Partial Estrogen Receptor Agonist Research Articles

Abstract P6-04-15: Oral selective estrogen receptor degrader AZD9496 demonstrated preclinical model specific differences to fulvestrant in estrogen receptor degradation, agonism and anti-tumour effects

Abstract Anti-hormonal therapy has been the mainstay for the treatment of estrogen receptor (ER) positive breast cancer for over 40 years. The selective ER degrader (SERD) fulvestrant (Faslodex) has been shown to provide clinical benefit over aromatase inhibitors and selective ER modulators. However, delivery of its maximum possible efficacy may be limited by the intra muscular route of administration. To overcome this, AstraZeneca developed the orally bioavailable SERD AZD9496. Here we report further preclinical characterisation of AZD9496 with regards to its ability to induce ERα degradation, antagonise and agonise ER in vitro and the impact on tumour xenografts. AZD9496 caused equivalent ERα degradation to fulvestrant in the MCF7 ER+ cell line. Interestingly, in other ER+ cell lines (e.g. CAMA1 and T47D), the maximal level of ERα degradation induced by AZD9496 was 54% of that induced by fulvestrant. RNAseq analysis of cell lines treated with AZD9496 or fulvestrant in the presence of estradiol did not reveal any differences in ER antagonism. Furthermore, no ER agonism was detected in ER+ breast cancer cell lines treated with AZD9496 in the absence of estradiol. However, unlike fulvestrant, AZD9496 was able to induce PR protein expression in the Ishikawa ER+ endometrial cancer cell line, demonstrating a potential to agonise ER. Also, in proliferation assays, while there was little difference in MCF7 cells between the maximal antiproliferative effect of fulvestrant and AZD9496, in CAMA1 and T47D cells the maximal antiproliferative effect of AZD9496 was 75% and 82% respectively of that caused by fulvestrant. In vivo, AZD9496 increased the uterine weight and induced PR expression in the uterus of juvenile rats, consistent with AZD9496 being a partial ER agonist albeit less than tamoxifen. AZD9496, at a maximally efficacious dose, did not induce the same degree of ER degradation or anti-tumour effect as a supraclinical dose of fulvestrant in CTC174, a patient derived xenograft that harbours a D538G ESR1 mutation and grows in the absence of supplemental estradiol. Taken together, these data suggest that, while AZD9496 is equivalent to fulvestrant in terms of ERα degradation and anti-proliferative/anti-tumour effects in some preclinical models, this is not the case in all models. However, it is not clear which, if any, model accurately reflects the clinical setting and how AZD9496 would compare to fulvestrant at exposures achievable in the clinic. Thus, testing AZD9496 vs fulvestrant in ER+ breast cancer patients is crucial to determine potential clinical benefit. Citation Format: Mandy Lawson, Natalie Cureton, Oona Delpuech, Pei Zhang, Azadeh Cheraghchi-Bashi-Astaneh, Sladjana Gagrica, Dawn Trueman, Gareth Maglennon, Daniel Sutton, Bairu Zhang, Jonathan Cairns, Teresa Klinowska, Christopher J Morrow. Oral selective estrogen receptor degrader AZD9496 demonstrated preclinical model specific differences to fulvestrant in estrogen receptor degradation, agonism and anti-tumour effects [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-04-15.

Abstract NG05: Not all “SERDs” are equal: Context-independent ER degradation and full ER antagonism define the next generation of ER therapeutics

Abstract NG05: Not all “SERDs” are equal: Context-independent ER degradation and full ER antagonism define the next generation of ER therapeutics

Hyper‐reactivity of cerebral arteries from ovariectomized rats: therapeutic benefit of tamoxifen

