CHAPTER 6: THE PHARMACOLOGIC MODULATION OF RECEPTOR ACTIVITY

Adriana Maggi, Ph.D.* and Rodolfo Paoletti, M.D.† Increased knowledge of the structure of ERs and KEY POINTSa of the mechanisms of 1. Cell specificity of estrogen action. Increased knowledge of the structure of ERs the receptors’ synthesis and of the mechanisms of the receptors’ synthesis and their interaction with key elements of the transcription apparatus is facilitating the synthesis of new phar- and their interaction macologically active molecules. with key elements of the 2. Pharmacologic modulation of ERs. Synthetic ER modulators can be classified as transcription apparatus steroidal or . Novel synthetic steroidal ER agonists hold promise for is facilitating the use in HRT because of agonist activity on the progesterone and androgen recep- tors. Of particular interest are the nonsteroidal agents that can act as SERMs by synthesis of new behaving as agonists in target tissues, such as bone and liver, and as antagonists pharmacologically or partial agonists in reproductive tissues [A and B]. active molecules. 1. INTRODUCTION

Natural modulate the activity of target cells Transcriptionally inactive ERs reside in the target by binding at least two intranuclear receptors. ERα cell nucleus; they are generally bound to inhibitory and ERβ belong to a large family of structurally proteins. Their activation can occur either by bind- related transcription factors that are well conserved ing of the cognate hormone (ligand-dependent acti- from the evolutionary and functional points of view.1 vation) or by posttranslational modifications (lig- The considerable progress made recently in com- and-independent activation) that enable them to prehending ER structure (figs. 6Ð1 and 6Ð2) and dissociate from the inhibitory proteins and associ- mechanism of action (fig. 6Ð2) provides a basis for ate with EREs, which are specific DNA sequences the development of new synthetic ligands aimed at in the promoters of target genes.2Ð7 Although cur- modulating ER functions in an organ-specific fashion. rently little is known about ligand-independent activation of ERs, analyses of crystal structures of

* From the Milano Molecular Pharmacology Laboratory, Department of Pharmacological Sciences, University of Milan, Milan, Italy. † From the Department of Pharmacological Sciences, University of Milan, Milan, Italy. a Evidence categories are given in square brackets. A = randomized controlled trials (rich body of data); B = randomized controlled trials (limited data); C = nonrandomized trials and observational epidemiologic studies; D = Panel expert judgement. (See also table 1Ð1).

103 FIGURE 6Ð1

Structural features of ERs. Mutation studies have identified six functional domains within the ER molecule (see ch. 5 for a more detailed description of ER functional domains): the N-terminal A/B domain, C domain, or DNA binding domain, D domain, the so-called “hinge region,” and E/F domain, or ligand-binding domain. Ligand-independent AF-1 is located in the A/B domain, while ligand-dependent AF-2 is harbored by C-terminal E/F domain. C domain mediates the interaction between the receptor and the DNA through “zinc finger” structures.

the hormone-binding domains of ERβ and Erα 2. CELL SPECIFICITY OF ESTROGEN complexed with agonists and antagonists provide RECEPTOR ACTION some insight into the intramolecular modifications It is now well known that estrogens produce a leading the ligand-receptor complexes to interact wide variety of physiologic effects and that ERs with the transcription machinery.8,9 In addition, are expressed in most mammalian tissues. To several coregulators involved in the generalized design drugs able to mimic estrogen activity in and tissue-specific activities of the receptors have only selected target tissues, it is mandatory to been identified.10Ð13 ERs may also influence the understand how the same hormone can exert so transcription of genes lacking EREs through bind- many different effects in the various cells targeted. ing other transcription factors (e.g., AP1) and One hypothesis is that the genes regulated by the thereby hindering these factors’ capability to act on hormone are different in each type of target cell. their responsive elements.14,15 Because the hormone-receptor complex acts through the binding of the same EREs in all the cells, the question arises as to the factors determin- ing the specificity of estrogen action. A number of

