol is readily formed from estrone and can also be formed directly by aromatization of testosterone. Hydroxylation of estrone at the 16 position results in the formation of estriol, the other biologically important estrogen in humans. These compounds are shown in Figure 37.3. The trivial (or common) and systematic names of selected steroidal hormones used for cancer therapy are shown in Figure 37.2, together with their structures. Table 37.1 provides a list of the derivative names for compounds available to manipulate estrogen action for therapeutic effect. Detailed chemical descriptions and steroid nomenclature are available from a number of sources.17, 18, 19, 20 The physiologic effects of estrogens are summarized in Table 37.2.

Estrogen Biosynthesis

The ultimate source of estrogens, like all endogenous steroid molecules, is cholesterol, which derives directly from the diet or via endogenous synthesis. All tissues, except possibly the adult brain, can synthesize cholesterol, although quantitatively the liver is the most important source. Cholesterol binds to lipoprotein receptors and is then taken up by steroid-producing cells, where it is transferred to the inner membrane of the mitochondria. In this location, the cytochrome P-450 enzymes begin to convert cholesterol to different steroid hormones via alteration of side chains on the molecule.21 Figure 37.4 provides a schematic of the enzymatic steps in steroid biosynthesis.

The final step in estrogen synthesis is aromatization, which is catalyzed by the P-450 aromatase monooxygenase enzyme complex located in the endoplasmic reticulum. The aromatase enzyme complex consists of the P-450 cytochrome, P-450 arom, and a flavoprotein, nicotinamide adenine dinucleotide phosphate cytochrome P-450 reductase, that regenerates active aromatase after completion of the aromatization reaction.21 The active site of aromatase contains a heme complex responsible for the nucleophilic attack on the androgenic precursor C19 methyl group that generates formic acid and an aromatized A ring characteristic of estrogenic steroids (Fig. 37.5).22

Figure 37.3 Endogenous and synthetic estrogens.

The gene encoding the cytochrome component of aromatase, cytochrome P-450 (CYP) 19, has low homology with other members of the CYP family and has been mapped to chromosome 15.23 The hormonal regulation of the CYP19 gene, which spans 120 kb, is intricate, primarily due to a complex promoter structure, with regulatory elements targeted by gonadotrophins, glucocorticoids, growth factors, cytokines, and the intracellular signaling molecule cAMP.24, 25,26 Examples of peptide growth factors that may increase local estrogen production are the insulin-like growth factors (IGF-I and IGF-II), key players in breast cancer pathogenesis and ER function.27 IGF-I and IGF-II promote aromatase activity in stromal cells and the conversion of estrone to the more active molecule estradiol.28 There are a number of unique promoters that direct expression of the gene in a tissue-specific manner. In ovarian tissue, the proximal promoter II drives CYP19 expression and is regulated by FSH through cyclic AMP.29 Aromatase is also regulated by LH during the menstrual cycle.30 In adipose tissue, on the other hand, the distal promoter I.4 regulates CYP19 expression under the control of glucocorticoids and TNFα.30In tumor tissues, a switch in the aromatase gene promoters, from promoter II to promoter I.4, can occur, leading to increased aromatase activity and possibly to tumor initiation and/or promotion.30




Estrogens and antiestrogens




   Estradiol benzoate

1,3,5-Estratriene-3,17β-diol-3 benzoate





   Ethinyl estradiol


   Faslodex (fulvestrant)

7α-[9-(4,4,5,5,5-Pentafluoropentylsulfinyl)nonyl]estra-1,3,5, (10)-triene-3,17β-diol







4-Chloro-1,2-diphenyl-1-{4-[2-N,N-dimethylamino)ethoxy]- phenyl}-1-butane


[6-Hydroxy-2(4-hydroxyphenyl)benzo[b]thien-3-yl]{4-[2- (1-piperidinyl)ethoxy]phenyl}methanone-hydrochloride


   Medroxyprogesterone acetate


   Megestrol acetate


   Aromatase inhibitors






2,2′-[5-(1H-1,2,4-Triazol-1-ylmethyl)-1,3-phenylene]bis (2-methyl-propiononitrile)




Growth andmaintenance of female genitalia
Pubertal expression of female secondary sex characteristics
Breast enlargement
Increase in size and pigmentation of nipple and areolae
Molding of body contour with alteration of subcutaneous fat deposition
Promotionof female psyche formation
Alteration in skin texture
Maintenance of pregnancy(in concert with progestins)
Sodium retention

The pathways of estrogen synthesis described above are carried out in a number of different tissues. In premenopausal women, the principle source of estrogens consists of the ovaries, which primarily produce estradiol. Total blood concentrations of estradiol range from a low of approximately 10 pg/mL in the early follicular phase to as high as 500 pg/mL during midcycle. This peak is quite sharp and usually precedes the ovulatory gonadotropin surge.31 A second rise in serum estrogen occurs during the luteal phase and is lower but more prolonged. The cyclic nature of ovarian steroidogenesis occurs due to the cyclic secretory patterns of FSH and LH established by GnRH (LHRH) release from the arcuate nucleus of the hypothalamus. Steroidogenesis in the ovary is initiated by the binding of LH to LH receptors on theca interna cells, with the activation of adenylate cyclase, the formation of cAMP, and the activation of PKA. Uniquely, ovarian estrogens are synthesized by the cooperative action of two cell types. Androgens (either androstenedione or testosterone) are synthesized by ovarian thecal cells and converted to estrogens in the neighboring granulosa cells by aromatase.

Figure 37.4 Enzymatic steps in the biosynthesis of the steroid hormone.

In postmenopausal women, estrogens are not directly produced by the ovaries but are instead formed from the extragonadal conversion of ovarian and adrenal androgens via aromatase. Androgens from the ovaries and adrenal glands are released into the circulation and converted to estrogens in tissues with aromatase activity, including adipose tissue and muscle.32 Thus, in the postmenopausal state, estrogens may act primarily as paracrine factors, with much higher concentrations in local environments with high aromatase activity. This model is supported by findings of high aromatase activity in breast tumors.30

Figure 37.5 Aromatase reaction. (NADPH, nicotinamide adenine dinucleotide phosphate.)


Estrogen Metabolism

Estrogen metabolism occurs primarily in the liver, where there is free interconversion between estrone and estradiol.33 Equilibrium slightly favors estrone, which probably serves as the main precursor for the hydroxylated estrogen metabolites in the urine.34 Endogenous estrogens are excreted in the urine predominantly as glucuronides and sulfates, although numerous other water-soluble metabolites have been identified.35 These conjugates undergo enterohepatic recirculation via hydrolysis by bacteria in the intestine and then reabsorption into the enteric blood supply. More than half of the estrogen metabolites and one third of the progesterone metabolites are excreted in the bile shortly after the administration of radioactive hormone. Eventually, 50 to 80% of an administered dose is excreted as metabolites in the urine within 4 to 6 days, and up to 18% may be found in the feces. In the liver, two systems for sulfation exist, one for the estrogens and the other for 3β-hydroxy steroids. Glucuronides are formed from diphosphoglucuronic acid by the microsomal enzyme glucuronyl transferase.35

Estrogens also undergo hydroxylation and methylation in the liver and other tissues, resulting in the formation of catechol and methoxylated estrogen metabolites. These metabolic pathways are important because the resultant products are active compounds with differing biologic properties. Hydroxylation of estrogens leads to the formation of 2-hydroxyestrogens (2-HE), 4-hydroxyestrogens (4-HE), and 16α-hydroxyestrogens (16α-HE).21, 36 Both 4-HE and 16α-HE are known to be estrogenic and are hypothesized to be carcinogenic due to their ability to form DNA adducts and create gene mutations.37 In contrast, methylation of 2-HEs and 4-HEs yields a number of anticarcinogenic methoxylated metabolites. Thus, the proportion of carcinogenic versus anticarcinogenic metabolites generated from estrogens appears to be a balance between the 2-HE and 16α-HE reactions. Genetic polymorphisms affecting these metabolic pathways have been linked to an alteration of breast cancer risk.36, 38, 39 Further details of estrogen metabolism and physiology can be found in other sources.40, 41

Estrogen Transport

Estrogens, like other steroids, circulate in the bloodstream predominantly bound to albumin and steroid-binding globulins. The estrogens and androgens are transported via testosterone and estradiol-binding globulin (TEBG) or sex steroid–binding globulin.41 The unbound, or free, hormone, which make up only 2 to 3% of total circulating estrogens, enters the cell by a non- energy-dependent process, the cell membrane providing a favorable lipid-rich environment for passage of the hormone by diffusion.21 Once inside the cell, estrogens bind to the estrogen receptor (ER), thereby starting a complex series of signaling events in the target cell.


ER Structure

The ER is a member of the nuclear hormone receptor superfamily that includes the progesterone receptor (PgR), androgen receptor (AR), glucocorticoid receptor (GR), and mineralocorticoid receptor. This receptor family also includes receptors for nonsteroidal nuclear hormones such as the retinoids (retinoid alpha receptor and retinoid X receptor), vitamin D or deltanoids, and thyroid hormone. The amino acid homologies that define this receptor family are illustrated in Figure 37.6.

The ER, like most nuclear hormone receptors, operates as a ligand-dependent transcription factor that binds to DNA at estrogen response elements to direct changes in gene expression in response to hormone binding.21, 42 The ER protein structure includes six domains, designated A to E (Fig. 37.6). Estradiol binds to the ligand-binding site in the E domain. The E domain also mediates ER dimerization, with assistance from residues in domain C. The sequence-specific DNA-binding function resides in domain C. Domain D contains a nuclear localization signal required for transfer of the ER from the cytoplasm to the nucleus. Domains that promote transcription, or activation functions (AFs), are present in domains A and B (AF1) and domain E (AF2). The basic structure and functional components of steroid hormones follow the same pattern, with a hormone-binding site, a dimerization domain, transactivation domain(s), and a nuclear localization signal.43

ER Subtypes: ERα and ERβ

The first identified ER, now known as “ERα,” was discovered in the late 1960s, and the gene was cloned in 1986. The second ER, named “ERβ,” was first reported in 199644 and has provided a further layer of complexity to our understanding of estrogen-regulated gene expression.45

Furthermore, each subtype of ER exists in several isoforms. While ERα has been studied more extensively, the role of ERβ in breast cancer and endocrine therapy sensitivity is beginning to be elucidated.45

Figure 37.6 Examples of amino acid homology between nuclear hormone receptors. (A/B, activation function 1 transactivation domain; C, DNA-binding and homodimerization domain; D, nuclear localization signal and heat shock protein 90–binding domain; E, activation function 2 transaction domain.)

At the amino acid level, ERα and ERβ are highly homologous in the DNA-binding domain (96%), but the homology in the ligand-binding domain (LBD) is only 58%. This structural comparison suggests that the two subtypes would recognize and bind to similar DNA sites. However, the differences in the LBD suggests that responses to different ligands may be more distinct than anticipated from primary sequence analysis,46 Experimental data have shown that affinities for estrogens and antiestrogens do vary significantly between ERα and ERβ.47

In contrast to the hormone- and DNA-binding domains, ERα and ERβ are not homologous in the N-terminal A and B (transactivation) domains, and, as a result, the transcriptional properties of ERα and ERβ are dissimilar, as discussed later in the “Hormonal Resistance” section.

Consensus on the clinical significance of ERβ expression in breast cancer has been elusive, with some conflicting findings in many different studies. However, Speirs et al. have observed that breast cancers that coexpress ERα and ERβ tend to be node-positive and a higher grade than tumors that express ERα alone,48 and Dotzlaw et al. noted a tendency for ERβ-expressing tumors to be PgR-negative.49 These correlations suggest an adverse effect of ERβ expression on prognosis. Other work, on the other hand, suggests that decreased levels of ERβ may be associated with increased tumorigenesis.45

Another major difference between ERα and ERβ is tissue expression. ERα is primarily expressed in classical estrogen target tissues such as the uterus, mammary gland, placenta, liver, CNS, cardiovascular system, and bone. ERβ is highly expressed in nonclassical estrogen target tissues such as prostate, testis, ovary, pineal gland, thyroid, parathyroid, adrenals, pancreas, gallbladder, skin, urinary tract, lymphoid, and erythroid tissues.50 As might be expected, ERβ is therefore less important than ERα for normal reproductive organ development and function in mouse models.51 As further study of this ER subtype progresses, however, additional actions of estrogens may be better understood.


Ligand-dependent ER Signaling

The well-described classical ER signaling pathway is illustrated in Figure 37.7. Estrogens bind to the LBD of the ER, leading to the release of the receptor from heat shock protein (HSP) 90. This ligand binding is then followed by phosphorylation of the receptor at specific serine residues, ER dimerization, and then sequence-specific DNA binding to a sequence referred to as an “estrogen response element” (ERE). In the presence of estrogen, messenger RNA (mRNA) transcription is promoted though AF2. Residues in AF1 also promote transcription, although the function of AF1 does not require the presence of estrogen.50The consensus sequence GGTCAnnnTGACC has been defined as the ERE. Like other steroid hormone response elements, this is a palindromic sequence separated by a spacer sequence, which varies in length according to the cognate receptor. The consensus DNA binding sites for each of the nuclear hormones are illustrated in Figure 37.8. While the ER binds most strongly to the ERE consensus sequence, it is also capable of promoting transcription through sequences that have only partial homology to a classic ERE. In these cases, nearby response elements for other transcription factors (e.g., SP-1) contribute to ER activity.50, 51, 52 The characteristics of the target gene promoter are critical to the specific nuclear actions of the activated ER. Other factors that are critical are the structure of the bound ligand and, as discussed later, the balance of coactivators and corepressors associated with the ER-ligand complex. In addition, ERα and ERβ can either homodimerize or heterodimerize, and this has an impact on their activity at the DNA binding site.53

Figure 37.7 Simplified operational details of nuclear hormone action, using the estrogen receptor as an example. (E, estrogen-occupied ligand-binding site; ER, estrogen receptor; ERE, estrogen response element; HSP, heat shock protein.)