1. An increased incidence of systemic hypertension has been documented in postmenopausal women and identified as an independent risk factor in the development of cerebrovascular stroke. The present study examined whether cerebrovascular reactivity was increased in the hypertensive ovariectomized rat, and explored the potential therapeutic benefit of the partial estrogen receptor agonist tamoxifen. 2. Female Sprague-Dawley rats were subjected to bilateral ovariectomy (OVX, n=16) or a sham operation (n=8). At 6-week postsurgery, rats were anesthetized to assess ventricular contractility and blood pressure. In a second series of experiments, OVX rats (n=8) were given tamoxifen starting 3 weeks postsurgery, and continued for 3 weeks. At the end of each protocol, the middle cerebral artery was harvested and rings were mounted in wire-myographs to measure isometric tension. 3. Systolic arterial pressure (SAP) was significantly increased (P<0.05) in the OVX rat (174+/-8 mmHg), as compared to sham (135+/-6 mmHg). The resting tension of isolated cerebral arteries from OVX rats (186+/-15 mg) was significantly elevated (P<0.05), as compared to sham (129+/-9 mg). Phenylephrine treatment did not elicit a constriction of cerebral arteries isolated from sham rats, whereas a potent response (P<0.05) was observed in OVX rats. Nitric oxide (NO) synthase inhibition with L-NNA led to a limited contraction in sham rats (8+/-3% of Emax), whereas a significant (P<0.05) increase was observed in OVX rats (34+/-12% of Emax). Lastly, vascular sensitivity (pD2) to sodium nitroprusside was significantly increased (P<0.05) in OVX rats, as compared to sham. 4. Tamoxifen therapy normalized the resting tension of isolated cerebral arteries from OVX rats, abrogated phenylephrine-mediated contraction, and modestly reduced SAP. By contrast, tamoxifen treatment of OVX rats did not attenuate L-NNA-mediated contractile response of cerebral arteries. 5. These data demonstrate that the cerebral artery isolated from the OVX rat was associated with an exaggerated vasoconstrictor response to phenylephrine, and altered NO-dependent vascular reactivity. The administration of tamoxifen to OVX rats normalized cerebral artery reactivity to phenylephrine. These findings provide the impetus to examine the potential therapeutic benefit of the partial estrogen receptor agonist tamoxifen to reduce the incidence of cerebrovascular stroke in postmenopausal women.

Role of anastrozole in adjuvant therapy for postmenopausal patients

For the past 25 years tamoxifen has been the mainstay for adjuvant treatment of postmenopausal patients with hormone-sensitive breast cancer. However, tamoxifen has some safety and tolerability issues, and its partial estrogen-receptor agonist activity may have efficacy implications. Highly specific aromatase inhibitors, of which anastrozole was the first, were introduced in the 1990s and have emerged as a potentially better tolerated and more effective class of agents targeting hormonally responsive breast cancer. This article provides a review of the clinical pharmacology of anastrozole (1 mg once daily) and reviews the first results of the ongoing Arimidex, Tamoxifen, Alone or in Combination early breast cancer trial, initiated in 1996. This randomized, double-blind multicenter trial compared tamoxifen (20 mg once daily) with anastrozole (1 mg once daily) alone and the combination of anastrozole plus tamoxifen, as adjuvant endocrine treatment for postmenopausal patients with operable, invasive, early breast cancer. The results of the Arimidex, Tamoxifen, Alone or in Combination trial show anastrozole to be an effective and well tolerated endocrine option for early breast cancer and provide evidence for its potential role in chemoprevention.

A Surveillance Program for Serious Adverse Events During Phase III Drug Development Studies

A new, simple approach to surveillance of serious adverse events during Phase III is suggested. The large-scale Phase III studies are typically double-blind comparisons of the drug with placebo, or a control, performed in order to assess the risk of frequent adverse events. The risk of rare and severe adverse events cannot be assessed with sufficient precision, but the events must be monitored in order to stop the trials if there is a major safety problem. This paper suggests a cumulative sum (CUSUM) approach, where the events in the treatment group are cumulated, adjusting for the expected numbers based upon the total number of serious adverse events. Thus, if there are many events in the treatment group compared to the control group, there will be an alarm. The procedure requires unblinding the treatment for serious adverse events, but no other information from the ongoing studies. Exact probability properties of this sequential Bernoulli procedure can be evaluated by means of Markov chain methods. Optimizing the surveillance program with respect to the mean time to alarm (the standard in CUSUM applications) leads to a design that depends upon the alternative considered, whereas the optimum solution based on the probability of alarm within the expected course of the study is independent of the alternative. The procedure was applied to adverse events for NNC 46-0020, a partial estrogen receptor agonist. The finding of too many adverse events led to closure of the study.

Resveratrol, a natural product derived from grape, exhibits antiestrogenic activity and inhibits the growth of human breast cancer cells.

Resveratrol is a natural phytoalexin compound found in grapes and other food products. In this study, the effect of resveratrol on the growth of human breast cancer cells was examined. Results show that resveratrol inhibits the growth of estrogen receptor(ER)-positive MCF-7 cells in a dose-dependent fashion. Detailed studies with MCF-7 cells demonstrate that resveratrol antagonized the growth-promoting effect of 17-beta-estradiol (E2) in a dose-dependent fashion at both the cellular (cell growth) and the molecular (gene activation) levels. At 5 x 10(-6) M, resveratrol abolished the growth-stimulatory effect mediated by concentrations of E2 up to 10(-9) M. The antiestrogenic effect of resveratrol could be observed at a concentration of 10(-6) M and above. The antiestrogenic effect of resveratrol was also demonstrated at the molecular level. Resveratrol in a dose-dependent fashion antagonized the stimulation by E2 of progesterone receptor gene expression in MCF-7 cells. Moreover, expression of transforming growth factor-alpha and insulin-like growth factor I receptor mRNA was inhibited while the expression of transforming growth factor beta2 mRNA was significantly elevated in MCF-7 cells cultivated in the presence of resveratrol (10(-5) M). In summary, our results show that resveratrol, a partial ER agonist itself, acts as an ER antagonist in the presence of estrogen leading to inhibition of human breast cancer cells.