104 factors listed below may contribute, although, in Tissue-specific Transcription Elements: As spite of very active research in the field, more mentioned above, an ER must interact with tran- studies are needed to clarify which of the mecha- scription factors to initiate the transcription of tar- nisms predominate in the various targets. get genes. The factors are in part ubiquitous and in part cell-specific. Therefore, the abundance of Developmental Cues: During the process of cell these cell-specific proteins will contribute to affect differentiation, fragments of the genome are modi- -ER action on individual promoters. fied enzymatically or are bound by specific pro- teins and become inaccessible to transcription fac- Receptor Dosage: Like other membrane recep- tors. The process will undoubtedly involve some of tors, ERs can be up- or down-regulated. It is most the ER-inducible promoters, which will become likely that higher concentrations of the receptor insensitive to the hormone activity. protein will allow the hormone to stimulate most of the target promoters, whereas lower concentra- Complexity of the Target Promoters: In complex tions will allow generation of subgroups of tran- promoters, in which different responsive elements scripts, for example, from genes whose promoters control the transcriptional activity of a single gene, possess multiple EREs. the activity of estrogens will be influenced by the presence of the other factors involved in gene tran- Ligand Characteristics: ERs bind estradiol and scription control. some of its metabolites. Depending on the ligand

FIGURE 6Ð2

Panel a. Graphic of the three-dimensional structure of the ER hormone-binding domain. The binding of the agonist induces a series of conformational changes that expose sites of the receptor capable of interaction with co-activators. The binding of antagonists induces a very different conformation of the receptor, mainly in the displacement of helix 12 (solid cylinder), and prevents the exposure of the domains involved in co-activator recruitment. Panel b. Mechanism of ligand-dependent activation of ERs. On binding to the receptor (ER), estradiol induces release of inhibitory proteins, which allow the recep- tor to dimerize and associate with the responsive elements in the promoters of target genes. Once bound to the target DNA, the receptor initiates a series of protein-protein interactions that result in the activation of the transcriptional apparatus.

105 bound, the conformation of the receptor differs, progestins are administered in combination with thus changing its functional activity. estrogens to limit the undesired side effects of estrogens on uterine cells. Receptor Subtype: The To design drugs able to type of ER expressed in a The ligands for the ER can be grouped as ER single cell type may also be agonists, ER antagonists and SERMs. Subgroups mimic estrogen activity important for the selectivity are based on chemical structure, categorized as in only selected target of estrogen action. In addi- steroidal and nonsteroidal compounds. tion to the different effects tissues, it is mandatory exerted by ERα and ERβ 3.1 Agonists to understand how the homodimers, it is possible Known agonists of the ER consist of both steroidal and nonsteroidal molecules, with the latter includ- same hormone can that ER heterodimers act differently and that the two ing examples of different chemical structures. exert so many different receptors exert mutual 3.1.1 Steroidal Agonists effects in the various control. That has been Natural human estrogens are the follicular hormone reported to occur with other cells targeted. 17β-estradiol and its main metabolites, , members of the intracellu- , and 2-hydroxyestradiol—together with lar receptor superfamily.16 their sulfated and glucuronidated counterparts. A Influence of Other Members of the Nuclear more extended definition of natural estrogens Receptor Superfamily: Orphan receptors highly includes the mare and . Critical homologous to ERα—for example, ER-related structural characteristics of this class of compounds receptors α, β, and χ may interfere with ER activi- include (1) a phenol at the CÐ3 position of the aro- ties, also by heterodimerization. matic A ring, (2) a relatively flat and rigid hydro- carbon core, and (3) a ketone or alcohol function at the CÐ17 position. A detailed pharmacophore 3. PHARMACOLOGIC MODULATION OF model suggests the important contribution of the β ESTROGEN RECEPTORS two hydroxyl groups of 17 -estradiol to receptor Pharmacologic treatment of endocrine dysfunction binding, with CÐ3 hydroxy acting as the major involves the control of (1) endocrine signaling, by contributor to the binding free energy. The model 8,9 acting on the synthesis, storage, or release, or is supported by recent X-crystallography data. intracellular transport and metabolism, of the spe- When natural estrogens cific hormone; (2) sensitivity of a single target tis- are administered orally, In replacement therapy, sue to the hormone, by blocking or enhancing syn- they undergo rapid thesis of the receptor of interest; or (3) activity of catabolism in the natural are the receptor, by the use of specific ligands with intestinal mucosa and currently preferred over agonist or antagonist activity. The synthetic com- liver; shortly after synthetic molecules. pounds generated and in use to date for ER modu- estradiol ingestion, lation belong to the third category, with the excep- there are high concen- tion of progestins. In several organs, progestins trations of the metabolites, predominantly estrone, antagonize the activity of estrogens by a dual in the systemic circulation. Peak concentrations are mechanism: down-regulation of the synthesis of observed at 1 to 4 hours after ingestion; subse- new ERs and control of the transcription of genes quently, concentrations rapidly decline. For this that interfere with estrogen action. Therefore, reason, major efforts have been made, particularly