ER Coactivators and Corepressors

Ligand-bound receptors interact with a family of “coactivator” and “corepressor” proteins that are sensitive to the conformational changes that occur in the LBD of each receptor. These coregulatory proteins interact with the ER to either increase or decrease transcriptional activity at a promoter site. One key mechanism for the coactivator and corepressor modulation of ER transcription likely involves alteration of histone acetylation.54 On ligand binding, a histone deacetylase–containing corepressor complex is displaced from the nuclear receptor in exchange for a histone acetyltransferase–containing coactivator complex. As their name implies, histone acetyltransferases catalyze the acetylation of histones, thereby altering chromatin structure. When histones become acetylated in the vicinity of the liganded nuclear receptor, DNA becomes unwound, or open, allowing access to the RNA polymerase complex, and transcription is initiated.55 In addition to alteration of chromatin structure and histone acetylation, coactivators may also promote interactions between the nuclear hormone receptor and the basal transcriptional machinery to activate gene transcription.43

Figure 37.8 Consensus core response element motifs for nuclear hormone receptors. (AR, androgen receptor; ARE, androgen receptor response element; DR, direct repeat; ER, estrogen receptor; ERE, estrogen response element; GR, glucocorticoid receptor; GRE, glucocorticoid response element; IP, inverted palindrome; n, any nucleotide; P, palindrome; PgR, progesterone receptor; PRE, progesterone receptor response element; RAR, retinoic acid receptor; RARE, retinoic acid receptor response element; T3RE, triiodothyronine receptor response element; VDRE, vitamin D receptor response element.)

An increasing number of proteins have been described with coactivator properties. Some ER coactivators actually possess intrinsic histone acetyltransferase activity. Other coactivators, such as p160 coactivators, augment ER-mediated transcription by recruiting other proteins with chromatin-modifying activity. There are three p160 coactivators, NCoA-1 (SRC-1), NCoA-2 (TIF2, GRIP1), and NCoA-3 (AIB1, ACTR, RAC3, p/CIP, TRAM-1).43 The p160 coactivators recruit other transcriptional coactivators and histone acetyltransferases such as p300, CBP (CREB–binding protein), and pCAF (p300/CBP-associated factor).56 The complex of proteins assembled around the ER promotes histone acetylation to allow gene transcription to occur. An additional group of coactivators, including TRAP220 (thyroid hormone receptor–associated protein, DRIP205), PGC-1, SNURF, PELP1, and NCoA-7, bind to the ER and allow interaction with transcriptional machinery.43

Corepressors have the opposite function and negatively regulate transcription via recruitment of histone deacetylases.57 The list of corepressors is shorter but is also growing and includes the nuclear receptor corepressor (N-CoR) and silencing mediator for retinoid and thyroid receptors (SMRT). Other recently identified corepressors, such as SHP, DAX-1, and ER-specific corepressor, act by competing with p160 coactivators for binding to the ER.58

In general, coactivators and corepressors appear to be expressed at similar levels in many different tissues, suggesting that the responses to estrogen agonists and antagonists are not determined simply by the relative abundance of these cofactors. Instead, it appears that differential regulation of coactivator activity occurs through other signal transduction pathways.54


ER Activation by Other Signal Transduction Pathways

The classical ER signaling pathway, as described earlier, is perhaps the best-studied mechanism of ER signal transduction, but recently other pathways have been described (Fig. 37.9). It is important to emphasize that nuclear hormones are not simply receptors for lipid-soluble hormones but are also critical signaling targets for protein phosphorylation–dependent second messenger pathways.59 These functional relationships, or cross-talk, with growth factor pathways suggest that nuclear hormone receptors have an integration function that ultimately determines the cellular response to a complex set of extracellular signals.

ER expression and function are strongly influenced by growth factor signaling (Fig. 37.9 B). As a result, ER expression levels correlate with distinct patterns of growth factor receptor overexpression. When ErbB2 or EGFR is activated in experimental systems, ER expression is suppressed. For example, chronic activation with heregulin, a ligand for the ErbB family of receptors, can lead to ER down-regulation and the acquisition of an ER-negative phenotype.60, 61These data suggest that EGFR and ErbB2 signaling can bypass the requirement for estrogen for breast cancer cell growth and drive breast cancer cells into an ER-negative, endocrine therapy–resistant state.62

Figure 37.9 More complex models of estrogen receptor (ER) models. A: Estrogen (E) promotes coactivator (CoA) interactions that activate the general transcription apparatus (GTA). Tamoxifen (T) promotes corepressor (CoR) interactions that prevent activation of the general transcription apparatus. B: Estrogen receptor is phosphorylated by protein kinases activated by growth factors and neurotransmitters. C: ER interacts with other DNA-binding transcription factors to modulate the transcription of genes that do not possess an ERE. (cAMP, cyclic adenosine monophosphate; ERE, estrogen response element; MAP, mitogen-activated protein kinase; P, phosphate.)

In other circumstances, EGFR and Erb2 signaling can activate the ER in a estrogen-independent manner. Signaling of these growth factors through the mitogen-activated protein (MAP) kinase cascade leads to phosphorylation of the Ser118 residue in the ERα AF-1 domain.63, 64 This, in turn, leads to recruitment of coactivators, allowing ligand-independent gene transcription by the ER.60, 61, 62

In a similar manner, signaling through the IGF-I receptor provides another example of a positive interaction between growth factor signaling and ER function. Several key components of the IGF system (e.g., the IGF-I receptor and the signaling intermediate insulin receptor substrate-I) are regulated by estrogen. As a result, IGF-I and estrogen synergistically promote the growth of breast cancer cells.65, 66 The angiogenic fibroblast growth factor family also regulates ER function, with induction of tamoxifen resistance, in an animal model.67 Finally, the neurotransmitter dopamine and the second messenger cAMP also influence ER function through phosphorylation.59 Interestingly, activation of cAMP leads to phosphorylation in the AF2 domain, altering the agonist and antagonist response to tamoxifen. This suggests a role for cAMP in modulating the tissue-specific effects of tamoxifen.59 As discussed in the “Hormonal Resistance” section, insights into the cross-talk between the ER and other signal transduction pathways provide a potential strategy for novel therapeutic combinations as well as an approach to new predictive tests for endocrine therapy sensitivity.

Interaction of the ER with Other Transcription Factors

In another departure from the classical ER signaling model, ER-mediated transcription can also occur without direct contact with DNA (Fig. 37.9 C). In these instances, the ER operates in conjunction with a second transcription factor that provides the sequence-specific DNA-binding function. Through this indirect mechanism, the ER can influence transcription through a greater variety of promoter sequences. Examples include the AP1 site (a target for many signals involved in cellular proliferation),68, 69 NFκB sites,70 and a polypurine tract in the transforming growth factor-beta promoter referred to as a “raloxifene response element.”44 Because AP1 transcription factors, such as c-fos and c-jun, are key regulators of cell growth, ER-dependent AP1 activation may be critical to estrogen-dependent cell cycle progression. Furthermore, new classes of antiestrogen do not have the same profile of activities at AP1 sites as tamoxifen, which may help to explain differences in the clinical activity between compounds.68

Plasma Membrane ER Signal Transduction

With classical ER signaling pathways, the induction of gene transcription by estrogen can take 30 to 45 minutes, with another few hours required to see protein translation.71, 72 The observation that some estrogen effects can occur within seconds to minutes suggests a nongenomic component to ER signaling. In 1977, Pietras and Szego demonstrated that a plasma membrane form of the ER is present in cells, and this receptor was shown to respond to estradiol.73, 74Over the past several years, this plasma membrane ER signaling pathway has received increasing attention.

Within a few seconds, estradiol can stimulate an intracellular calcium increase, adenylate cyclase activation, and phospholipase C activation.71 These events occur through activation of the membrane ER, leading to association with G proteins. In endothelial cells, this interaction leads to generation of nitric oxide, with resulting vasodilation.71 In addition, plasma membrane ER signaling can lead to the activation of EGFR, with subsequent activation of the Ras-Raf-MAPK signaling cascade.75 The observation that the membrane ER can activate the EGFR-Ras-Raf-MAPK cascade is particularly interesting and suggests a feed-forward signaling circuit where the ER on the plasma membrane activates the EGFR pathway, which then increases ER activity in the nucleus. This interlaced complexity of estrogen signaling suggests the possibility of potentiating hormonal therapy through concomitant signal transduction inhibition.



High-dose estrogen therapy was the first medical treatment for metastatic breast and prostate carcinoma and has been used for nearly 6 decades.76 While the antiandrogenic actions of estrogen explain the activity of estrogen against prostate cancer, the mechanism for antitumor activity in breast cancer remains unclear. Physiologic dosages of estradiol stimulate receptor-positive tumor cell growth in vitro77, 78 and in vivo.79 However, high dosages inhibit breast cancer growth in vitro80 and in vivo.81 There are a number of possible explanations for the paradoxical inhibitory effects of high-dose estrogens. One possibility is that excess estrogen causes an imbalance of estrogen-ER complex and coactivators that are critical to ER activity. Thus the altered stoichiometry of the estrogen-ER complex with coactivators causes the number of active ER complexes to decrease.82 In addition, it has been observed that high-dose estrogens cause a decrease in the level of ER in the cell; this also may explain the inhibitory effect on breast cancer cells.78 Finally, one study suggested that estrogens promote chromosomal nondisjunction, which could cause loss of cellular viability.83


Estrogen Preparations

Several different estrogens have been used in clinical trials in the treatment of breast and prostate cancer. These include polyestradiol phosphate, micronized 17β-estradiol, ethinyl estradiol, conjugated equine estrogenic hormones, DES diphosphate, estradiol undecylate, stilbestrol, and DES.84 The most widely used agent has been DES, a nonsteroidal synthetic estrogen; however, this agent is no longer freely available in the United States. Nonetheless, these agents are briefly discussed, as the therapeutic value of high-dose estrogen continues to be explored even after exhausting the many different hormonal therapies for metastatic breast cancer.85

The relative potency of several estrogens has been assayed by determining their effects on plasma FSH, a measure of the systemic effect, and by measuring increases in SHBG, CBG, and angiotensinogen, all of which indicate the hepatic effect. Piperazine estrone sulfate and micronized estradiol were equipotent with respect to increases in SHBG, whereas conjugated estrogens were 3.2-fold more potent, DES was 28.4-fold more potent, and ethinyl estradiol was 600-fold more potent. With respect to decreased FSH, conjugated estrogens were 1.4-fold, DES was 3.8-fold, and ethinyl estradiol was 80- to 200-fold more potent than piperazine estrone sulfate. The dose equivalents for ethinyl estradiol (50 µg) and DES (1 mg) reflect these relative potencies.86 Intravaginal administration of creams containing either conjugated estrogens or 17β-estradiol results in substantial pharmacologic levels of estradiol and estrone, with subsequent decreases in LH and FSH.87 Therefore, vaginal creams containing estrogens should be used with caution for patients who are at high risk of developing breast cancer or who have a history of early breast cancer.


DES, a potent synthetic estrogen (see Fig. 37.12), is absorbed well after an oral dosage. Patients given 1 mg of DES daily had plasma concentrations at 20 hours ranging from 0.9 to 1.9 ng/mL. The initial half-life of DES is 80 minutes, with a secondary half-life of 24 hours.88 The principal pathways of metabolism are conversion to the glucuronide and oxidation. The oxidative pathways include aromatic hydroxylation of the ethyl side chains and dehydrogenation to (Z, Z)-dienestrol, producing transient quinone-like intermediates that react with cellular macromolecules and cause genetic damage in eukaryotic cells.89Metabolic activation of DES may explain its well-established carcinogenic properties.90

Ethinyl Estradiol

Ethinyl estradiol, a more potent estrogen than DES, has a biologic half-life in plasma of approximately 28 hours. It is excreted in urine as a glucuronide and as unchanged drug. It is 600-fold more potent than piperazine estrone sulfate and 22-fold more effective than DES in increasing sex hormone–binding globulin (SHBG), a parameter of estrogen potency.86 The usual dosage is 1 mg three times a day for female breast cancer and 150 µg per day for prostate cancer.