Maternal genistein exposure mimics the effects of estrogen on mammary gland development in female mouse offspring.

Human and animal data indicate that a high maternal estrogen exposure during pregnancy increases breast cancer risk among daughters. This may reflect an increase in the epithelial structures that are the sites for malignant transformation, i.e., terminal end buds (TEBs), and a reduction in epithelial differentiation in the mammary gland. Some phytoestrogens, such as genistein which is a major component in soy-based foods, and zearalenone, a mycotoxin found in agricultural products, have estrogenic effects on the reproductive system, breast and brain. The present study examined whether in utero exposure to genistein or zearalenone influences mammary gland development. Pregnant mice were injected daily with i) 20 ng estradiol (E2); ii) 20 microg genistein; iii) 2 microg zearalenone; iv) 2 microg tamoxifen (TAM), a partial estrogen receptor agonist; or v) oil-vehicle between days 15 and 20 of gestation. E2, genistein, zearalenone, and tamoxifen all increased the density of TEBs in the mammary glands. Genistein reduced, and zearalenone increased, epithelial differentiation. Zearalenone also increased epithelial density, when compared with the vehicle-controls. None of the treatments had permanent effects on circulating E2 levels. Maternal exposure to E2 accelerated body weight gain, physical maturation (eyelid opening), and puberty onset (vaginal opening) in the female offspring. Genistein and tamoxifen had similar effects on puberty onset than E2. Zearalenone caused persistent cornification of the estrus smears. These findings indicate that maternal exposure to physiological doses of genistein mimics the effects of E2 on the mammary gland and reproductive systems in the offspring. Thus, our results suggest that genistein acts as an estrogen in utero, and may increase the incidence of mammary tumors if given through a pregnant mother. The estrogenic effects of zearalenone on the mammary gland, in contrast, are probably counteracted by the permanent changes in estrus cycling.

Estrogen- and antiestrogen-responsiveness of HEC1A endometrial adenocarcinoma cells in culture

HEC1A endometrial cancer cells express the wild-type form of the estrogen receptor (ER) and 17 β-estradiol (E2) induces proliferation of these cells. In contrast, tamoxifen only causes a minimal increase (<20%) in cell proliferation. In HEC1A cells transiently transfected with the C3-Luc plasmid derived from the complement C3 gene, both E2 and tamoxifen exhibited ER agonist activity and tamoxifen was also a partial antagonist for this response. The relative ER agonist/antagonist activities of E2, tamoxifen and ICI 182,780 were also investigated in HEC1A1 cells transiently transfected with two E2-responsive plasmids, pCATHD-CAT and pCKB-CAT which contain 5′-promoter inserts from the cathepsin D and creatine kinase B genes, respectively. The results showed that E2 and tamoxifen induced reporter gene activity in cells transiently transfected with both constructs. ICI 182,780 exhibited partial ER agonist activity only in cells transiently transfected with pCKB-CAT and antagonized E2-induced reporter gene activity using both the CKB- and CATHD-derived constructs. These results demonstrate that HEC1A endometrial cancer cells are E2-responsive and represent a useful cell culture model for understanding hormone/antihormone-induced endometrial cell responses.

A partial estrogen receptor agonist with strong antiatherogenic properties without noticeable effect on reproductive tissue in cholesterol-fed female and male rabbits.

Estrogen replacement therapy retards the development of cardiovascular disease and osteoporosis in postmenopausal women. However, long-term unopposed use increases the risk of cancer in endometrium and possibly in breast. The racemic compound ormeloxifene, widely used in India as an antifertility agent, is a partial estrogen receptor agonist with antiosteoporotic properties. The present study was undertaken to investigate the effect of the L-enantiomer (levormeloxifene) and the d-enantiomer (d-ormeloxifene) on the development of atherosclerosis. In a short-term experiment (6 weeks), 4 x 10 ovariectomized female rabbits were fed a 0.25% cholesterol-enriched diet and the effect on plasma cholesterol levels was studied. In a long-term experiment (13 weeks), 4 x 15 ovariectomized female and 4 x 15 shamoperated male rabbits were maintained at a similar plasma cholesterol level of 25 mmol/L and the effect on undamaged and balloon-injured arterial wall was studied. In both experiments, the rabbits were treated with levormeloxifene, d-ormeloxifene, 17 beta-estradiol, or placebo, respectively. In the short-term experiment, levormeloxifene, in contrast to d-ormeloxifene, significantly reduced plasma cholesterol by 30% compared with the placebo group. In the long-term experiment, levormeloxifene, in contrast to d-ormeloxifene, significantly reduced atherosclerosis by 50% in the undamaged arterial wall of both female and male rabbits. Because these rabbits were cholesterol-clamped, the antiatherogenic effect was not mediated via plasma cholesterol lowering. Like estrogen, levormeloxifene did not inhibit atherosclerosis in the endothelium-denuded site of aorta. The antiatherogenic effects of levormeloxifene were thus similar to those of estrogen, but produced in the absence of any noticeable estrogenic effect on uterine or testicular tissue.