106 in the past, to synthesize analogues of allow the rate-controlled delivery of estrogen. The estrogens with longer half-lives. All the synthetic hormone is suspended in an ethanol solution or, in molecules developed are far more potent than the second-generation TTS, in a matrix, which ensures natural estrogens and have, when administered the programmed release of the hormone for several orally, much longer half-lives. Some, like DES are consecutive days. no longer used in clinical practice. The finding of a More recently, high incidence of clear cell adenocarcinoma of the novel synthetic Observational epidemiologic vagina in daughters born to DES-treated mothers molecules such as led to the hypothesis that the compound was a car- studies indicate that women (7α, 17α)-17- cinogen. However, further studies did not show a hydroxy-7-methyl- who ingest , significant increase in in the DES- 19-norpregn- treated mothers, and studies of male offspring particularly in soy products, 5(10)en- showed genital abnormalities, suggesting that DES in large amounts seem to 20-yn-3-one should be considered more as teratogenic than can- () have have lower rates of CVD, cerogenic. Other synthetic estrogens, such as raised considerable ethinyl estradiol, have been and are largely used in breast cancer, and uterine interest because oral contraception. Ethinyl estradiol is obtained by they combine with cancer as well as fewer the addition of an ethinyl radical in position CÐ17 the estrogenic climacteric symptoms than and is much more resistant than natural estrogens activity progesto- to liver metabolism. Orally administered ethinyl women consuming typical genic and andro- estradiol has a half-life of about 48 hours. genic properties western diets. In replacement therapy, natural steroids are cur- that relieve climac- rently preferred over synthetic molecules. In the teric symptoms without stimulation of the United States, the most commonly used natural endometrium.18,19 estrogens are CEEs, extracted from the urine of pregnant mares. CEEs are mainly composed of 3.1.2 Nonsteroidal Agonists estrone and ; other components Pioneering studies published more than 60 years include the ring-B unsaturated sulphoconjugated ago showed the effects of subcutaneous adminis- estrogens equilin, equilenin, and their 17α-deriva- tration of nonsteroidal compounds on the onset of tives. The metabolites account for a great part of estrus in mammals and on uterine growth. They the estrogenic effects of CEEs. In addition, equilin enabled the identification of several nonsteroidal can be stored in adipose tissue and released for molecules with estrogenic activity. The studies several weeks after withdrawal of the treatment. were also instrumental in the subsequent develop- 17β-estradiol is also used in HRT, particularly in ment of and of partial agonists- Europe. Preparations of or antagonists, including SERMs. micronized estradiol were shown to have half-lives The major categories of the nonsteroidal agonists compatible with therapeutic effects when adminis- are (1) 1,2-diarylethanes and ethylenes; (2) tered orally.17 To avoid the intensive first-pass flavones, isoflavones, , and ; (3) metabolism, nonoral routes of administration of macrolactones; (4) alkylphenols and arylphenols; 17β-estradiol have been studied. Several parenteral and (5) nonaromatic estrogens. delivery systems are available: 17β-estradiol can 1,2-Diarylethanes and Ethylenes: DES and be administered through injections, vaginal rings, (fig. 6Ð3) represent a milestone in the percutaneous gels, or transdermal therapeutic sys- identification of orally active nonsteroidal agents tems (TTS). Of particular interest are the TTS that

