The selective oestrogen receptor modulation: evolution and clinical applications

Steroids are widely dispersed throughout the biosphere where they fulfil a plethora of roles as bioregulators in both plants and animals. Indeed the simplest organism yet discovered to be utilizing them is a water mold which deploys a pheromone, oogoniol, which is a steroid. Within the animal kingdom, true hormonal use of the oestrogens appears to be restricted to the phyla Arthropoda and Chordata whereas their elaboration appears to have originated as early as ≈400 million years ago with the phyla Mollusca and Echinodermata which developed the cytochrome P450arom necessary to convert androstenedione to oestrone and testosterone to oestradiol. Until relatively recently it was assumed that, in the human, the endocrine oestrogens were largely if not exclusively involved in reproductive physiology. They were known to be responsible, with other agents, for the complex signalling and signal transduction which prepares the female organism for the key events of ovulation, blastocyst implantation, maternal adaptation to pregnancy and lactation—all of which, being central to the survival of the species, are highly conserved. However, the last half century has seen a major expansion in our understanding of the range of oestrogenic activity in physiology. The physico-chemical characteristics of the oestrogens and their receptors provide a signal and signal transduction system of high utility which has found a role in many systems unrelated to reproduction. It was just over half a century ago that Fuller Albright [ 1] breached the reproductive enclosure with a report that osteoporotic women frequently reported a history of surgical oophorectomy. This observation led to the investigations which have now placed the oestrogens at the very heart of bone physiology where they play a pivotal role in the maintenance of structural and functional integrity of the human adult female skeleton. This is perhaps best illustrated by summarizing the effect of their withdrawal at the time of menopause when climacteric ovarian failure, following functional oocyte exhaustion, leads to an order of magnitude decline in the level of plasma E2 from ≈500 pmol l−1 to ≈50 pmol l−1. The latter is a level below that seen in many age-matched men and results in profound changes in bone homeostasis. The principal net effect of E2 loss is observed at the Bone Multicellular Unit (BMU) which is responsible for bone turnover. In oestrogen replete women, the unvarying 5 month BMU cycle of bone resorption—pause—bone formation, results in new bone replacing old with no significant deficit in bone tissue. In other words, bone resorption and formation are securely coupled and oestrogen has a major role on maintaining this balance. This state of affairs is lost in oestrogen deficiency [ 2]. The amount of bone excavated by the osteoclast cooperative at each site is increased and, crucially, is not matched by osteoblastic replacement. The minute loss of bone mass at each BMU is subjected to a multiplier effect in that the absolute number of BMUs also rises thereby increasing turnover. The loss of oestrogen removes a restraint to the still unknown primary signal for osteoclasts to assemble at a bone surface to commence a BMU cycle (Figure 1). Unbalanced bone turnover. Certain of the intermediate steps in the translation of oestrogen deficiency into derangement of bone remodelling have now been elucidated and involve parathyroid hormone [ 3], certain growth factors including insulin-like growth factor-I [ 4] and the cytokines IL-I and IL-6 [ 5]. In men also, oestrogen is now known to be essential to bone health. Precise documentation has been published of men who are profoundly oestrogen deficient, either through absence of the oestrogen receptor [ 6] or through the absence of cytochrome P450arom [ 7]. These men exhibit giantism, unfused epiphyses and, in the case of aromatase deficiency, rapidly respond to oestrogen replacement with epiphyseal closure and bone density gain. The cardiovascular system was the next in which a physiological role for oestrogen was described. The Framingham study reported premenopausal women who had been deprived of endogenous oestrogen exhibited a significant excess of cardiovascular disease over oestrogen replete age-matched controls [ 8]. There followed a multitude of clinical and laboratory-based studies which have assigned to the oestrogens a significant role in cardiovascular physiology. In particular, they operate through direct vascular effects including stimulation of NO synthase and inhibition of endothelin-1 production, whose net effect is to restrain the process of atherogenesis [ 9]. This protective activity is aided by the favourable influence of oestrogen on the plasma lipid profile where they reduce total and LDL-cholesterol and increase HDL-cholesterol, a process whose clinical advantage is attested by observational [ 10] though not yet by interventional studies [ 11]. Data from randomized controlled trials such as the Women’s Health Initiative (WHI) in the US will be necessary to establish a causative