Despite substantial response rates in breast cancer, serious complications of estrogen therapy, including exacerbation of ischemic heart disease, hypertension, congestive heart failure, venous thromboembolic disease, and cerebral ischemia, have limited the value of this treatment approach.14, 91 These side effects are dose-dependent and most troublesome with DES doses of 5 mg daily. In addition, estrogens induce increased platelet aggregation and increases in factor VII and plasminogen, as well as decreased antithrombin III.92, 93, 94 Hypertension is believed to result from estrogen-related fluid retention. Other side effects of exogenous estrogens include nausea and vomiting, diarrhea, abdominal cramps, anorexia, and glucose intolerance. Chloasma, erythema multiforme, erythema nodosum, hirsutism, and alopecia have been reported.94, 95 Estrogens cause various central nervous system side effects, including dizziness, headache, and depression. Keratoconus, or change in the corneal curvature, has been noted, with resultant intolerance to contact lenses for patients treated with estrogens. Hypercalcemia and increased bone pain are associated with a greater likelihood of subsequent antitumor response. Women may develop pigmentation of the nipples, vaginal bleeding, urinary urgency, and incontinence. Premenopausal women report mastodynia, venous dilatation of the breast, amenorrhea, and dysmenorrhea. Men develop gynecomastia and mastodynia. The risk of gallbladder disease is higher in postmenopausal women taking conjugated estrogens.96Elevated liver function tests and cholestatic jaundice have been seen. Hepatocellular adenomas have been reported in oral contraceptive users.97 The use of conjugated estrogens without a progestational agent is associated with an increased risk of endometrial carcinoma.98 Estrogens are rarely used to treat premenopausal breast cancer, but they should never be administered to pregnant patients. Vaginal adenosis is found in 66.8%, and vaginal or cervical ridges are found in 40% of DES-exposed offspring.99 Also, the occurrence of clear cell adenocarcinoma of the vagina has been determined to correlate with DES exposure in utero.100 A recent analysis of DES-exposed offspring reveals a risk through age 34 of 1 case per 1,000 women. In addition there is an association between intrauterine DES exposure and testicular cancer in men.101, 102

Drug Interactions

Inducers of the hepatic microsomal enzymes, such as rifampin, barbiturates, carbamazepine, phenylbutazone, phenytoin, and primidone, enhance the metabolism of estrogen and decrease estrogenic activity. Estrogens have been reported to decrease the activity of oral anticoagulants because of the induction of the synthesis of clotting factors. Estrogens increase the half-life and pharmacologic effects of glucocorticoids but induce the metabolism and anticonvulsant activity of phenytoin and other hydantoin anticonvulsants.



Tamoxifen is a nonsteroidal triphenylethylene first synthesized in 1966. Activity in metastatic breast cancer was first described in the early 1970s, and tamoxifen rapidly became the drug of choice for advanced disease, with response rates ranging from 16 to 56%.103, 104, 105 Tamoxifen became the preferred drug not because it proved better than contemporary alternatives but because it was found to be safe and easy to tolerate.14, 106, 107 In fact, the tolerability of tamoxifen was one of the chief reasons for the success of tamoxifen adjuvant and prevention trials, as patients are able to take the drug for prolonged periods of time with acceptable levels of toxicity.

Tamoxifen: The First Selective Estrogen Receptor Modulator (SERM)

Tamoxifen affects organ systems besides the breast. Organs affected by tamoxifen administration include the endometrium (endometrial cancer and hypertrophy),108, 109, 110 the coagulation system (thrombosis),111, 112 bone (modulation of mineral density),113, 114 and liver (alterations of blood lipid profile).115, 116 In the organ systems listed here, tamoxifen generally acts as an agonist, mimicking the effect of estrogen, in contrast to its action on breast epithelial cells, where it generally acts as an antagonist. Therefore, tamoxifen is correctly described as a selective estrogen receptor modulator (SERM) with organ site–specific mixed agonist and antagonist effects. The agonist properties of tamoxifen also manifest in the treatment of advanced breast cancer. Flare reactions, withdrawal responses, and the experimental demonstration of breast tumor growth stimulated by tamoxifen (see the “Hormonal Resistance” section) are evidence that tamoxifen can operate as an agonist in breast tissue under certain circumstances.

Figure 37.10 Tamoxifen and metabolites. (Adapted with permission from Lyman SD, Jordan VC. Metabolism of nonsteroidal antiestrogens. In: Jordan VC, ed. Estrogen/Antiestrogen Action and Breast Cancer Therapy. Madison: University of Wisconsin Press, 1986:191.)

Tamoxifen Metabolism

The metabolism of tamoxifen is complex and has been extensively studied by thin-layer chromatography, high-pressure liquid chromatography, and gas chromatography.117 Ten major metabolites have been identified in patient sera (Fig. 37.10 and Table 37.3).118, 119, 120 An excellent review of tamoxifen and its metabolism is available that presents in schematic form the proposed metabolic pathways of tamoxifen.121 Originally, it was believed that 4-hydroxytamoxifen was the major metabolite,117 but Adam et al., by using a different solvent system, found that the major plasma metabolite was N-desmethyltamoxifen.122 Metabolism of tamoxifen is mediated in the liver by cytochrome P-450–dependent oxidases. The metabolites are excreted largely in the bile as conjugates, with little tamoxifen eliminated in the unchanged form. In two subjects given 14C-labeled tamoxifen, Fromson et al. found that 74 to 78% of the radioactivity was recovered in the feces and 9 to 14% was recovered in the urine.123 Sutherland et al. reported that impaired renal function does not result in elevated blood levels of tamoxifen.124 Also, metabolite BX, or 4-hydroxy-N-desmethyl-tamoxifen, has been detected in serum from patients receiving tamoxifen; its biologic significance is unknown at the present time.125, 126


Mechanism of action

Changes folding of the steroid-binding domain, preventing gene activation of estrogen receptor

Blocks cellsin mid-G1, may be mediated by cyclin D1

Blocks estrogen-stimulated progesterone receptor synthesis, 52-K protein synthesis, DNA polymerase activity, and tritiated thymidine incorporation

Stimulates production of T-cell growth factor β, decreases insulin- like growth factor-I


Metabolite B: 4-hydroxytamoxifen

Metabolite E: (loss of basic side chain)-phenol

Tamoxifen N-oxide

Metabolite X: N-desmethyltamoxifen

Metabolite Y: primary alcohol

Metabolite Z: desdimethyltamoxifen

Metabolite BX: 4-hydroxy-N-desmethyltamoxifen

Metabolites A, C, D, F: (tentative)

Relative binding affinity for estrogen receptor

17 β-Estradiol = 100

Tamoxifen = 6

Metabolite B = 280

Metabolite E = 3

Tamoxifen N-oxide = 6

Metabolite X = 4

Metabolite Y = 0.5



t1/2α4–14 hr

t1/2α > 7 d

Steady state, 4–16 wk


t1/2 9.8–14 d

Steady state,8 wk


Conjugation with biliary excretion

Drug interactions

Potentiates mild hepatotoxicity induced by chronic allopurinol treatment

Medroxyprogesterone acetate and aminoglutethimide affect metabolism

? Inhibition of warfarin metabolism


Rat on cogenicity study—hepatocellular carcinoma at doses of 5–35 mg/kg/d and increased incidence of cataracts

Intraretinal opacities, primarily paramacular, and optic neuritis

Venous thrombosis and pulmonary embolism

Hepatitis, fatty liver, two cases of hepatic carcinoma

Thrombocytopenia (2–5%)

Increased incidence of endometrial carcinoma

Severe lipemiain patients with prior history of hypertriglyceridemia


t1/2, half-life.

The estrogen agonist and antagonist effects of the metabolites of tamoxifen have been tested in the rat and mouse systems.118, 127, 128 Agonist effects are seen as positive uterotropic effects, and antagonist actions as blocking the uterotropic effects of estrogen.118 It must be mentioned, however, that tamoxifen acts as an estrogen agonist in mice but as a partial agonist in rats and that the metabolite effects differ in these species. Metabolite B, or 4-hydroxytamoxifen, is an estrogen agonist in mice but a mixed agonist and antagonist in rat uteri. Metabolite D is an estrogen antagonist in rat uteri and a mixed agonist and antagonist in mice. Metabolite E is the only metabolite that has no antagonist properties in any system but acts as a pure agonist. Metabolites X and Y are mixed estrogen agonists and antagonists. Metabolite A has antiuterotrophic effects in rats. Finally, tamoxifen-N-oxide acts as a pure estrogen antagonist for MCF-7 cell proliferation.128

Tamoxifen Pharmacokinetics

Data concerning steady-state plasma levels and the relative potency of binding to the ER suggest that the biologic actions of tamoxifen are exerted by the parent compound and its 4-hydroxy metabolite.129, 130, 131, 132, 133, 134 Estradiol is present in plasma in the range of 15 to 48 pg/mL. Tamoxifen equilibrates at levels of 300 ng/mL, its N-desmethyl metabolite at 470 ng/mL, and 4-hydroxytamoxifen at 7 ng/mL.127, 134 Because 4-hydroxytamoxifen binds to the ER with 25 to 50 times the affinity of tamoxifen and with an affinity equal to that of estradiol, tamoxifen and 4-hydroxy-tamoxifen may exert an equal biologic action. N-desmethyltamoxifen is probably a minor contributor to the therapeutic effect of tamoxifen, because, despite a higher plasma concentration in plasma, its binding affinity for ER is 1,250 less than that of 4-hydroxytamoxifen. Another active metabolite, 4-hydroxy-N-desmethyl-tamoxifen, was recently identified which is present in the plasma at an average of 12.4 ng/mL in women on chronic tamoxifen therapy.126 The plasma levels of tamoxifen and metabolites remain roughly proportional to dosage over the therapeutic dosage range, indicating no saturation of metabolic pathways.133, 135

The initial plasma half-life of tamoxifen ranges from 4 to 14 hours, depending on the study, with a secondary half-life of approximately 7 days.117, 123, 130, 136Steady-state concentrations of tamoxifen are achieved after 4 to 16 weeks of treatment.130 The biologic half-life of the metabolite N-desmethyltamoxifen is 14 days, with a steady-state concentration reached at 8 weeks. These long half-lives reflect the high level of plasma binding to protein (greater than 99%) and enterohepatic recirculation.130 Only free tamoxifen or metabolites can bind to ERs. Tamoxifen persists in the plasma of patients for at least 6 weeks after discontinuation of treatment.136 Because of the long plasma half-life of tamoxifen, at least 4 weeks are required to reach steady-state levels in plasma, leading several investigators to explore the use of loading doses. In general, these investigators aimed at achieving plasma levels of 150 ng/mL, the lowest steady-state concentration observed for patients responding to the drug. Loading doses of 80 mg/m2 twice daily yielded levels of 225 ng/mL at 3 hours after the first dose, whereas 50 mg/m2 twice daily yielded proportionately lower levels, barely exceeding 150 ng/mL. Another study using 100 mg/m2 over 24 hours on day 1 confirmed that peak concentrations of tamoxifen exceeded 150 ng/mL by the end of day 1 and could be maintained above that level by a daily dose of 20 mg.136, 137 Although studies of loading doses are of theoretical interest, their clinical relevance is uncertain. It is likely that tamoxifen and its 4-hydroxy metabolite are present in excess at all dosage levels used clinically. A thorough analysis indicates that a single dose of 20 mg a day is the most appropriate approach to tamoxifen administration.136 Because most tamoxifen is bound to serum proteins, tamoxifen is present in low concentrations in the cerebrospinal fluid,138 suggesting the response to tamoxifen is likely to be poor in leptomeningeal disease and central nervous system metastasis.

Intratumoral Tamoxifen Metabolism

Osborne et al. measured tamoxifen metabolites in 14 breast tumors.139 The metabolite 4-hydroxytamoxifen exists in the trans (potent antiestrogen) and cis (weak antiestrogen) forms. In nonresponding patients, all except one had reduced tumor tamoxifen levels or a high cis-trans ratio of the metabolite. It was suggested that these metabolic abnormalities might contribute to tamoxifen resistance. Another group investigated this hypothesis in an animal system; it concluded that the metabolism and isomerization of tamoxifen to more estrogenic compounds were not mechanisms of tamoxifen resistance.140 Levels have been measured in tumor biopsy specimens after a daily dose of 40 mg.134 The mean concentration of tamoxifen was 25.1 ng; of N-desmethyltamoxifen, 52 ng; and of 4-hydroxytamoxifen, 0.53 ng per mg of protein. These concentrations are sufficient to prevent specific binding of estradiol to ER in human tumors in vitro.

Drug Interactions

The CYP3A family is responsible for N-demethylation of tamoxifen. Many other drugs are substrates for this enzyme family, such as erythromycin, nifedipine, cyclosporine, testosterone, diltiazem, and cortisol. The combination of tamoxifen with any of these drugs could potentially interfere with tamoxifen metabolism.141 Mani et al. found tamoxifen N-demethylation is catalyzed in humans by CYP3A enzymes, whereas 4-hydroxylation is catalyzed by CYP2D6.142,143 None of the inducers of P-450 tested was able to elevate the rate of 4-hydroxylation. Inducers of P-450 enzymes do enhance N-demethylation, but the clinical relevance of this information is unknown at this time. CYP2D6 is commonly involved in drug hydroxylation. There is an absence of hepatic CYP2D6 in 8% of Caucasians, defining a group in which 4-hydroxytamoxifen would not be produced efficiently.

The activity of CYP2D6 is also reduced by serotonin selective reuptake inhibitor antidepressants (SSRIs).144 In view of the widespread use of SSRIs in patients with a history of breast cancer, a detailed study was done to determine the effects on tamoxifen metabolites.145 Levels of 4-hydroxy-N-desmethyl-tamoxifen, a metabolite generated via CYP3A4 and CYP2D6 activity, was 64% lower in women taking SSRIs. While the clinical significance of this finding is unclear, it does raise the possibility that a common combination of drugs may alter clinical results. Tamoxifen also lowers plasma levels of the aromatase inhibitor letrozole, indicating that these two agents should not be administered together.146 Medroxyprogesterone acetate has been found to alter the metabolism of tamoxifen.147 Also, serum concentrations of tamoxifen and the metabolites Y, B, BX, X, and Z are reduced with the use of aminoglutethimide combined with tamoxifen.