107 with extremely potent estrogenic activity. The genic activity of flavones and isoflavones is depen- medical uses of compounds with 1,2-diarylethyl- dent on ER binding affinity, which is determined ene and 1,2-diaryl ethane include maintenance of by the presence of the aromatic ring as well as pregnancy, HRT, suppression of lactation, post- hydroxyl groups at specific sites. coital contraception, and cancer treatment, as has Compared with estradiol, and been reviewed.20 bind to ERs with significantly less affinity.23 Flavones, Isoflavones, Coumestans, and Nevertheless, in the quantities that can be con- Lignans: The best studied plant-derived estrogens, sumed in the diet, isoflavones can have biologic or phytoestrogens, belong to these classes of effects. Soybean isoflavones do not have feminiz- chemicals with estrogenic activity. A significant ing effects in male primates as reflected by source of isoflavones is soybeans. The most abun- unchanged weights of the reproductive organs in dant and active components of isoflavones are the animals,24 although prenatal exposure of rats genistein (fig. 6Ð3) and daidzein, which appear to resulted in diminished weights of ovaries and have selective estrogenic actions. In some tissues, uteri.25 Observational epidemiologic studies indi- they provoke proestrogenic responses; in others, cate that women who ingest phytoestrogens, par- they inhibit estrogenic effects. This finding is per- ticularly in soy products, in large amounts seem to haps explained by different affinities for the two have (1) lower rates of CVD, breast cancer, and described ERs. Indeed, genistein has a thirtyfold uterine cancer and (2) fewer climacteric symptoms higher affinity for ERβ than for ERα.21,22 The estro- than women consuming typical western diets.26 (See ch. 3, 8, and 10.) Preclinical and clinical stud- FIGURE 6Ð3 ies have shown that isoflavones can improve plas- ma lipid profiles as well as the ability to inhibit Structural Characteristics of oxidation of low-density lipoproteins.26 Isoflavones Estrogen Receptor Agonists have been shown to normalize vascular reactivity in estrogen-deprived primates.27 Antineoplastic activity of the compounds has been postulated on the basis of their inhibitory activity on angiogene- sis.28 In addition to climacteric symptoms, bone density appears to be favorably influenced by phytoestrogens.26 (See also ch. 9.) Other known phytoestrogens are coumestans (e.g., ), found in alfalfa sprouts, and lignans (e.g., ), found in cereals and oil seeds such as flaxseed.

Macrolactones: Macrolactones were first identi- fied when hyperestrogenicity was seen to develop in swine fed mold-infected corn. Katzenellenbogen and colleagues extensively studied these mycotox- ins, exemplified by (fig. 6Ð3), which was shown to bind ERs with high affinity and induce uterotropic responses in rats.29 Zearalenone has been used to relieve the incidence and severity of hot flushes in women.30

108 Alkylphenols and Arylphenols: Alkylphenols are 3.2 Estrogen Receptor Antagonists used in the synthesis of detergents and as antioxi- Antiestrogens can be grouped according to basic dants. The detergents are not estrogenic; upon structure as steroidal or nonsteroidal. degradation during sewage treatment, however, they can release estrogenic compounds, such as 3.2.1 Steroidal Antagonists para-octyl phenol and para-nonyl phenol, which The difficulty in modifying the basic steroid struc- have been shown to possess uterotropic activity in ture discouraged research into steroidal antagonists vivo and in vitro.31 The structural requirements of of ERs until Raynaud et al. demonstrated that the α alkylphenols indicate that both the position (para > 7 -estradiol derivatives with a long, unbranched 35 meta > ortho) and the branching (tertiary > sec- alkyl chain retained high affinity for ERs. The ondary) of the alkyl group dramatically affect compound ICI 164,384 was developed and shown estrogenicity.32 In addition to alkyl substitution to act as a pure ER antagonist, stimulating further at the para position of the halogenated or the research. The most active compounds that emerged hydroxy-substituted aromatic ring, aryl sub- from the studies are ICI 164,384 and ICI 182,780 stituents have estrogenic activity. Compounds in (fig. 6Ð4a). Both avidly bind to ERs and retain a pure the class include ; polychlorinated activity in all tissues studied to date. biphenyls; diphenylmethanes, such as DDT (dichlorodiphenyltrichloroethane); and tri- FIGURE 6Ð4a 33 cyclic aromatic hydrocarbons, such as dioxin. Structural Characteristics of Many of these compounds are used in the manu- Estrogen Receptor Antagonists facture of plastics, and their estrogenic activity was discovered by accident because they are released by polystyrene and polycarbonate test tubes used in laboratory experiments. Bisphenol A was found to contaminate the content of canned food because tin cans are lined with polycarbonate. Bisphenol A is also used in dental sealants and composites.