association between hormone replacement therapy (HRT) use and the primary prevention of atherosclerotic disease. Intriguing, but even less well established are the effects of oestrogen lack on the central nervous system. The excess of Alzheimer’s disease in women, the ability of oestrogen to promote cerebral blood low and neurotransmitter production have led to observational case-control and cohort studies which suggest that the incidence and clinical course of Alzheimer’s disease may be adversely affected by oestrogen deficiency [ 12]. Complementary in vitro data suggest that the elaboration of amyloid from amyloid precursor protein may be inhibited by oestrogen at nanomolar concentrations [ 13]. The list continues to grow. Other conditions in which quality observational evidence now suggests an aetiological role for oestrogen deficiency include adenocarcinoma of the colon [ 14], cataract [ 15] and certain auto-immune diseases such as rheumatoid arthritis and multiple sclerosis [ 16]. It must be emphasized, however, that observational studies, however, well conducted, can only show association and are subject to the play of chance, the intrusion of bias and the operation of confounding variables. Interventional studies with appropriate randomization blinding and rigorous controlling are the current gold standard for establishing causation. For all the nonreproductive systems utilizing oestrogen for optimal function, the midlife climacteric with its functional and endocrine ovarian failure, poses a problem of adaptation. The postmenopausal reduction in circulating oestradiol brings in its train a set of symptoms which are well known and described and a set of metabolic changes which are not. High prevalence of menopause is a relatively new human female phenomenon. The present longevity of the female population—≈81.5 years in the UK—means that the prevalence of the postmenopausal state in developed countries is high and its clinical consequences are now of high relevance not only to individual welfare but also to the public health. The realization that the symptoms reported by perimenopausal women were capable of amelioration by oestrogens has led, over half a century, to the present highly developed and researched field of hormone (oestrogen) replacement therapy (HRT). It is now beyond scientific debate that exogenous oestrogens will resolve such menopausal symptoms as hot flushes, night sweats, vaginal dryness and irritability. However, there is a major problem which centres on acceptability. Many women believe that the menopause is a natural phenomenon which should not attract medical intervention, whereas the counter argument, that the oestrogen deficiency state is itself profoundly unnatural in evolutionary terms, has failed to gain lay credence. Some women feel that postmenopausal stimulation of the oestrogen sensitive reproductive organs is not only unnatural but likely to be harmful in the longer term. To support this view it is accepted that unopposed oestrogen stimulation of the endometrium is clearly associated with an excess of endometrial adenocarcinoma and the incidence of breast cancer rises by a small but quantifiable amount for each year of HRT therapy [ 17]. A further major difficulty in the acceptance of HRT in the postmenopause is the return of cyclic or unscheduled bleeding cited by many women as a prime reason for a refusal to embark on HRT or to discontinue it. Conventional cyclic HRT regimes usually impose a withdrawal bleed which is either monthly or quarterly. The so-called ‘bleed-free’ preparations containing oestrogen+progestogen taken daily, avoid cyclic scheduled bleeding but may cause unscheduled bleeding especially in the first 6 months of treatment. For these reasons it has been repeatedly observed that the acceptance of HRT is poor even among properly counselled women likely to benefit materially from its effects [ 18]. Hence as deliberate policy, a pharmacological research drive has now resulted in the first selective oestrogen receptor modulator (SERM) licensed for the prevention of bone loss, an effect apparently unaccompanied by the stimulation of such classical oestrogen-sensitive tissues as breast and endometrium. The title of SERM, cumbersome but accurate, describes an agent capable of effecting differential responses via the oestrogen receptor in specific tissues. The path leading to SERM development originates in 1896 when Beatson reported regression of advanced mammary carcinoma after oophorectomy in some but not all patients [ 19]. This differential response would eventually be tracked to the variability in the tumour of the oestrogen receptor (ER) which was described by Jensen & Jacobson in 1962 [ 20]. Four years earlier, however, Lerner [ 21] reported that a nonsteroidal antioestrogen MER-25 (ethamioxytriphetol) inhibited the action of oestradiol on endometrium without itself causing endometrial stimulation. Thereafter, and roughly contemporous with the discovery of the ER, the triphenytethylene clomiphene citrate was reported to be capable of inducing ovulation by blocking the negative feedback of oestrogen upon the hypothalamic-pituitary