Finally, reports of an interaction between warfarin and tamoxifen have prompted the manufacturer to list concurrent warfarin use as a contraindication.148Supratherapeutic effects of warfarin have been reported with tamoxifen use; however, the number of cases is small. The mechanism of this interaction is likely inhibition of hepatic metabolism of warfarin by tamoxifen. While in some clinical settings, the potential benefits of tamoxifen may outweigh the risks, it is critical to monitor coagulation indices closely when warfarin and tamoxifen are prescribed together.148

Tamoxifen Side Effects

Although tamoxifen remains the first-line endocrine therapy for early-stage and advanced breast cancer, important side effects should be considered. The tamoxifen chemoprevention trial, National Surgical Adjuvant Breast Project (NSABP) P01, established one of the most accurate sources of information on tamoxifen toxicity, because the true incidence of tamoxifen side effects was not obscured by tumor-related medical problems.149 In NSABP P01, the excess incidence of serious adverse events (pulmonary embolus, deep venous thrombosis, cerebrovascular accident, cataract, and endometrial cancer) for patients receiving tamoxifen therapy was five to six events per 1,000 patient years of treatment. Less serious but troublesome side effects of tamoxifen included hot flashes, nausea, and vaginal discharge. Depression is also considered a side effect of tamoxifen, although there was no clear evidence of this association for women who received tamoxifen or placebo in NSABP P01. In summary, tamoxifen therapy is usually well tolerated and safe, and serious side effects occur in approximately 1 in 200 patients annually.

Ophthalmologic Side Effects

Tamoxifen retinopathy, with macular edema and loss of visual acuity, was first reported in patients receiving 120 to 160 mg per day. Three of the four originally reported patients also had corneal opacities.150 The retinal lesions are superficial, white, refractile bodies 3 to 10 µm in diameter in the macula and 30 to 35 µm in diameter in the paramacular tissues, and they occur in the nerve fiber layer, suggesting that they are products of axonal degeneration.151Additional cases have been described among patients receiving 30 to 180 mg per day.152 Other ophthalmologic findings reported include optic neuritis, macular edema, crystalline macular deposits with reduced visual acuity, intraretinal crystals with noncystoid macular edema, refractile deposits in the paramacular areas with progressive retinal pigment atrophy, bilateral optic disc edema with visual impairment and retinal hemorrhages, tapetoretinal degeneration, and two cases of superior ophthalmic vein thrombosis.152, 153, 154, 155 It is worth emphasizing that, in the chemoprevention study, the only significant optic toxicity at increased incidence compared with placebo was cataract.149


The increased risk of thrombosis associated with tamoxifen therapy may be associated with decreased levels of antithrombin III levels. Enck and Rios found a decreased functional activity of antithrombin III in 42% of tamoxifen-treated patients.111 Pemberton et al. found reduced antithrombin III and protein C levels in women taking tamoxifen.156 The incidence of venous thrombosis in this study was 5.62%, compared with 0% in controls. The generally accepted rate is lower. In the P01 prevention trial, the average annual rates per 1,000 women of stroke, pulmonary embolism, and deep venous thrombosis were increased by 0.53, 0.46, and 0.50 cases respectively.149 The NSABP B-14 study reported thromboembolic events in 12 patients (0.9%), with one fatal pulmonary embolus, compared with two thromboemboli (0.2%) in controls,157 a rate similar to that occurring in the P01 prevention trial.149

Hematologic Side Effects

Thrombocytopenia occurs in 5% of patients and is usually transient, resolving after the first week of treatment. Leukopenia is less frequent and is also transient.158


Changes in serum lipoproteins have been noted in patients taking tamoxifen. Changes occur that are indicative of an estrogenic effect of tamoxifen: an increase in total triglycerides, a decrease in total cholesterol, an increase in low-density lipoprotein (LDL) triglycerides, and a decrease in LDL cholesterol. Many other studies substantiate the effect of tamoxifen on lowering total cholesterol and, in most cases, LDL cholesterol and apolipoprotein B.159, 160, 161Despite these potentially favorable effects, there was no evidence of an improvement in the rate of cardiovascular mortality in either the Oxford metaanalysis15 or the NSABP P01 prevention trial.149


Hepatic Toxicity

Tamoxifen has been associated with various hepatic abnormalities, including cholestasis, jaundice, peliosis hepatitis, and hepatitis. Carcinogenicity studies in rats reveal hepatocellular carcinoma in dosages ranging from 5 to 35 mg/kg per day.162 A dose of 20 to 40 mg given to humans is 5 to 10 times less than the 5 mg/kg dose, and an increase in hepatocellular carcinoma in humans taking tamoxifen has not been reported. This is significant because tamoxifen forms adducts with DNA and could be mutagenic and carcinogenic.163

Bone Side Effects

The effect of tamoxifen on bone mineral content in postmenopausal women can be considered an advantageous side effect, with substantial data supporting an estrogen agonistic activity of tamoxifen on bone.164, 165 In the P01 study, a nonsignificant decrease in fracture rate was documented; further follow-up of this study should clarify this issue.149 In premenopausal women, bone mineral density is decreased. Presumably in a high-estrogen environment, tamoxifen operates predominantly as an antagonist.166, 164

Gynecologic Side Effects

Many premenopausal patients notice a change in the duration of menses or heaviness of flow, and there have been suggestions of an increased incidence of ovarian cysts in these patients.167 In postmenopausal women, endometrial cancer is increased, but screening for endometrial cancer is complicated by tamoxifen-induced benign endometrial hyperplasia and polyp formation. Data from the pilot British Breast Cancer Prevention Trial found that 39% of women taking tamoxifen had histologic endometrial changes, 16% with atypical hyperplasia and 8% with endometrial polyps. These results compared with 10% abnormalities in placebo-treated patients, with no atypical hyperplasia and 2% with polyps. Transvaginal ultrasonography was used, with an endometrial thickness of 8 mm or more predictive for atypical hyperplasia or polyps.168 The risk of endometrial cancer relates to age and duration of tamoxifen treatment. In the original Swedish report, 13 endometrial cancers were diagnosed in 695 tamoxifen patients, for a frequency of 0.9% among patients receiving 2 years of drug treatment versus 3% among those receiving 5 years of treatment. The frequency for the controls was similar to that for patients receiving 2 years of treatment, whereas the highest frequency occurred among those treated for 5 years. The increased frequency appeared in the third and fourth years of follow-up. It should be noted that the Swedish patients received the high dose of 40 mg daily. The same investigators later described the histology of 17 cases of tamoxifen-linked endometrial cancer, 16 of which were grade 1 or 2, although three patients from the group died of the uterine malignancy.168 These frequencies are similar to those reported for the P01 study, with an increase in endometrial cancer but overall a low rate of death from the disease. Recently a case-control study found that exposure to tamoxifen interacts with other risk factors for endometrial cancer, namely, a history of hormone replacement therapy and higher body mass index.169

In addition to endometrial cancer, recent reports shown that the risk of uterine sarcoma, a very rare malignancy, is also increased by tamoxifen. A recent update from six NSABP trials in which over 17,000 women were randomized to tamoxifen or placebo shows that the rate of uterine sarcoma was increase to 0.17 per 1,000 women-years versus 0 per 1,000 women years in women taking tamoxifen versus placebo.170

Recommendations for gynecologic follow-up of patients taking tamoxifen have varied from observation to yearly vaginal ultrasound to yearly endometrial biopsy, with no solid data supporting any of these approaches. Yearly pelvic examinations and rapid investigation of postmenopausal bleeding, but not radiologic or biopsy screening, are currently recommended. Particular attention should be given to patients at higher risk.169 Cyclic progestins have been considered to obviate the estrogenic effect of tamoxifen on the endometrium. Preclinical data suggest that progesterone may reverse the antitumor effect of tamoxifen in the dimethylbenzanthracene-induced rat mammary tumor model.171 There continues to be concern about the safety of cyclic progestins. Furthermore, many postmenopausal women find regular “withdrawal” bleeding inconvenient.

Endocrine Effects

The effects of tamoxifen on circulating hormones through feedback effects on the pituitary-hypothalamus axis, through effects on plasma steroid-binding proteins, or through end-organ effects vary according to gender and menopausal status. In postmenopausal women, most investigators have found a decrease in prolactin, LH, and FSH, although all three remain within the normal range.172, 173, 174, 175 Thyrotropin-releasing hormone induction of prolactin secretion is suppressed.176 Plasma estrone and estradiol remain unchanged in most studies, although one study reported a minor decrease in plasma estrogens and an increased urinary excretion of glucuronide conjugates, suggesting an increase in metabolism or renal clearance of endogenous estrogens.177 In premenopausal women, estradiol, estrogen, and progesterone have been reported to be increased after tamoxifen.175 Many women continue to ovulate while taking tamoxifen, and in those who do, supraphysiologic levels of estradiol have been seen.178 Tamoxifen causes an elevation of serum cortisol due to an increase in transcortin,179 as well as increases in sex hormone–binding globulin, thyroxine-binding globulin, and apolipoprotein AI.180, 181, 182 Because tamoxifen is a weak estrogen, it binds to ER and has estrogenic effects on normal hormone-responsive organs. In postmenopausal women, it causes an increase in cornification of the vaginal epithelium183 and induces PgR in the endometrium184 and in breast tumors.185 In men, the only consistent finding seems to be an increase in progesterone in plasma,132 whereas levels of LH, estradiol, and other hormones remain unchanged in most studies.132, 134 IGF-I or somatomedin C levels have been shown to be decreased with the use of tamoxifen.186, 187, 188 Because exogenous estrogens are known to decrease IGF-I levels in postmenopausal women, it is possible that the effect seen with tamoxifen is an estrogen agonistic effect.189


The effect of tamoxifen on offspring is unknown. There was one case report of a mother on tamoxifen giving birth to a baby with Goldenhar's syndrome, an oculoauriculovertebral syndrome.190 This woman also had smoked marijuana and ingested cocaine, so the effect of tamoxifen is unclear. In the same case report, there is a discussion of 50 pregnancies on file at AstraZeneca Pharmaceuticals in women taking tamoxifen. These resulted in 19 normal births, 8 terminated pregnancies, 13 unknown outcomes, and 10 infants with a fetal and neonatal disorder, 2 of which had craniofacial defects. In another report, 85 women taking tamoxifen for prevention of breast cancer became pregnant; none of these pregnancies resulted in fetal abnormalities.191 Another case of an infant born with ambiguous genitalia after in utero exposure through 20 weeks has also been reported.192 The evidence for the teratogenicity of tamoxifen is primarily derived from animal studies, in which reproductive organ abnormalities and increased susceptibility to carcinogens were found.193 Despite this evidence in animals, in humans only anecdotal reports of abnormalities, without an established definitive causal link to tamoxifen, have been reported. However, given the possibility of harmful effects, women should be told to use mechanical contraception while on this drug.

Male Breast Cancer

There have been case reports of impotence194 and of nocturnal priapism195 in male breast cancer patients receiving tamoxifen. Another series reported a decrease in libido in 29.2% of male breast cancer patients.196 The issue of impotence in men treated with tamoxifen has been studied in men treated with tamoxifen for male infertility. One paper reports a loss of libido in four cases (9%),197 whereas studies that reported an increase in testosterone levels with tamoxifen did not report an increase in impotence.198 These data suggest that the effect of tamoxifen on sexual function is minimal and is probably not the cause of impotence.

Flare Reactions

A transient exacerbation of symptoms, or “flare reaction,” was first observed during the treatment of postmenopausal women with high-dose estrogen. A clinical flare reaction is characterized by a dramatic increase in bone pain and an increase in the size and number of metastatic skin nodules with erythema.199Typically, symptoms occur from 2 days to 3 weeks after starting treatment and can be accompanied by hypercalcemia, which occurs in approximately 5% of patients.200 Tumor regression may occur as the reaction subsides.

In this context, two further manifestations of flare need to be taken into consideration, tumor marker flare and bone scintigraphic flare. Tumor marker analysis should be interpreted with caution in the first months after starting tamoxifen, because up to 75% of patients beginning a new therapy for metastatic disease will exhibit a rise in tumor marker levels that subsequently return to baseline or below. Bone scans can also show flare, with an increase in uptake 2 to 4 months after the initiation of systemic therapy, which may confuse a radiographic evaluation of flarelike symptoms.201, 202 Plain film radiography is helpful here, as the presence of sclerosis, suggesting healing, may be documented in previously purely lytic lesions.

Withdrawal Response

Between 25% and 35% of patients treated with estrogens have a secondary response if estrogen is stopped when disease progression is diagnosed.14, 203 The same phenomena can be observed on withdrawal of tamoxifen and progestins.204 Convincing withdrawal responses only occur for patients who have experienced tumor regression followed by subsequent recurrence of tumor.



The structures of SERMs and antiestrogens that have been recently approved or are under investigation are provided in Figure 37.11. Although pure antiestrogen therapy, as discussed later with fulvestrant, is a logical approach to the treatment of advanced breast cancer, drugs devoid of all estrogenic activities might be problematic in the adjuvant and prevention settings, because secondary hormone replacement therapy–like benefits of tamoxifen are considered worthwhile for postmenopausal women. This concern stimulated the development of alternative antiestrogens with a modified mixed agonist and antagonist profile. Ideally, these drugs are antiestrogenic in the breast and endometrial but retain beneficial effects on bone mineralization. Although, historically, estrogenic effects on lipids and other cardiovascular markers were felt to be beneficial, a recent large randomized study, the Women's Health Initiative, has demonstrated that hormone replacement did not improve cardiovascular outcomes.205 As a result, the optimal balance of agonism versus antagonism for cardiovascular endpoints remains a issue of debate.