Nonaromatic Estrogens: Interest in nonaromatic estrogens has increased because of this class can derive from commercial sources, such as . Most of the agents are halo- genated carbocycles, such as hexachlorocyclohexa- ne, (Kepone) (fig. 6Ð3), chlorobor- nane (Toxaphene), dieldren, and . Studies with hexachlorocyclohexane showed it to have estrogen agonist activity in human breast can- cer cell lines. It and others of the class have not 3.2.2 Nonsteroidal Antagonists been shown to bind to ERs. Therefore, the origin For the rational design of nonsteroidal antiestrogens, of the estrogen activity of the compounds is not essentially two basic structures were systematically clear; it is believed to occur through nonclassic altered: the and stilbene struc- pathways.34 tures. The first nonsteroidal antiestrogen discov- ered was (MERÐ25) (fig. 6Ð4b).

109 Its potency as an antiestrogen was rather low, and FIGURE 6Ð4b serious side effects in the CNS were found during clinical development.36 The antagonist activity Structural Characteristics of of the molecule may be Estrogen Receptor Antagonists The activity of due to the hydrophilic hydroxy group in the in postmenopausal center of the molecule, patients with hormone- which most likely inter- feres with the receptor dependent breast cancer binding. Clomiphene has been demonstrated (fig. 6Ð4b), the second in many clinical studies, ER antagonist developed, lacks the central hydroxy and the agent has group, and a double bond become a treatment of with a chloro substituent choice for the malignancy. improves its lipophilicity. Clomiphene is used as a gonad-stimulating agent in subfertile women. With the identification of the ER antagonist activity of nafoxidin (fig. 6Ð4b), it became evident that a certain steric arrangement at the double bond favors the antiestrogenic activity. The most prominent among the group of drugs is tamoxifen, which was developed by ICI and is marketed as a citrate salt. The activity of tamox- ifen in postmenopausal patients with hormone- dependent breast cancer has been demonstrated in many clinical studies, and the agent has become a treatment of choice for the malignancy. Tamoxifen shown to have minimal uterotropic activity, to is used in patients with advanced disease, as well reduce plasma concentrations, to stimu- as in the adjuvant setting after surgical removal of late osteoclast apoptosis, and to block gene expres- 37 the primary tumor. sion in endometrial cells. Despite the fact that the basic side chain is one of tamoxifen’s most Other triphenylethylene derivatives were synthe- important structural elements, only a very limited sized with the objective of obtaining an antiestro- number of variations of that part of the molecule gen that would have a lower rate of metabolism have been synthesized. Interestingly, when the and a different pharmacodynamic profile compared 2-(dimethylamino)ethoxy fragment was replaced with tamoxifen and would not present its cis/trans by acrylic acid, the agonist activity was lost in the isomerization. The knowledge that 4-hydroxyta- uterus but was retained in bone and the cardiovas- moxifene is more potent in vitro than the parent cular system.38 drug but is more readily catabolized provided the basis for the development of the 3-hydroxy deriva- Studies carried out principally at Eli Lilly and tive and of the iodo derivative idox- Company demonstrated that the geminal arrange- ifene (fig. 6Ð4b). Droloxifene and were ment of the two phenyl rings of tamoxifen is not

110 essential and can be replaced by structures in FIGURE 6Ð4c which all three phenyl groups are located at differ- ent atom groups. Several compounds were generat- Structural Characteristics of ed (fig. 6Ð4c), among them LY 117,018, the first to Estrogen Receptor Antagonists undergo detailed study of its endocrine activities.39 A series of modifications of LY 117,018 led to the synthesis of keoxifene (LY 156,758), which is now known as .40 In raloxifene, the presence of two free hydroxy groups in the benzothiophene and phenyl rings gives rise to an ER-binding affin- ity higher than in compounds lacking those polar functions. Another basic structure that has been utilized for the rational design of antiestrogen drugs is 2-phenylindole. (fig. 6Ð4c) belongs to the compounds synthesized with this structure. Its endocrine profile is that of an antiestrogen with partial agonist activity. The preclinical and clinical data thus far obtained suggest that zindoxifene is a new lead structure of interest for the design of drugs acting through ERs.