Effects of estrus cycle, ovariectomy, and treatment with estrogen, tamoxifen, and progesterone on apolipoprotein(a) gene expression in transgenic mice.

Plasma levels of lipoprotein(a) (Lp(a)), are regulated by the synthetic rate of apolipoprotein(a) (apo(a)), a major protein component of this atherogenic lipoprotein. Exogenously administered sex steroid hormones are potent regulators of plasma Lp(a) concentrations. We utilized a recently developed apo(a) yeast artificial chromosome (YAC) transgenic mouse model to study the effects of ovariectomy, estrus cycle, and exogenous administration of ethinyl-estradiol, the partial estrogen receptor agonist, tamoxifen, and progesterone on circulating apo(a) plasma levels. Analysis of liver RNA revealed that estrogen and tamoxifen exerts their plasma apo(a) lowering effect at the level of apo(a) mRNA. This action of estrogen and tamoxifen may contribute to their antiatherosclerotic and cardiovascular protective effect.

Significant reduction of the antiatherogenic effect of estrogen by long-term inhibition of nitric oxide synthesis in cholesterol-clamped rabbits.

The purpose of this study was to investigate whether endothelium-derived nitric oxide (NO) is involved in the plasma lipid-independent antiatherogenic effect of estrogen and levormeloxifene, a partial estrogen receptor agonist. 85 rabbits were ovariectomized and balloon-injured in the middle thoracic aorta. The rabbits were fed a cholesterol-enriched diet supplemented with 17beta-estradiol, levormeloxifene, or placebo, either alone, or together with 160 microg/ml NG-nitro- -arginine methyl ester (-NAME), an NO synthase inhibitor, in their drinking water for 12 wk. Plasma cholesterol was maintained at 25-30 mmol/liter by individualized cholesterol feeding. In the undamaged aorta, the extent of atherosclerosis in the estrogen group was only one-third that in the placebo group. Simultaneous administration of -NAME, however, significantly reduced the antiatherogenic effect of estrogen (P < 0.01). There was no significant difference between the placebo group given -NAME and the group treated with placebo alone. At the previously endothelium-denuded site, estrogen had no effect on atherosclerosis development, whereas -NAME combined with estrogen significantly increased atherogenesis (P < 0.05). The effects of levormeloxifene were almost similar to those of estrogen. Active vascular concentrations of -NAME were demonstrated in an additional study, in which maximal aortic/coronary endothelium-dependent relaxation was significantly inhibited in rabbits given -NAME. Thus, in this study a considerable part of the plasma lipid-independent antiatherogenic effect of estrogen was mediated through its effect on endothelial NO in cholesterol-fed rabbits. The results for levormeloxifene suggest a common mechanism of action for estrogen and partial estrogen receptor agonists on atherogenesis.

Effect of antagonists on DNA binding properties of the human estrogen receptor in vitro and in vivo.

Functional analyses, performed with the estrogen receptor (ER) isolated from different sources or produced with various expression systems, led to contradictory results concerning the role of estrogen (E2) and antiestrogens in ER DNA binding. Here we report the DNA-binding properties of the human ER and show that the wild type ER (HEG0) binds in vitro to an estrogen response element (ERE) as a dimer, irrespective of the presence or absence of estrogen. We also show that the two antihormones, 4-hydroxytamoxifen (OHT, a partial ER agonist) and ICI 164,384 (a pure antagonist) do not impair HEG0 dimerization and DNA binding in vitro. Exposure of HEG0 to elevated temperature (37 C) in vitro results in a much faster reduction of its binding capacity to an ERE in the absence of ligand or in the presence of ICI 164,384 than in the presence of either E2 or OHT. The Gly to Val mutation at amino acid 400 present in the human ER that we initially cloned (HE0), is responsible for an even faster heat inactivation of unliganded receptor compared with HEG0 and largely accounts for the previously observed in vitro ligand-dependent DNA binding of ER. We also show that, as previously observed for OHT, ICI 164,384 does not prevent ER binding to an ERE in vivo, even though ICI 164,384 acts as a pure antagonist for transcriptional activation by ER. We discuss these results in the context of a ligand-dependent interaction between the C-terminal region E, which contains the ligand-binding domain, and the N-terminal A/B region, which contains the activation function AF-1.