Figure 37.11 Antiestrogens. (SERM, selective estrogen receptor modulator.)

It is also important to emphasize that although ideal SERMs may represent a small advance in terms of safety, they are not necessarily more efficacious. In general, antiestrogens that exhibit a mixed agonist and antagonist profile, even if modified in a way that improves tissue-specific toxicities, are likely to exhibit resistance and toxicity profiles that overlap those of tamoxifen.206 This may be because any antiestrogen that triggers ER dimerization and DNA binding is prone to the same coactivator-based resistance mechanisms that may limit the activity of tamoxifen.


Raloxifene [6-hydroxy-2-(4-hydroxyphenyl)-benzo[b]thien-3-yl]–[4-[2-(1-piperidinyl) ethoxy] phenyl] is the first approved drug to exhibit a “modified” SERM profile; however, the indication for raloxifene is osteoporosis, not breast cancer.207 An early evaluation of activity in tamoxifen-resistant breast cancer (when the drug was referred to as “keoxifene” or “LY156758”) was disappointing.208 Consequently, raloxifene should not be used for the treatment of either early stage or advanced breast cancer. However, there is continued interest in this drug in the prevention setting, because a decrease in breast cancer incidence was seen in raloxifene osteoporosis trials.209 The current NSABP prevention study compares raloxifene to tamoxifen in women at high risk of the disease.210 A raloxifene-related drug, currently referred to as “arzoxifene,” has a profile similar to that of raloxifene but with greater ER antagonist activity in breast cancer models.

Mechanism of Action

While raloxifene has agonist and antagonist activity that is similar to tamoxifen in breast tissue and bones, its antagonist effects in the endometrial provide a distinct advantage. The molecular mechanisms of this endometrial activity are now fairly well understood. Raloxifene is a benzothiophene with a structure that includes a flexible “hinge” region that results in a nearly orthogonal orientation of its side chains. This is markedly different from tamoxifen, which has a rigid triphenylethylene structure.211 Although both drugs bind to the ER, their structural differences lead to dissimilar conformations of the ER-ligand complex212; this conformation difference may partly account for differential recruitment of co activators and co repressors. In the Ishikawa endometrial carcinoma cell line, tamoxifen, but not raloxifene, induces recruitment of coactivators SRC-1, AIB1, and CBP to the c-Myc and IGF-1 promoters.213 This recruitment of coactivators is critical to tamoxifen's transactivating ability and agonist activity in the endometrium. The comparative inability of raloxifene to assemble this coactivator complex appears to account for its antagonist activity in the endometrium.

Pharmacology and Metabolism

Raloxifene binds to the ER with a Kd of about 50 pmol/L, which is comparable to the value for estradiol.214 The drug is given at 60 mg per day, and after oral administration it is rapidly absorbed, reaching its maximal concentration in about 30 minutes.215 While absorption of 60% is seen, first-pass glucuronidation limits the drug's bioavailability to 2%. After absorption, raloxifene is distributed widely throughout the body and is bound to plasma proteins, including albumin. The half-life of the drug is 27.7 hours.215

Metabolism of raloxifene, as mentioned, is primarily via first-pass metabolism in the liver, where it is glucuronidated. The agent does not appear to be metabolized by the CYP enzyme systems, and there are no other known metabolites.215 Elimination occurs primarily through bile and feces, with just a small amount found in the urine.

Drug Interactions

Cholestyramine causes a 60% reduction in raloxifene absorption and enterohepatic circulation.215 Unlike tamoxifen, raloxifene has a minor interaction with warfarin, with just a 10% decrease in prothrombin time. Concomitant therapy with ampicillin reduced the raloxifene peak concentration by 28%, which was felt to be clinically insignificant.216

Skeletal Effects

Raloxifene has been demonstrated to have estrogen agonist activity in bone in both animal models and in clinical trials. Promotion of bone density may occur through inhibition of osteoclast activity and stimulation of osteoblasts. In an ovariectomized rat model, raloxifene protected against bone loss and resulted in augmentation of bone strength.211 Furthermore, in rats with intact ovarian function, the drug does not antagonize estrogen effects on bone density.

Results in human clinical trials have demonstrated similar effects. Bone remodeling studies show that raloxifene inhibits bone resorption.216 This activity results in increased bone density in postmenopausal women with and without osteoporosis. The largest raloxifene trial reported to date, the MORE study, enrolled 7,705 women with osteoporosis to determine if the drug could decrease the rate of fractures.217 While vertebral fractures were reduced by 30%, nonvertebral fracture were not significantly different. However, bone mineral density was increased in both the spine and femoral neck.

Endometrial Effects

Unlike estrogen and tamoxifen, raloxifene does not have stimulatory activity in the uterus. In rat models, uterine weight and endothelial height were unchanged with raloxifene exposure.218 Similarly, a number of clinical trials have carefully evaluated endometrial thickness, endometrial hyperplasia, vaginal bleeding, and endometrial cancer. All studies have found that raloxifene does not promote uterine proliferation.218 The 4-year update of the MORE trial further confirmed this finding over a longer time frame. In this study, the rates of vaginal bleeding and endometrial cancer in patients taking raloxifene have been the same as those on placebo.219

Cardiovascular Effects

Raloxifene has also been shown to have estrogen agonist effects on the cardiovascular system, with reduction of serum cholesterol levels by 70% in postmenopausal rat models.218 In clinical trials, raloxifene has been shown to reduce serum LDL, fibrinogen, lipoprotein (a), homocysteine, and C-reactive protein. Unlike tamoxifen, however, raloxifene does not affect triglycerides or HDL. Despite these findings, which would argue for a possible cardiovascular protective effect, there is only limited data indicating that raloxifene has clinical benefit in this regard.218 While in the MORE trial, there was no difference in cardiovascular events and cerebrovascular events in the overall cohort, subgroup analysis with multiple comparisons revealed that women with increased cardiovascular risk had a relative risk reduction in cardiac events of 40% when taking raloxifene.209 This finding, however, must be confirmed in other trials, given the exploratory nature of the analysis. In addition, the Women's Health Initiative, a large prospective, randomized trial that assigned women to hormone-replacement therapy or placebo, showed no decrease in cardiovascular disease with estrogen alone, so it is possible that similar results may be found with raloxifene.205 The RUTH (Raloxifene Use for the Heart) trial is currently enrolling patients and was specifically designed to evaluate cardiovascular endpoints.220

Other Side Effects

Other major side effects of raloxifene compared to placebo in the MORE trial included hot flashes (9.7% vs. 6.4%), leg cramps (5.5% vs. 1.9%), and thromboembolic events (1.4% vs. 0.47%).217 The etiology of leg cramps is unclear, but they were generally mild and did not result in discontinuation.211 The increased risk of thromboembolic events is similar to that seen with tamoxifen and is perhaps the side effect of most concern when the drug is used in clinical practice. In addition, raloxifene has been demonstrated to be teratogenic in animal studies.211 Given this risk and the lack of clinical data in the premenopausal setting, raloxifene should only be used in postmenopausal women outside of a clinical trial.


Toremifene was the first new antiestrogen since tamoxifen to be approved in the United States, and it provides an alternative to tamoxifen for first-line treatment of advanced breast cancer.221 Toremifene is reported to have antiestrogenic activity similar to that of tamoxifen.222 The drug acts as almost a pure antiestrogen in rats and is partially agonistic in mice. Unlike tamoxifen, however, this drug has no hepatocarcinogenicity or DNA adduct-forming ability in rats.222 It was hoped, therefore, that toremifene would provide a safer SERM for long-term use, such as in the adjuvant setting, although studies comparing the two drugs have shown no clear advantage over tamoxifen. As a result, toremifene's use in the clinic has been somewhat limited. The major metabolites of toremifene are N-demethyl-toremifene and 4-hydroxytoremifene. The mean terminal half-life of toremifene and these metabolites is from 5 to 6 days.223 The side effects are similar to those of tamoxifen, with hot flashes being the most common. Five patients developed leukopenia, with the lowest white blood cell count being 2,500. One patient had to discontinue therapy because of tremor. Toremifene is cross-resistant with tamoxifen and should not be used in tamoxifen-resistant disease.222



Given findings that resistance to tamoxifen may sometimes be due to its partial agonist effects, pure antiestrogens without agonist activity were developed as hormonal agents. Fulvestrant, which is the only approved pure antiestrogen, is a 7α-alkylamide analog of 17β-estradiol (Fig. 37.11). This drug is also often called a “selective estrogen receptor down-regulator” (SERD), because it has been demonstrated to decrease the level of ER protein in the cell.224

Mechanism of Action

Fulvestrant is a steroidal antiestrogen that acts in a distinctly different manner than tamoxifen and other SERMs. The drug binds the ER, like SERMs, but its long, bulky alkylamide side chain at the 7α position prevents ER dimerization due to steric effects. The result is that the ER cannot bind DNA, and in addition it is eliminated more rapidly via proteosome degradation, leading to a marked reduction in ER protein levels.225, 226 In this way, fulvestrant blocks ER-mediated gene transcription completely. In vitro, this results in inhibition of tamoxifen-resistant breast cancer cell lines by fulvestrant. In addition, the drug has no growth stimulatory effect in a tamoxifen-stimulated MCF-7 breast cancer xenograft model.225 In primate studies, fulvestrant also acts as a pure antiestrogen outside of the breast, with complete inhibition of estrogen stimulation of uterine tissue.227

The antiproliferative effects of fulvestrant may be augmented by its ability to suppress insulin-like growth factor receptor signaling. This appears to occur both in vitro and in animal models. Finally, fulvestrant also has activity as an aromatase inhibitor, although it is not known how much this property contributes to the clinical activity of the drug.228

The effects of fulvestrant on ER, PgR, proliferation, and apoptosis have been examined in benign endometrial tissue and malignant breast tissue. When fulvestrant was given for a week before hysterectomy, it was found to decrease a Ki67-based proliferation assay; however, ER and PgR levels were not affected. In contrast, short-term exposure to fulvestrant before breast surgery decreased ER and PgR expression and proliferation and increased apoptosis.229,230 These data suggest that, as in the case of other antiestrogens, there may be differences in the action of fulvestrant at different organ sites.

While current experience with fulvestrant in breast cancer is limited to postmenopausal women, there have been trials with benign gynecologic conditions that demonstrate its antiestrogenic properties in premenopausal women.225, 231


In vitro, fulvestrant has an ER-binding affinity that is 100-fold greater than that of tamoxifen.225 Because the drug is not reliably absorbed orally, it is formulated as a monthly depot intramuscular injection. With this preparation, peak levels of fulvestrant occur at a median of 8 to 9 days after dosing and decline thereafter, but the levels remain above the projected therapeutic threshold at day 28. In pharmacokinetic studies, the AUC was 140 ng per day in the first month and 208 ng per day after 6 months, suggesting some drug accumulation.232 Because a single injection of the 5-mL volume can be difficult in some patients, the drug is often given as two 2.5-mL injections; this alternative method of delivery has no effect on the pharmacokinetics of the drug.233


While published data on the metabolism of fulvestrant are sparse, the drug is known to undergo oxidation, hydroxylation, and conjugation with glucuronic acid and/or sulphate in the liver, with negligible renal excretion. The half-life of the drug is approximately 40 days.234

Side Effects

In preclinical studies, fulvestrant has been reported to cause decreased bone mineral density in adult female rats, suggesting that osteoporosis might be a side effect.235 Clinically, there is not yet enough data to determine if fulvestrant leads to osteoporosis. Preclinical results also show that fulvestrant differs from tamoxifen in its effects on bovine lenses maintained in vitro. While these experiments suggest that fulvestrant might have decreased ocular effects as compared with tamoxifen, large randomized trials would be required to determine differences in this rare complication.225

In other preclinical studies, fulvestrant was found to allow the uptake of [3H]-estradiol in the brain, suggesting that the drug might have decreased CNS effects compared with other hormonal approaches. Interestingly, in early human studies, no clear effect on FSH levels and LH levels have been documented, indicating that fulvestrant has no impact on pituitary function.225 Despite these findings, the frequency of hot flashes with fulvestrant in clinical trials has been similar to the frequency with aromatase inhibitors. However, a recent trial comparing fulvestrant and tamoxifen in the metastatic setting suggested decreased hot flashes with fulvestrant.236 There were no other side effect in this trial that were statistically different between the two drugs.

In early clinical studies, fulvestrant did not cause a change in sex hormone–binding globulin, prolactin, or lipids.224 In addition, there was no increase in endometrial thickness in patients undergoing hysterectomy.231 In more recent clinical trials for metastatic breast cancer, fulvestrant has been well tolerated, with hot flashes and gastrointestinal disturbances as the most common adverse events. Tolerability was similar between fulvestrant and anastrozole in two phase III trials, with a treatment-related withdrawal rate of 2.5%.225

Fulvestrant in Premenopausal Women

While breast cancer trials with fulvestrant have focused on postmenopausal women, the drug has also been used in premenopausal women with uterine fibroids and endometriosis. In these studies, fulvestrant had pure antagonizing effects in endometrial tissue.225

Other Pure Antiestrogens

Clinical development programs have recently been activated for other “pure” antiestrogens. EM 652 has a nonsteroidal structure and is theoretically interesting because of an inhibitory action against ERα and ERβ whether these receptors are operating through a classic ERE or an AP1 site.237

ERA923 is another nonsteroidal antiestrogen in early clinical trials. No preclinical information on this compound has been released.