axis [ 22]. It thus promotes gonadotrophic secretion—a function which it performs therapeutically in certain anovulatory subfertile women to this day. This was followed in 1974 by the landmark paper by Jordan which showed that tamoxifen (ICI 46 474) inhibited the dimethylbenzanthracene (DMBA) induced mammary tumours in the rat [ 22]. That these agents had a more varied portfolio of action was shown by Beall et al. [ 23] using clomiphene and Jordan [ 24] using tamoxifen and raloxifene, the latter then known as keoxifen. These authors demonstrated that the two agents were capable of restraining bone loss in the oophorectomised rat model. These were remarkable observations since a pure antioestrogen would have been expected to promote rather than inhibit bone loss. Clomiphene is a racemic mixture of enclomiphene and zuclomiphene and hence tamoxifen must be accorded primacy in the SERM field. However, the discovery that tamoxifen was, in some patients, capable of endometrial stimulation leading to hyperplasia or neoplasia [ 26] precluded its development as a bone antiresorptive agent which would be bleed-free and, hopefully, breast-safe as well. This agent is a benzothiophene (Figure 2) which was first examined in the Indianapolis Laboratories of Eli Lilly as a potential inhibitor of breast tumour cell lines including MCF-7 [ 27]. Although performing well in vitro and proving inhibitory also to MCF-7 invasiveness, it was reported that raloxifene did not match the efficacy of tamoxifen in vivo. Similarly, raloxifene was shown not to stimulate endometrial tumours in nude mice [ 28] and also inhibited such tumours which had been stimulated by tamoxifen. With regard to bone, Sato et al. [ 29] again using the OVX rat model, showed that raloxifene prevented bone loss to a degree compatible with a parallel OVX group treated with oestradiol 17β. In the human, raloxifene has now been subjected to extensive phase III trials. Raloxifene [6-hydroxy-2-(4-hydroxyphenyl)-benzo[b])thien-3-yl][4-[2-(1-piperidinyl)ethoxy]pheny]methanone;keoxifene:[LY-139481]. The ultimate site of raloxifene action is the oestrogen receptor. The receptor (ER) is a member of the steroid-thyroid receptor superfamily and is most concisely described as a nuclear ligand-inducible transcription factor. The family includes the receptor for tri-iodothyronine, cortisol, progesterone, testosterone and 9-cis retinoic acid and exhibits a modular construction with domains coded A to F as shown in Figure 3. Schematic representation of the oestrogen receptor with six domains (A–F). The arrival of ligand leads to the discard of heat-shock proteins which cloak the inactive receptor which then undergoes conformational change. The ER dimerizes and its new configuration permits access to the AF-2 domain of coactivators which shepherd the activated receptor to the promoter sequences of those genes which are oestrogen responsive. The AF-1 domain is believed to be constitutive and to operate independently of ligand. The process then proceeds to the activation of RNA transcriptase and message is produced. The above is a necessary simplification but elucidates the essential steps which may be disabled by raloxifene. As Brzozowski et al. have graphically demonstrated using electron crystallography (Figure 4). Raloxifene enters the binding pocket of the ER but the activation process is inhibited due to the very molecular construction of this ligand [ 30]. Raloxifene has an alkylaminoethoxy side chain which physically protrudes from the binding pocket. It has been shown by Levenson & Jordan [ 31] that a nitrogen atom in the side chain binds to AA residue 351 (aspartate) in the ligand-binding domain of the ER. This binding splints the key helix 12 which is thus unable to undergo its normal wild-type rotation to seal the pocket and configure the AF-2 domain for the adherence of coactivators. The process thus cannot proceed to transcription and raloxifene would hence be more precisely designated as an AF-2 inhibitor. Indeed, it is clear that mutation of aspartate 351 to a tyrosine residue will convert raloxifene from an oestrogen antgonist to an agonist. Thus, at those tissues such as breast and endometrium where intact AF-2 activity is required for oestrogen activity, raloxifene acts as an antagonist. Conversely, the other activation site, AF-1, proceeds with transcription in the presence of raloxifene. This is believed to be the basis for the drug’s action on bone and in lipid physiology. It may thus be speculated that a raloxifene inhibitory action should be anticipated at any other sites where AF-2 integrity is required. In addition to the above specific actions of raloxifene a more general selectivity may apply, in that the drug may be preferentially taken up by certain tissues and not by others. Further studies are required to finally delineate the means whereby the action of raloxifene its of the ligand binding domain of the oestrogen receptor with oestradiol and raloxifene shown in Brzozowski et with As the central of bone physiology after menopause is an increase in BMU with the of bone formation and resorption in of the Thus, data