3.2.3 Estrogen Receptor SubtypeÐ tional activity of which may significantly differ Specific Antagonists from the homodimers.45,46 Finally, in vitro studies α The substantial structural homology between ER evaluating the response of reporter genes or more β and ER has raised the question of whether the physiologic parameters of pharmacologic activa- two receptors are, indeed, responsible for different tion of the two receptors have demonstrated signif- physiologic responses. Localization studies per- icant differences between ERα and ERβ activities. formed in mature and developing mammals sug- In spite of the remarkable similarity in their gest different roles for the two receptors. In fact, response to synthetic ligands, the two receptors α β ER and ER are often differentially expressed in seem to activate distinct target genes, with diver- developing and mature tissues (e.g., mammary gent consequences for cell physiology.47,48 The dif- gland and brain) and seldom colocalize in a given ferential activity can be attributed to the significant 41 cell type. Ablation of one or the other of the two structural diversity of the A/B region carrying the receptors in gene knockout experiments further ligand-independent, N-terminal activation function supports the hypothesis of a different effect of the AFÐ1. This hypothesis is supported by studies with α two receptors. ER activity seems to predominate a chimera receptor in which the A/B domain of β in the uterus and breast, whereas ER may have ERα was substituted to the same domain of ERβ.49 significant roles in the CNS, cardiovascular sys- Thus, because of the potential physiologic differ- tem, immune system, urogenital tract, kidney, and ences in the activities of the two receptors, differ- β lung. (See also ch. 5.) ER appears to be the only ences in ligand interaction or activity could trans- 42Ð44 form expressed in the embryonic CNS. On the late into important differences in their biological other hand, the two receptors can co-localize; it is and pharmacological profiles. possible that they form heterodimers, the transcrip-

111 The observation that the degree of homology of plex seems to be slower than that of agonist-ER the two hormone-binding domains of ERα and complex. Dissociation of the former complex is ERβ is approximately 56 percent suggests the pos- much slower than dissociation of the latter. sibility of developing ligands with different agonist Protein-Protein Interaction: The availability of or antagonist characteristics with regard to the two crystals of the ERα and ERβ hormone-binding receptor subtypes. Recently, novel compounds peptides bound to 4OH-tamoxifen and raloxifene with pronounced subtype-selective differences in provides important new information about the binding affinity and transcriptional potency or effi- mechanism of these agents’ activity. Studies show cacy were identified.50 An aryl-substituted pyrazole that the additional side chain of the antagonists was shown to be an ERα potency-selective ago- protrudes from the ligand-binding cavity, so that nist: in fact, it showed a higher binding affinity for helix 12 is translocated to a position in which the ERα than for ERβ and, in transactivation assays, a co-activator binding side is obscured. As a conse- potency of ERα of about two orders of magnitude quence, the antagonist-ER complex can still dimer- higher than ERβ. ize and bind to DNA, but this event cannot be fol- Another compound, a tetrahydrochrysene (THC), lowed by the series of protein-protein interactions was shown to have a fourfold preferential binding indispensable for AFÐ2-dependent transcription affinity for ERβ; it was an agonist for ERα and a initiation, mediated through ligand-dependent, complete antagonist for ERβ. Interestingly, the C-terminal activation function. antagonist activity appeared to be associated with Those findings underline the importance of pro- the R,R-enantiomer (R,R-THC); the S,S-THC was tein-protein interactions (ER with co-activators or an agonist for both ERα and ERβ but had a twen- co-repressors) in the mechanism of action of ER tyfold lower affinity for ERβ than R,R-THC. antagonists. Furthermore, they provide an explana- tion for the mixed agonist-antagonist activity 3.2.4 Molecular Mechanisms of Antiestrogen Action reported for both raloxifene and tamoxifen bound The molecular mechanisms of antiestrogen block- to ERα. (See fig. 6Ð5.) In fact, the binding of this ade of ER activities have been the object of several antagonist prevents interactions of AFÐ2, but not studies addressing all the steps necessary for ER AFÐ1, with the transcription machinery. Therefore, transcriptional activation. the receptor, once bound to the ERE, may still attract transcription factors with the AFÐ1 region Receptor Binding: Generally, the affinity of and allow the transcription of selected genes. antagonists for ERs is similar to or even higher Deletion and mutation studies have demonstrated than the affinity of estradiol. It was initially pro- that the full transcriptional activation of ERα is posed that the binding of an antagonist, in particu- observed when both AFÐ1 and AFÐ2 are present lar the binding of pure antagonists,51 prevents the and activated.53 When access to AFÐ2 is prevented dimerization and limits the affinity of the complex by the presence of an antagonist, the activity of the for DNA. Those studies were highly controversial, receptor is limited to those cells and those promot- and the mechanism cannot be extended to tamox- ers that contain factors that activate AFÐ1. (See ifen, which was clearly shown to allow the forma- also ch. 5 for mechanisms of differential actions of 52 tion of ER dimers. antagonists on ER subtypes α and β.) DNA Binding: Most in vitro studies have shown Receptor Turnover: ER antagonists may affect that DNA binding activity of the ER complexed the kinetics of ER degradation. This represents with agonists and antagonists is very similar, even another potential mechanism of action of antago- though the DNA binding of antagonist-ER com-