Aromatase Background

Postmenopausal Estrogen

The therapeutic effect of reducing estrogen levels for patients with breast cancer was originally restricted to patients with functioning ovaries. However, as discussed earlier, postmenopausal women still produce significant amounts of estrogen through aromatization of circulating adrenal androgens in peripheral normal tissues, such as fat, muscle, liver, and the epithelial and stromal components of the breast.237, 238 Peripheral aromatization is increased in certain medical conditions, including obesity, hepatic disease, and hyperthyroidism, but is independent of pituitary hormone secretion. The relative proportion of estrogens synthesized in extragonadal sites increases with age, and eventually nonovarian estrogens predominate in the circulation.239

Intratumoral Aromatase

Expression of aromatase in the breast led to the hypothesis that local synthesis of estrogens contributes to breast cancer growth in postmenopausal women.240, 241 In support of this theory, the decline in estrogen concentrations after menopause is less marked in breast tissue than in plasma due to a combination of aromatase activity and preferential estrogen uptake from the circulation.28 Furthermore, aromatase activity has been shown to correlate with a marker of breast cancer cell proliferation, and quadrants of the breast bearing a breast cancer have more aromatase expression than those not bearing tumors.242 It is unclear whether an increase in aromatase expression precedes breast cancer development or is a direct consequence of the presence of a tumor. However, aromatase activity in the breast is an attractive resolution to the paradox that breast cancer increases with age although overall estrogen levels decline.28

Development of Aromatase Inhibitors

Steroidal versus Nonsteroidal

The pivotal role of aromatase in the development of breast cancer defines this enzyme as a key therapeutic target. Two distinct solutions evolved to the problem of designing potent, specific, and safe aromatase inhibitors.243 One strategy was to develop “steroidal” aromatase inhibitors that are resistant to aromatase action and that bind aromatase and block conversion of androgenic substrates (Type 1 inhibitors). An alternative was to develop a family of “nonsteroidal” inhibitors that disrupt the aromatase active site by coordinating within the heme complex without affecting the active sites of other steroidogenic enzymes (Type 2 inhibitors). Both approaches led to the successful introduction into clinical practice of potent and specific aromatase inhibitors.

Early Aromatase Inhibitors

In 1973, Griffiths et al. first demonstrated the activity of aminoglutethimide, an inhibitor of cholesterol conversion to pregnenolone in the treatment of metastatic breast cancer.244 Subsequently, it was appreciated that inhibition of aromatase, rather than suppression of general steroidogenesis, was key to the therapeutic action of aminoglutethimide.245, 246 Although the drug has well-documented efficacy in the metastatic setting, its side effect profile is troublesome. Unfortunately, even at the lowest dosages effective against breast cancer, aminoglutethimide inhibits the formation of corticosteroids by blocking P-450 enzymes involved in cholesterol side chain cleavage.247 This lack of specificity exposes patients to the risk of glucocorticoid deficiency. Furthermore, the clinical utility of aminoglutethimide is limited by troublesome side effects, including rash, nausea, somnolence (aminoglutethimide was originally developed as a sedative), and blood dyscrasias.247, 248

These observations provided a strong rationale for the development of more potent and selective aromatase inhibitors. While second-generation aromatase inhibitors, such as fadrozole and formestane, had improved potency and selectivity, the third-generation aromatase inhibitors were soon found to provide superior clinical results. These newer aromatase inhibitors have now rendered both aminoglutethimide and the second-generation drugs obsolete in breast cancer therapeutics.

Exemestane: Steroidal Aromatase Inhibitor


A large number of androstenedione derivatives were screened in aromatase inhibition assays, and two compounds, formestane (4-hydroxyandrostenedione) and exemestane (6-methylenandrosta-1, 4-diene-3, 17-dione), emerged as drugs suitable for clinical development.249 Given the superior potency and clinical activity of exemestane, the use of formestane, which is available in Europe but not the United States, has declined precipitously. As shown in Figure 37.12, the structures of these compounds retain androgenic properties but have side chain substitutions that prevent conversion to estrogenic metabolites. Although not intrinsically reactive, exemestane exhibits tight or even irreversible binding to the aromatase active site.28 The compound is therefore considered a “mechanism-based” or “suicide” inhibitor because it permanently inactivate aromatase. Recovery of aromatase activity after treatment with a suicide inhibitor therefore requires the synthesis of new aromatase protein. In vivo, the pharmacokinetics of suicide inhibition is characterized by persistently low aromatase activity despite complete drug clearance. Because suicide inhibition prolongs drug action, intermittent dosing should be possible, potentially improving the side effect to benefit ratio. However, in clinical practice, this benefit remains largely theoretical, because exemestane has been administered daily with a favorable toxicity profile.28


Exemestane (Fig. 37.12) has potent aromatase activity with a Ki of 26 nM and no cholesterol side chain cleavage (desmolase) or 5-reductase activity. An oral dose is rapidly absorbed, with peak plasma concentrations reached within 2 hours of administration. The absorption of exemestane is enhanced by high-fat foods, and it is recommended that the drug be taken after eating.250 Plasma concentrations fall below the limit of detection 4 hours later (for the approved 25-mg dose), although inhibition of the enzyme persists for at least 5 days.250 Steady-state levels are achieved within 7 days with daily dosing, and the time to maximal estradiol suppression is 3 to 7 days.251 The smallest dosage found to have maximal suppression of plasma estrone, estradiol, and estrone sulfate and urinary estrone and estradiol was 25 mg, now the recommended daily dosage. This dosage inactivates peripheral aromatase by 98% and reduces basal plasma estrone, estradiol, and estrone sulphate levels by 85 to 95%.252 Other endocrine parameters, such as cortisol, aldosterone, dehydroepiandrosterones, 17-OH-progesterone, FSH, and LH, were not significantly affected by 25 mg of exemestane.253


Exemestane is extensively metabolized, with rapid oxidation of the methylene group at position 6 and reduction of the 17-keto group, along with subsequent formation of many secondary metabolites. The drug is excreted in both the urine and feces. As a consequence, clearance is affected by both renal and hepatic insufficiency, with threefold elevations in the AUC under either condition. Metabolism occurs through CYP3A4 and aldoketoreductases, and the activity of the major CYP enzymes is unaffected.251, 254 While exemestane does not bind to the ER and only weakly binds to the AR, with an affinity of 0.28% relative to DHT, the binding affinity of the 17-dihydrometabolite is 100 times that of the parent compound.255 As a result, there is slight androgenic activity in the rat with this drug. A screen of potential metabolites for aromatase activity did not reveal any compounds with inhibitory activity greater than exemestane.256

Figure 37.12 Aromatase inhibitors.


In clinical studies, exemestane has been well tolerated and has had a treatment-related discontinuation rate of less than 3%.254 At high doses in rat studies, exemestane has been observed to have androgenic effects. Similarly, at high doses in clinical trials, androgenic side effects, including hypertrichosis, hair loss, hoarseness, and acne, have been reported in 4% of patients.254 Other reported side effects include hot flashes, increased sweating, and nausea.

With all aromatase inhibitors, bone loss is a significant concern. However, the androgenic properties of exemestane metabolites might mitigate this effect. In ovariectomized rat studies, exemestane treatment provided protection from bone loss in comparison with untreated animals.257 Initial studies in postmenopausal women using bone turnover biomarkers suggest that exemestane has a significantly smaller impact on bone formation and bone resorption than letrozole.258 Despite these findings, results of the Intergroup Exemestane Study showed that postmenopausal patients on exemestane experience more osteoporosis than those on tamoxifen.259

Other side effects that were increased in the exemestane group in this trial included visual disturbances, arthralgia, and diarrhea. Conversely, patients on tamoxifen experienced more gynecologic symptoms, vaginal bleeding, thromboembolic disease, and cramps.259

Drug Interactions

Given that exemestane is extensively metabolized by CYP3A4, interference with other drugs affected by this P-450 enzyme may occur. Interestingly, however, ketoconazole does not significantly influence the pharmacokinetics of exemestane, suggesting that with CYP3A4 inhibition exemestane may be metabolized via a different route.251

Nonsteroidal Aromatase Inhibitors

The nonsteroidal approach to aromatase inhibition has yielded two compounds with significant clinical impact, anastrozole and letrozole. In attempts to find potent nonsteroidal inhibitors, research focused on a series of imidazole and triazole derivatives with “molecular shapes” that efficiently coordinate within the aromatase heme complex. Progress was assisted by new systems for rapid drug screening, and out of these screens came three drugs with the desired profile: anastrozole, letrozole, and vorozole (Fig. 37.12).260 Because vorozole is not available for further clinical trials despite good clinical activity,261 our discussion focuses on letrozole and anastrozole.



Anastrozole [2,2′-[5-(1H-1,2,4-triazol-1-ylmethyl)-1, 3-phenylene]bis(2-methyl-propiononitrile)] is a competitive aromatase inhibitor with high potency and was the first selective aromatase inhibitor approved in North America and Europe. It inhibits human placental aromatase and has an IC50 (the concentration that inhibits enzyme activity by 50%) of 15 nM.262 Anastrozole is rapidly absorbed, with the maximum level occurring within 2 hours after administration, and it is 40% bound to plasma proteins. The mean peak plasma concentration at the 1-mg dose was 13.1 ng/mL.262 Pharmacodynamic studies reveal that subjects receiving 1 mg per day orally achieved 96.7% aromatase inhibition, with maximal estradiol suppression and a decrease of estradiol ranging from 78 to 86% from baseline. Suppression is maintained long term, and there is no compensatory rise in androstenedione levels. The drug has a half-life of 40.6 hours and reaches steady-state levels in 7 days, with maximal estrogen suppression within 3 to 4 hours.251 There is no effect on glucocorticoid or mineralocorticoid secretion as tested by ACTH stimulation.262 In addition, anastrozole at a dosage of 1 mg daily does not have an effect on gonadotrophins or follicle-stimulating hormone. Importantly, even at higher doses (5 to 10 mg), anastrozole administration does not affect basal or ACTH-stimulated cortisol and aldosterone levels.251


Elimination of anastrozole is primarily by metabolic degradation, and less than 10% of the drug is cleared as unchanged drug. Degradation occurs through N-dealkylation, hydroxylation, and glucuronidation, and metabolites are excreted predominantly in the urine. The plasma elimination half-life with the 0.5- and 1.0-mg multiple doses ranges from 38 to 61 hours.262

Anastrozole has no effect on CYP2A6 but does inhibit CYP1A2 and CYP2C8, with an IC50 of 30 µmol/L and 48 µmol/L, respectively. In clinical use, the drug is present at much lower levels than are required to inhibit these enzymes.251


Growing clinical experience with anastrozole in the metastatic and adjuvant settings shows that it is well tolerated even with extended use.263 In comparison with megestrol acetate in metastatic disease, anastrozole is associated with less weight gain. In contrast, nausea and vomiting were more common with anastrozole. However, gastrointestinal toxicities were less troublesome with the 1-mg dose, and gastrointestinal problems rarely led to interruption of therapy.264, 265 In the ATAC (Arimidex, Tamoxifen Alone or in Combination) trial, a large adjuvant trial comparing anastrozole versus tamoxifen versus their combination, anastrozole demonstrated a favorable toxicity profile with prolonged use.263 Compared with patients taking tamoxifen, patients in the anastrozole arm of the study experienced less vaginal bleeding (4.8% vs. 8.7%), vaginal discharge (3.0% vs. 12.2%), endometrial malignancy (0.1% vs. 0.7%), ischemic cerebrovascular events (1.1% vs. 2.3%), and venous thromboembolic events (2.2% vs. 3.8%). Anastrozole, however, did result in an increase in fractures (7.1% vs. 4.4%) and in musculoskeletal complaints (30.3% vs. 23.7%). Given that there was no placebo control in this trial, it is not clear if the increased incidence of fractures is due to deleterious effects of anastrozole or protective effects of tamoxifen.

Drug Interactions

To date, clinically significant drug interactions with anastrozole have been minimal. Coadministration with tamoxifen has been shown to lead to a 27% decrease in steady-state levels of anastrozole.251 However, given the ATAC trial findings that combining tamoxifen and anastrozole provides inferior clinical outcomes compared with anastrozole alone, the combination of these drugs should not be used clinically in the future. It is unclear whether this pharmacokinetic interaction might partially account for the inferiority of the combination arm in ATAC.



Letrozole (4,4′-[(1H-1,2,4-triazol-1-yl)methylene]bisbenzonitrile) was the second nonsteroidal selective aromatase inhibitor approved for the treatment of advanced breast cancer. The drug has a profile similar to anastrozole, combining high potency, selectivity, and good clinical activity against breast cancer.260The IC50 for placental aromatase is 11.5 nM, so it is marginally more potent than anastrozole or exemestane. For comparison, the IC50 for aminoglutethimide is 1,900 nM. Oral absorption is rapid, with high bioavailability that is only minimally affected by food.251 Aromatase inhibition at the recommended 2.5-mg dose of letrozole reaches 98.9%,251 and both letrozole doses examined in phase III trials, 0.5 mg and 2.5 mg, suppress estrogen levels over 90% (less than 0.5 pmol/L).266 Letrozole reaches peak concentration within 2 hours, and steady-state levels are achieved within 14 to 40 days.251 Maximal estrogen suppression occurs within 2 to 3 days, and the half-life of the drug is 2 to 4 days.251

Although letrozole has high specificity for aromatase, two studies have shown effects on either cortisol levels or aldosterone levels; however, these two studies had conflicting results.267, 268 In addition, changes in glucocorticoid and mineralocorticoid levels were small and do not appear to be clinically meaningful. In these studies, no changes were found in 11-deoxycortisol, 17-hydroxyprogesterone, ACTH, or plasma renin levels. In addition, plasma androgens, thyroid function, LH, and FSH remained unchanged.