were by & [ who studies using the in postmenopausal These authors found that both raloxifene and a parallel group oestrogen oestrogen exhibited a significant to which was at and at The cause of the was a reduction in which was itself tracked to a reduction in bone raloxifene oestrogen affected and raloxifene, did not reduce bone formation at the The authors that the activity of raloxifene was to bone by inhibition of bone of the early of raloxifene reported on the use of up to However, since the standard due has been set at this report will on studies utilizing this The randomized controlled published to was reported by and [ Some women were who were between and years postmenopausal and whose bone density was above the for which is set at standard below that of the normal In other words, were Raloxifene evidence of a in bone with of bone formation and resorption being from by This of was by significant in means in of at at total at total these were significant when with treated controls data were reported in this and [ reported a randomized controlled in osteoporotic postmenopausal women who at at of bone formation and as did a number of bone resorption was found in or in There were significant in at total and at the These authors did not significant over controls in at or total and at there was no reduction in using either a or reduction in the was well with no over of breast or These data a activity for raloxifene, though of a than that expected with standard antiresorptive oestrogen The lack of effect is even more when to the effects observed with oestrogen therapy [ Interventional study data have been published [ which that the of new in raloxifene treated osteoporotic women with controls was With to the cardiovascular raloxifene resulted at months in a significant of in total and a in LDL-cholesterol [ A reduction in plasma failed to and were clinical data have yet been The effect of raloxifene on the of the human endometrium was examined in a study of postmenopausal women in which the SERM, in of or for was to standard oestrogen and [ A system was to delineate oestrogenic effects on endometrial cell and Similarly, density and were The raloxifene treated up to the clinical failed to any oestrogenic effect on these sites of oestrogen This was a study but in the clinical trials the lack of vaginal bleeding at years [ to suggest that the endometrium is to raloxifene and that patients may be that their of bleeding is not above A study in premenopausal women found that raloxifene for from of the cycle did not ovulation and effect on cycle or on endometrial clinical utility in this is and the licensed the use of raloxifene to the prevention of osteoporotic in postmenopausal trials of raloxifene, however, will to data on in on endometrial as by and on raloxifene who postmenopausal bleeding should be to raloxifene is though not to the such as tamoxifen and since its observed action was an inhibition of in MCF-7 breast cancer there has been clinical in the incidence of breast cancer in treated patients on raloxifene did not report any excess of breast or when with controls [ the of in reported from a of osteoporotic postmenopausal women assigned in a of to [ a months of breast had in raloxifene treated women and in those These data have now been published [ The in the raloxifene group was the Jordan et al. data from a range of some years of In this group of studies there had been of breast on raloxifene, on The of breast cancer in raloxifene treated patients was There was a for the new to decline after 12 months on raloxifene due to the of in breast cancer was present at data that breast cancer is to with no effect on the is required in the above results since the number of is low but the of the expected number of tumours among the and the significant reduction only in in treated for It should be that an osteoporotic no excess of breast cancer and a study is now in the US which will the effects of raloxifene and tamoxifen in a and The drug was well and in there was no excess of in the raloxifene group of vaginal bleeding when with treated It should be that breast and are the principal means for of HRT therapy [ 18]. with raloxifene have been and and include hot flushes, and In this it is of that raloxifene not the symptoms of the of such symptoms will to the use of HRT The most report to is an excess of including of which the in treated patients is of Eli Lilly & as This is an to that reported by [ and by [ with The absolute of a with raloxifene is of the order of The favourable profile led to a at years in raloxifene treated patients in the clinical trials. As with the and the of in and function of the oestrogen receptor is a not a to The SERM are at present are not HRT replacement Until they are capable of menopausal symptoms and of of the action of this oestrogen will to play a major role in postmenopausal With the of the of the ER and the realization that cell the may respond to specific it is to a range of These will provide oestrogenic support for those tissues where it is an individual the ability to the receptors of breast and where postmenopausal has been for by and