112 FIGURE 6Ð5

Mechanism of action of SERMs in different cell settings. Binding with a SERM results in blockage of activity of the AFÐ2 region of the ER; the AFÐ1 region, however, is free to interact with other proteins, which allows the interaction of the DNA-bound ER with other elements of the transcription apparatus. Thus, depending on the proteins expressed in the host cell, the SERM-bound receptor will be able to act as a full agonist (cell A) or a partial agonist (cell B). In cells that lack proteins able to interact with the AFÐ1 region of the receptor, the SERM will completely prevent the activation by estrogens and, therefore, will be a complete antagonist (cell C).

nists; it has been shown to be of importance for itself causing endometrial stimulation. Later, the pure ER antagonists, such as ICI 164,884 and ICI nonsteroidal triphenylethylene compounds 182,780, but not for nonsteroidal antagonists.54 clomiphene citrate and tamoxifen were reported to be capable of inducing ovulation by blocking the 3.3 Selective Estrogen Receptor Modulators negative feedback of estradiol on the HPA56,57 and (SERMs) to block the development of dimethylbenzan- A SERM is defined as a compound that has estro- thracene-induced mammary tumors in rats58 gen agonism in one or more of desired target (tamoxifen). The results clearly demonstrated the tissues, such as bone and liver, and antagonism antiestrogenic activity of the molecules and stimu- and/or minimal agonism in reproductive tissues lated interest in their development for antineoplas- such as breast and uterus.55 tic activity. In the 1980s, two seminal studies, by The path leading to SERM development began Beall et al.59 and Jordan et al.,60 demonstrated that with the synthesis of the first clomiphene and tamoxifen had a more varied port- antagonist, MERÐ25, which was shown to inhibit folio of action in that the agents were able to the action of estradiol in the endometrium without decrease bone loss in ovariectomized rats. The

113 findings were remarkable because a pure antiestro- The challenge for the future in the development of gen would have been expected to promote rather ligands for the ERs will be to refine the target than inhibit bone loss. Because clomiphene is a specificity of SERMs and, it is hoped, amplify racemic mixture of enclomiphene and their scope of action. For instance, a SERM with a zuclomiphene, primacy in the SERM field must be profile similar to idoxifene or raloxifene in the accorded to tamoxifen. However, the discovery peripheral organs and CNS might protect against that tamoxifen was, in some patients, capable of Alzheimer’s disease and hot flushes, while helping endometrial stimulation leading to hyperplasia or prevent , CAD, and breast and neoplasia61 precluded its development as a bone endometrial cancers. antiresorptive agent that would be free of uterine bleeding and, it was hoped, breast safe. The concept of selective ER modulation has been 4. FUTURE NEEDS demonstrated subsequently for a number of com- The availability of drugs selective for ERα and pounds, including raloxifene, droloxifene, GW5638, ERβ will constitute a major milestone toward the idoxifene, and FCÐ1271. and idoxifene development of drugs with specificity of action. are molecules similar to tamoxifen; despite their On the other hand, the acquired knowledge of the potential as SERMs, they have been targeted for molecular mechanisms of estradiol activity in the treatment of advanced breast cancer. different cells might suggest novel paths to be Droloxifene is known to have efficacy as a breast followed for the development of molecules active cancer drug but is also a SERM at bone sites.62 through the ERs. For example, ER turnover is Raloxifene was developed because it held promise differentially regulated in various organs (e.g., for multiple applications. It is used in the preven- following gonadectomy, ERα mRNA is increased tion of bone loss and fracture in postmenopausal in the uterus but significantly decreased in the women and is being tested against tamoxifen in the liver66). Drugs aimed at modulating the synthesis Study of Tamoxifen and Raloxifene (STAR) for of ERs in specific organs may enable fine tuning the prevention of breast cancer in high-risk post- of the activity of the hormone. Antisense oligonu- menopausal women.63 Additionally, raloxifene cleotides have been able to block ER synthesis in reduces circulating cholesterol concentrations64 and cells in culture. The development of such mole- may be efficacious to reduce risk for endometrial cules as drugs might allow their future clinical cancer.55 The main drawback of the drug is that it use.67 Alternatively, novel preparations of estradiol does not reduce the occurrence of hot flushes, in which its bioavailability is modified might have which could hinder compliance. Similar pharmaco- pharmacologic profiles of interest. For instance, a logic profiles are shared by idoxifene and formulation allowing a very rapid intraplasmic FCÐ1271, which were shown to have beneficial release of estradiol and its rapid clearance (e.g., effects on bone metabolism in rat models and to transnasal administration of appropriately modified act like estrogens in bone and on circulating estrogens) could have a quite different profile of lipoprotein concentrations, with little estrogenic action compared with estradiol administered orally activity in the uterus.37,65 A number of SERMs are or transdermally. Such a drug could rapidly acti- in preclinical or clinical development (table 6Ð1). vate the receptors but, because of its rapid clear- ance from the blood, might have little effect on ER The molecular mechanisms of the different effects down-regulation or might affect it differently in the of SERMs in different organs are summarized various targets. above (“Molecular Mechanisms of Antiestrogen Action”) and in figure 6–5.