Comparison with Anastrozole

While letrozole appears to have characteristics similar to those of anastrozole, comparison of the drugs at the recommended doses has revealed minor differences in potency. In a preclinical model of aromatase-dependent breast cancer growth, letrozole had greater antitumor activity than did anastrozole and tamoxifen.269 In a double-blind, cross-over study, letrozole was found to suppress aromatization by greater than 99.1%, versus 97.3% for anastrozole (P = .0022). In addition, suppression of plasma estrone sulfate was greater with letrozole treatment.270 The clinical implications of these differences is not yet clear; however, preliminary data from a study in metastatic disease revealed no difference between the two drugs.271


As with anastrozole, metabolic clearance of letrozole is mainly through the liver, and the half-life of 50 hours allows a once-a-day dosing schedule. CYP3A4 and CYP2A6 catalyze letrozole to its major metabolite, 4,4′-methanol-bisbenzonitrile, which is then subjected to glucuronidation. Letrozole can be safely prescribed for patients with renal insufficiency, because only 5% of the drug is cleared in the urine. However, the drug should be used with caution in treating patients with severe liver impairment.251 There are no known drug interactions with erythromycin, warfarin, or cimetidine. As with anastrozole, coadministration of tamoxifen reduces letrozole levels by 37%; in the case of letrozole, this is likely due to the induction of CYP3A4.146


Like anastrozole, letrozole is extremely well tolerated, with only 2% of patients discontinuing the drug due to adverse events; in comparison, megestrol acetate has an 8% discontinuation rate.272 A recently reported large adjuvant trial using letrozole versus placebo in patients who had completed 5 years of tamoxifen treatment has provided data on toxicities associated with long-term letrozole use.273 With a median follow-up of 2.4 years, patients on letrozole experienced an increased incidence of hot flashes (47.2% vs. 40.5%), arthritis (5.6% vs. 3.5%), arthralgias (21.3% vs. 16.6%), and myalgias (11.8% vs. 9.5%). In addition, there was a trend towards increased osteoporosis (5.8% vs. 4.5%). Compared with placebo, patients on letrozole had less vaginal bleeding (4.3% vs 6.0%). Discontinuations rates were nearly identical for placebo and letrozole.

Aromatase Inhibitors in Premenopausal Women

Hereditary aromatase deficiency, due to inherited loss-of-function mutations in the aromatase gene, is associated with a syndrome of hypergonadotropic hypogonadism, multicystic ovaries, virilism, and bone demineralization in childhood. These problems are reversible with low-dose estrogens.274, 275 Polycystic ovary syndrome in adult women has a less well defined etiology, although low aromatase activity is believed to play a part.276 Treatment of premenopausal women with aromatase inhibitors may therefore be complicated by polycystic ovaries and virilization. To circumvent these problems, the treatment of premenopausal women with advanced breast cancer with an LHRH analog and selective aromatase inhibitor combination is under investigation. Aminoglutethimide treatment of premenopausal women does not suppress estrogen levels, so the indication for this drug was restricted to patients without ovarian function.277 However, more potent aromatase inhibitors are able to suppress ovarian aromatase activity. For example, in a small pharmacokinetic study, a combination of formestane and an LHRH agonist produced more effective inhibition of premenopausal estradiol levels than the LHRH agonist alone.278 This is also the case for the nonsteroidal aromatase vorozole, because the combination of vorozole and goserelin was markedly more effective in reducing estrogen levels than goserelin alone.279 Further investigations will be required to determine whether the extremely low estrogen levels achieved with an LHRH-A and a third-generation aromatase inhibitor will translate into increased clinical benefit. Outside of clinical trials, the use of selective aromatase inhibitors continues to be restricted to postmenopausal women for adjuvant treatment or metastatic disease.


Historical Perspective

In 1897, Beard postulated that the corpus luteum was essential for pregnancy.280 Support for this hypothesis was provided by Fraenkel in 1905, who demonstrated that destruction of the corpora lutea in pregnant rabbits caused abortion, an event that could be prevented by the injection of luteal extracts.281Although progesterone, the active principle in corpus lutea extracts, was isolated in 1929 from the corpora lutea of sows by Corner and Allen,282 the limited amounts of available hormone hampered further studies until the 1950s, when progesterones with prolonged activity were synthesized. Since that time, progestins have been widely used as oral contraceptives. High-dose progestin therapy is the last synthetic sex steroid routinely used in the treatment of advanced breast cancer. Until recently, megestrol acetate was a favored second-line therapy for patients with tamoxifen-resistant disease. However, megestrol acetate has been relegated to third- or fourth-line therapy, with the emergence of third-generation aromatase inhibitors and fulvestrant, which both have better side effect and efficacy profiles.22, 265

Progesterone Physiology

Progesterone is secreted mainly by the corpus luteum of the ovary during the second half of the menstrual cycle. The principal physiologic target organ for progesterone is the uterine endometrium. Progesterone secretion begins just before ovulation, coincident with the LH surge, and derives from the follicle that becomes the corpus luteum once the ovum is released.48 Progesterone is synthesized from cholesterol and pregnenolone in all steroid-producing tissues: the ovary, testis, adrenal cortex, and placenta. Although the luteotroph varies with the species, in humans LH is the primary stimulator of progesterone synthesis.49 The production rate of progesterone varies from a few milligrams per day during the follicular phase to 10 to 20 mg per day during the luteal phase (reaching blood levels of 10 ng/mL), increasing to several hundred milligrams daily during the latter parts of pregnancy. Rates of 1 to 5 mg per day have been measured in men and are comparable to the values in women during the follicular phase of the cycle.283 Once secreted into the bloodstream, progesterone is either bound to CBG (with an affinity roughly equal to that of cortisol) or rapidly cleared from the circulation within a few minutes, predominantly by the liver, where glucuronidation or sulfation occurs before excretion in the urine. The isomers of pregnanediol are the principal metabolites.35, 284 In total, 50 to 60% of the progesterone C-14 given is excreted in the urine. A small and probably physiologically insignificant proportion is stored in body fat, from which it is slowly released. The enhanced biologic potency of such synthetic progestins as medroxyprogesterone acetate (6α-methyl 17α-hydroxyprogesterone acetate) may be explained by a lower metabolic clearance rate than progesterone285 and by the greater affinity for the PgR.286 The biologic roles of progesterone are listed in Table 37.4. Although progestins act principally through the PgR, these agents have some antiandrogenic (or sometimes weakly androgenic) and antimineralocorticoid-like properties on the basis of their low affinity for androgen and mineralocorticoid receptors.

Mechanism of Action in Breast Cancer Therapy

A variety of progestins have been used for patients with hormone-dependent cancer. These include the C-21, 17-acetoxysteroids, such as medroxyprogesterone acetate and megestrol acetate, and the 19-carbon steroids, such as norethisterone (Fig. 37.13). Historically, the rate of response to progestins was quoted as approximately 27 to 35% and as positively correlated with estrogen and PgR expression.158 This has been confirmed in large phase III studies in comparison with selective aromatase inhibitors, if patients who experience disease stabilization (lack of progression at 24 weeks) are included.265, 272

The mechanism of the anticancer action of the progestins is not established, in part because these agents act not only through the PgR but also through the AR and ER. Although progestins may have a direct antitumor action,287 progestins also suppress basal and GnRH-simulated gonadotropin secretion, cortisol, dehydroepiandrosterone, and estradiol in a dose-dependent manner.288 In the normal menstrual cycle, breast epithelial cell proliferation is maximal during the luteal phase (days 20 to 28), when progesterone levels are highest.289 These data suggest that progestins stimulate the growth of normal mammary epithelium. Antiprogestins induce apoptosis in mammary tumor models,290 suggesting that progesterone is an important stimulatory factor during mammary tumorigenesis. Also, progestins regulate metastasis-related cell surface receptors, such as the laminin receptor for adhesion molecules,291 which may result in increased metastases. A direct antitumor action is supported by data, suggesting that progestins alter signaling through peptide growth factor receptors. Vignon et al. showed that R5020, a progestin, decreases the production of a mitogenic 52-kd glycoprotein.


Establishment and maintenance of pregnancy
Promotion of development of secretory epithelium of uterine endometrium after estrogen priming
Alterations in vaginal epithelium, causing change from abundant watery secretions to scant viscid material
Proliferation and engorgement of mammary acini (in concert with estrogens)
Weakly antiandrogenic
Weakly antimineralocorticoid

Figure 37.13 Progestins.

Progestin treatment results in a decrease in FSH and LH levels293, 294 Also, cortisol and ACTH levels are depressed, and the ACTH response to metyrapone is blunted.295 The cortisol decrease is ACTH-dependent. Estrone levels are decreased in postmenopausal women by 82% compared with pretreatment,294 as are the levels of dehydroepiandrostenedione sulfate and androstenedione.296 Suppression of postmenopausal estrogen is therefore likely to be a predominant mode of action of progestins.

Medroxyprogesterone Acetate

Medroxyprogesterone acetate (MPA) was once commonly used for treating breast and endometrial cancer. The usual route of administration was intramuscular (i.m.). As a result, when oral progestins such as megestrol acetate became available, the use of MPA declined. MPA is extensively metabolized, and less than 1% of an intravenous (i.v.) dosage is recovered intact in the urine.284, 297 However, there were no clear correlations between plasma MPA and efficacy or between dosage and efficacy. There has been at least one study in which MPA was administered orally at a dose of 400 mg per day, with a 53% response rate in 30 patients.158, 298

Megestrol Acetate

Megestrol acetate (MA) is the synthetic progestin most commonly prescribed for advanced breast cancer. The pharmacokinetics differ from MPA, because MA has a shorter half-life (4 hours), greater renal excretion (56 to 78%, with 12% excreted as parent compound), and higher plasma levels.284, 299 The metabolites of MA are shown in Table 37.5. With oral dosages of 400 mg per day, plasma concentrations reach 400 ng/mL, with considerable interpatient variation. The recommended dosage is 40 mg four times each day or a single daily dose of 160 mg. MPA and MA probably have equivalent activities in breast cancer, but patients on MPA have a higher incidence of side effects300


Pannuti et al. noted increased toxicity with high i.m. dosages of MPA, with little increase in efficacy.301 These side effects include gluteal abscesses in 15% of patients receiving 1,500 mg intramuscularly per day, moon-shaped facies in 11%, fine tremors in 19%, and leg cramps in 19%; there is a much lower incidence of each of these effects with the 500-mg i.m. dose. Other side effects with MPA include sweating, vaginal discharge, and amenorrhea.158 Another significant side effect is weight gain, largely due to increased adipose tissue in the abdominal and cervicodorsal regions.299 Weight gain ranged from 3 to 10 kg in 56% of patients treated with 1,000 to 1,500 mg intramuscularly per day. Because there is no difference in efficacy with different dosages or routes of administration, 400 to 500 mg orally per is probably an adequate dosage. Crona et al. reported decreases in high-density lipoprotein (HDL) cholesterol and apolipoprotein A1 and an increase in triglycerides in patients receiving 1,000 mg intramuscularly per week.302 These results suggest that the risk of cardiovascular disease could be increased in patients taking MPA. Side effects with MA include increased appetite, weight gain of 5 to 20 kg, elevated liver function tests, thromboembolism, vaginal bleeding, hot flashes, fluid retention, nausea and vomiting, hypercalcemia and flare, and rash.299 In addition, megestrol acetate has shown activity in cancer patients as a treatment for anorexia and weight loss.303 This activity appears to result from suppression of cytokines that induce tumor cachexia.304 Megestrol acetate in low dosages (20 mg per day) is also active in the treatment of postmenopausal hot flashes in breast cancer patients.305


Mechanism of action









Half-life = 4 hr


Renal excretion, 56–78%, fecal excretion, 7.7–30.0%



Oophorectomy has been a standard therapy option for breast cancer for more than 100 years. As adjuvant therapy for premenopausal women, oophorectomy is associated with a marked reduction in relapse and death from breast cancer.306 The development of LHRH agonists has provided the option of reversible medical ovarian ablation. The only randomized comparison between ovariectomy and LHRH agonist therapy was underpowered, but it did not reveal significant differences between these two approaches to estrogen deprivation.307 Because premenopausal women treated with LHRH agonists have plasma estradiol concentrations typical for postmenopausal women, surgical oophorectomy and treatment with LHRH agonists are generally held to be equivalent.307, 308, 309Premenopausal women with breast cancer treated with LHRH agonists have objective response rates of between 36% and 44%.309 Medical castration using LHRH agonists is currently used in premenopausal women in both the adjuvant and metastatic setting, with or without tamoxifen. Despite the fact that a number of LHRH agonists have been tested in breast cancer, goserelin acetate (Zoladex, AstraZeneca) is the only one approved for this disease and is therefore the LHRH agonist of choice for breast cancer patients. The general characteristics and amino acid sequence of different LHRH agonists are detailed in Table 37.6 and Figure 37.14.


Mechanisms of action

Inhibition of gonadotropin secretion with resultant castration levels of testosteronein men and estrogens in women

Direct inhibitory effects on steroidogenesis through blockade of 17,20-desmolase and 17α-hydroxylase enzymes

Direct antitumor effect on human mammary cancer in vitro


Gonadotropin-releasing hormone

t1/2a, 2–8 min

t1/2b, 15–60 min

Goserelin s.c.

4.9 h

Triptorelin s.c.