114 TABLE 6Ð1 Selective Estrogen Receptor Modulators Under Development

Drug Pharmaceutical Company Phase of Development Therapeutic Class

SERM 339 Aventis Phase IIa

HM 144 Hormos Medical Preclinical Anti-Alzheimer

LY 139,478 Eli Lilly and Company Preclinical Bone calcium regulation

LY 326,391 Eli Lilly and Company Preclinical Cytostatic, bone calcium regulation

ERA 932 Ligand Pharmaceuticals Phase I Cytostatic

NNC 450,320 Novo Nordisk Preclinical Bone calcium regulation

NNC 450,781 Novo Nordisk Preclinical Bone calcium regulation

M.D.L 101,986 Aventis Pharma Preclinical Cytostatic, bone calcium regulation

Fc1271A Hormos Medical Phase II Other drugs for the musculoskeletal system

Arzoxifene Eli Lilly and Company Phase II Cytostatic

Research program Signal Pharmaceutical Preclinical Cytostatic, SERMs bone calcium regulation

Lasozifene Pfizer Phase II Bone calcium regulation

Research Program Ligand Pharmaceuticals /Pfizer Preclinical Cytostatic, SERMs bone calcium regulation, anti-Alzheimer

SR 1,634 SRI International Preclinical Cytostatic

115 Recently, the use of phage display has allowed the These and other molecular developments will in identification of peptides that interact specifically the near future represent starting points for the pre- with estradiol- or tamoxifen-activated ERα. Some clinical and clinical development of estrogen com- peptides were shown to regulate ER transcriptional pounds, of both natural and synthetic origins, with activities.68 Such studies demonstrate that ER selective spectrums of action, as the following activity can be regulated even by targeting sites points summarize: that are outside the ligand-binding pocket of the ¥ Developing drugs with receptor specificity protein. This is not surprising within the current (ERα or ERβ) view of ER interactions with other proteins rele- vant in transcription. Such results have pharmaco- ¥ Modulating the activity of the hormone interfer- logic implications of interest because they provide ing with the synthesis of ERs new targets for drug activities and more opportuni- ¥ Attaining different profiles of action for exoge- ties for the development of drugs that will modu- nous estradiol through use of different formula- late the activity of ERs in selected cells or even at tions specific genes. Finally, increased knowledge of the physiologic relevance of unliganded activation of ¥ Obtaining higher specificity of action by identi- ERs might lead to the development of pharmaco- fication of new target molecules involved in logic compounds that will activate ERs through gene transcription membrane receptors. ¥ Increasing knowledge of the mechanisms involved in ER activation through membrane receptors to develop new pharmacologic com- pounds acting along these pathways

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