2.8 h


Enzymatic degradation by pyroglutamate aminopeptidase, endopeptidase, and post-proline-cleaving enzymes and renal excretion

Drug interactions

None known


Hot flashes

Reduced libido


Local irritation at the injection site

Polyuria and polydipsia

Gastrointestinal problems, such as indigestion, nausea and vomiting, and constipation

Taste sensations

Peripheral edema


Gynecomastia and mastodynia

Disease flare

General allergic reaction


Because there is an initial surge of luteinizing hormone, follicle-stimulating hormone, and testosterone, an acute exacerbation of disease may be seen

t1/2, half-life.

Mechanism of Action

LHRH agonists reduce circulating concentrations of estrogens in premenopausal women via an inhibitory effect on the hypothalamic-pituitary-gonadal axis. The drugs bind to the LHRH receptors on the pituitary gland, leading to an initial stimulation of FSH and LH production during the first few days of treatment.308After this initial stimulation, the ligand-receptor complexes cluster and get sequestered in the cell; this leads to a reduction in the number of active receptors on the surface. This mechanism accounts for the paradoxical inhibition of pituitary gonadotropic cells with continuous instead of pulsatile LHRH agonism.310 As a result, after a few days of stimulation, FSH and LH levels fall and remain persistently suppressed, reaching levels comparable to the postmenopausal state within 21 days.310 Plasma progesterone, estrone, estrone sulfate, and estradiol levels decrease to postmenopausal levels after 6 weeks of treatment. There is no change in androstenedione, prolactin, or cortisol levels.

Figure 37.14 Gonadotropin-releasing hormone (GnRH) and analogs. (Arg, arginine; Glu, glucose; Gly, glycerol; His, histidine; Leu, leucine; Pro, proline; Ser, serine; Trp, tryptophan; Tyr, tyrosine.)

Goserelin: Pharmacology and Metabolism

Goserelin is a synthetic decapeptide analog of LHRH that is administered as a subcutaneous depot, providing gradual release of the drug. A 3.6-mg depot formulation is given once a month. In addition, a 10.8-mg depot is given once every 3 months, although this formulation is approved only for prostate cancer, not breast cancer.

When administered as a solution subcutaneously, goserelin has been observed to have a half-life of about 4 hours.311 After administration of the 3.6-mg depot, there is an initial, short-duration peak of goserelin in the serum at 8 hours. A more prolonged peak then occurs at about 14 days. There is no accumulation of the drug with multiple administration of the depots. After administration of the 10.8-mg depot, the serum concentration profile is notably different than for the 3.6-mg depot. After an initial release over a few days, the profile shows a downward trend, with a shallow second peak at 5 weeks.

Elimination of goserelin is primarily in the urine (93%), with only 2% found in the feces. Excretion occurs fairly rapidly, with greater than 75% of a dose being excreted within 12 hours.311 In the urine, approximately 20% of the drug is excreted unchanged, and the remaining products are fragments of the decapeptide. With renal impairment, goserelin clearance decreases, and there is a corresponding increase in half-life. With hepatic impairment, differences in the pharmacokinetic parameters of goserelin were minimal, with a small increase in Cmax but not AUC as compared with controls.311 Given the wide therapeutic window of goserelin, it is not considered necessary to alter dosing in patients with either renal or hepatic insufficiency.

Following an initial dose of the 3.6-mg depot, LH and FSH levels rise to a peak at about 48 hours.311 In women with benign gynecologic conditions, the levels then rapidly decline to below baseline by day 3. The levels are greatly reduced after day 8. Estradiol levels rise transiently at 3 days, followed by a decrease of estradiol; similar results are seen with progesterone. Estradiol levels return to normal within 3 months of stopping goserelin. With the 10.8-mg depot, there is an initial increase in LH and FSH in the first week, and castrate range is achieved within 21 days.

While ovarian ablation is achieved in most patients treated with LHRH agonists, there are reports of failure with normal treatment doses.312 In a study with three different LHRH analogs, residual ovarian estrogen production was detected in 5 of 40 patients at 3 months despite profound suppression of LH in all patients.313 In addition, up to 9% of patients treated for endometriosis had persistence of uterine bleeding episodes at 6 months.314

Triptorelin: Pharmacology and Metabolism

Triptorelin is a synthetic decapeptide analog of LHRH that is not yet approved for use in breast cancer treatment; however, there are currently two large adjuvant studies using this drug in premenopausal women.315 Both trials, the IBCSG Tamoxifen and Exemestane Trial (TEXT) and the IBCSG Suppression of Ovarian Function Trial (SOFT), will use the drug in combination with either exemestane or tamoxifen.

The drug can be administered as an intramuscular injection of a suspension of microspheres at a dose of 3, 3.2, or 3.75 mg once a month or 11.25 mg every 3 months.316 With the monthly formulation, peak serum levels of the drug occur during the first week and then remain at a detectable plateau level for about 4 weeks. As with goserelin, initially levels of LH and FSH increase but fall to low levels after 1 to 2 weeks,317 and estradiol levels reach nadir levels by 21 days.316 Studies in patients with hepatic and renal insufficiency show that clearance of the drug decreases by about half under both conditions. Given the generous therapeutic window of triptorelin, however, dose modification does not appear to be necessary.318


LHRH Agonist Toxicity

Side effects in women include hot flashes, nausea and vomiting, headache, dizziness, vaginitis, sweating, emotional lability, breast atrophy, tumor flare, diarrhea, local reaction, irritability, hives, and severe polydipsia and polyuria in one patient.131, 308, 319 Also, amenorrhea is induced in all women. Bone mineral density is reduced in the lumbar spine and femur after 4 months of treatment with goserelin and does not normalize after discontinuation of treatment.320 In the adjuvant breast cancer setting, this may be of concern, and treatment with bisphosphonates may be appropriate. Total serum cholesterol, LDL cholesterol, and LDL to HDL cholesterol ratios were higher, whereas HDL cholesterol was lower in polycystic ovary patients treated with goserelin for 6 months.321 Measurement of antithrombin III concentrations after treatment with goserelin revealed no change.322 This suggests there may be no increased risk of thromboembolic episodes with this therapy. There are limited data available on known drug-drug interactions.


Despite the proven clinical efficacy of hormonal therapy in all stages of breast cancer, hormone-refractory breast cancer remains a formidable challenge. Many ER-positive tumors are intrinsically resistant, and all patients with ER-positive metastatic disease ultimately become refractory to hormonal treatments. From a clinical standpoint, hormone-refractory breast cancer can be considered to exhibit either primary resistance (no response to hormones) or secondary resistance (progression after disease regression or stability). Approximately one third of patients with secondary resistance obtain clinical benefit from subsequent endocrine therapy. The molecular mechanisms of resistance can be divided into three groups: (1) ER mutation, splice variants, and isoform ratio; (2) coactivator and corepressor effects; and (3) activation of other signaling pathways.

ER Mutation, Splice Variants, and Isoform Ratio

One hypothesis to explain the variable clinical response to hormones in ER-positive breast cancer invokes ER gene mutation or splice variants.323, 324, 325 ER mutations that allow ligand-independent activity have been described in human tumors. Specifically, a missense mutation substituting tyrosine 537 in the ligand-binding domain for asparagine was equally active in the absence of ligand, with tamoxifen or with estradiol.326 However, evaluation of primary breast tumors has established that somatic mutations in ER are rare, occurring in fewer than 1% of either ER-positive or ER-negative breast cancers.327 Therefore, it does not appear that mutations in ER explain the majority of instances of tamoxifen resistance.323, 328

Another potential alteration of ER function may occur through changes in the ERα-ERβ ratio. Steroidal antiestrogens have been shown to activate ERβ-mediated transcription while they repress ERα activity.68 Although the biologic significance of this remains unclear, this observation suggests that increased ERβ might counter inhibitory effects of antiestrogens, but there is conflicting data regarding this issue. In support of this hypothesis, a relatively small study demonstrated that ERβ mRNA levels were twice as high as ERα mRNA levels in tamoxifen-resistant tumors compared with tamoxifen-responsive tumors.329Other studies, however, suggest that ERβ-positive tumors are less likely to be responsive to tamoxifen.330

ERα and ERβ splice variants may also play a role in hormonal resistance. Alternatively spliced ER mRNA variants have also been commonly identified in normal and malignant breast tissues.45 An ERα transcript that has received particular attention lacks exon 5; exon 4 directly splices into exon 6, with preservation of the reading frame.331 The exon 5–deleted variant binds to DNA but not to estrogen, and it activates transcription in an estrogen-independent manner (a dominant positive receptor). These properties imply a role in estrogen-independent growth. However, coexpression of the exon 5–deleted variant with an intact ER did not alter the transcriptional response to estrogen, arguing against a critical role in breast cancer pathogenesis.332

At least five isoforms and one tyrosine mutant of ERβ have been identified; however their role in hormone-resistance is not clear.45 The most interesting ERβ variant is ERβcx, which lacks amino acid residues important for ligand binding. Although ERβcx does not bind estrogen, is does heterodimerize with ERα and inhibits DNA binding. This, in turn, results in a dominant negative effect on ligand-dependent transcription of the ER.50 Higher levels of ERβcx in tumors have been correlated with resistance to tamoxifen; however, confirmatory studies are required.45

Coactivators and Corepressors

Given the role of coactivators and corepressors in regulating ER activity, it is not surprising that they might also contribute to hormonal response. NCoA3 (AIB1) was initially identified based on its amplification in breast cancer.328 When expressed in cultured cells, AIB1 promotes tamoxifen's agonist activity, suggesting a role in hormonal resistance.333 Overexpression of another coactivator, NCoA1, similarly increases tamoxifen's agonist activity, indicating a role in tamoxifen resistance.334

Conversely, decreased activity of corepressors has also been implicated as a mechanism of resistance. In a mouse model of tamoxifen resistance, NCoR1 levels were decreased, suggesting a role in hormonal response.328 Together, these findings suggest that alteration of the coactivator-corepressor balance can result in conversion of tamoxifen from an antagonist to an agonist.

Growth Factor Signaling Pathways

As previously discussed, ER function is strongly influenced by peptide growth factor signaling, including EGFR, ERBB2, and IGF-1-R, that leads to stimulation of ER activity in the absence of estrogen. These mitogenic pathways lead to downstream activation of kinases that phosphorylate the ER and/or ER coregulators.335 In this way, these growth factor pathways can allow the ER to function in the absence of ligand.

EGF has been shown to mimic the effect of estrogens on uterine cell proliferation in ovariectomized mice, and tumors overexpressing EGFR are less likely to be sensitive to hormonal therapy.335 Similar results have been demonstrated with overexpression of ERBB2.335 Several groups have evaluated response to hormonal therapy with respect to ErbB2 tumor status or circulating ErbB2 levels.336, 337, 338 These studies have found a markedly decreased response to hormones in patients with ErbB2-overexpressing tumors.

Strategies to Combat Hormonal Resistance

There has been remarkable growth in the number of hormone therapies available for the treatment of breast cancer. With more hormonal options, the clinical efficacy of hormonal therapy has improved; however, the improvement has been incremental in nature. While hormone resistance remains the primary obstacle, the combination of hormonal agents with new signal transduction inhibitors may provide new avenues of success. The inhibition of multiple growth pathways simultaneously could potentially combat the redundancy and cross-talk of growth signals, thereby providing effective, well-tolerated therapy.


With the advent of advanced techniques, such as DNA microarrays and proteomic technologies, the characterization of different types of tumors will continue to make major strides. With regard to hormonal therapy, significant achievements have already been made. Several groups have demonstrated that supervised cluster analysis of DNA microarray data allows identification of a set of genes that can distinguish between ER-positive and ER-negative breast tumors.339, 340The marked difference in the gene expression profile of these breast cancer subtypes suggests the possibility of distinct precursor cells. Interestingly, only a small number of genes that discriminate between ER-positive and ER-negative tumors are involved in ER signaling.341

Further classification of tumors will continue to be achieved. Recently, genes that predict patient benefit from adjuvant tamoxifen treatment have been identified.342 Given that resistance to hormonal agents does not entirely overlap, further work with other agents will likely show different resistance and sensitivity patterns associated with various hormonal therapies. This will undoubtedly help define how to use different hormonal agents. Furthermore, as signal transduction inhibitors are tested in combination with hormonal therapy, DNA microarrays will be critical in determining which targeted drug cocktail to use against a specific tumor.

In addition to predicting the ER status of tumors and their response to therapy, gene expression profiling has the potential to help elucidate which signaling pathways are critical in tumors and thus provide strategies to improve treatment. It is important to note that, with DNA microarray analysis, the delineation of differences in signaling pathways between tumors is only inferential. While these advanced analytic procedures can help generate hypotheses, the findings from these studies still require testing with more traditional laboratory techniques. A number of laboratories have identified and characterized changes in gene expression that occur with various hormonal agents.343, 344 Ontology mapping, whereby genes are classified into various functional categories, has been helpful in describing major cellular events that occur. However, constructing a detailed and integrated picture of differences in signaling pathways will require further work. Proteomic technologies, which are beginning to allow large-scale interrogation of signaling pathways, will be critical in achieving this goal. Integration of these technologies into clinical trial design is essential.

The determination of predictive biomarkers for targeted therapy is currently a major goal in the oncology community. Interestingly, the use of hormonal therapy for ER/PR-positive breast cancer provides the oldest paradigm for this approach. In addition, this history gives a glimpse of the promise and limitations of targeted therapy. Hormonal resistance remains a problem that we are only beginning to address in the clinic. However, with the development of new technologies and new drugs, hormonal therapy can be a foundation on which to build more effectively tailored and targeted treatments.


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