Long-term inhibitory effect of the orally active and pure antiestrogen EM-800 on the growth of human breast cancer xenografts in nude mice

The antiproliferative effect of the new antiestrogen EM-800 has been studied during 40 weeks of treatment on human breast carcinoma ZR-75-1 xenografts in ovariectomized nude mice supplemented with estrone (0.5 microg, s.c. daily). At the daily 50 microg (approximately 2.5 mg/kg) oral dose, EM-800 caused a complete inhibition of the 680% stimulatory effect of estrone on the growth of the ZR-75-1 human breast cancer xenografts. Complete response, defined as the complete disappearance of the tumors, was observed in 41% of tumors following treatment with the 50 microg dose of the antiestrogen, while a value of 26% was found in ovariectomized animals. The proportion of tumors showing progression at the end of 40 weeks of treatment decreased from 94% in the estrone-supplemented animals to 62%, 61% and 19% in the animals receiving the 5 microg, 20 microg and 50 microg daily doses of the antiestrogen, respectively. None of the tumors that showed a complete or a partial response progressed at later time intervals. The 50 microg daily dose of EM-800 nearly completely (93%) or completely (28% below the value in ovariectomized animals) reversed the stimulatory effect of estrone on uterine and vaginal weight, respectively. The disappearance of 41% of tumors in the group of animals that received the 50 microg daily dose of EM-800 indicates that the antiestrogen induces cell death or apoptosis in ZR-75-1 human breast cancer cells and that its action is cytotoxic and not only cytostatic.     

A Novel Pure SERM Achieves Complete Regression of the Majority of Human Breast Cancer Tumors in Nude Mice

Background. The objective was to determine if EM-652, a novel selective estrogen receptor modulator (SERM) having highly potent and pure antiestrogenic activity in the mammary gland could cause complete regression of the majority of human breast cancer xenografts in nude mice. Methods. Human breast cancer ZR-75-1 xenografts were used as model in nude mice. Results. EM-652 not only prevented estrogen-induced tumor growth but it reduced tumor size to 20% of the pretreatment value. Complete disappearance of the tumors was observed in 65% (106/163) of tumors. No tumor progressed. Most importantly, 93% of the tumors which had become undetectable under EM-652 treatment did not reappear when exposed to estrogen challenge for 12 weeks, thus achieving an overall 61% cure rate. Conclusions. The present data demonstrate that EM-652 is strongly cytotoxic or tumorocidal and not only cytostatic or tumorostatic in estrogen-sensitive breast cancer, thus changing the paradigm of a tumorostatic role of estrogen blockade established with tamoxifen. These findings support the use of such a compound for more efficient breast cancer prevention and therapy.     

The interaction of raloxifene and the active metabolite of the antiestrogen EM-800 (SC 5705) with the human estrogen receptor.

A naturally occurring mutation at amino acid 351 (D351Y) in the human estrogen receptor (ER) can change the pharmacology of antiestrogens. Raloxifene is converted from an antiestrogen to an estrogen, whereas the biological properties of the steroidal pure antiestrogen ICI 182,780 are not affected by the D351Y ER (Levenson, A. S., and Jordan, V. C. Cancer Res., 58: 1872-1875, 1998). We propose an assay system that can be used to classify antiestrogens by determining their ability to up-regulate transforming growth factor alpha (TGF-alpha) mRNA in MDA-MB-231 cells stably transfected with either wild-type or D351Y ER. The novel compound EM-800 and its active metabolite, EM-652, have been reported to be p.o. active nonsteroidal pure antiestrogens. Using the D351Y cell line, EM-652 is able to up-regulate TGF-alpha mRNA in a dose-dependent manner and to a similar extent as estradiol, whereas in the wild-type cell line, it acts as an antiestrogen. In addition, the pure antiestrogen ICI 182,780 is capable of inhibiting EM-652-induced TGF-alpha mRNA expression at the D351Y ER. In MCF-7 cells expressing wild-type ER, it has previously been shown that ICI 182,780 decreases ER only at the protein level. EM-652 treatment does not decrease ER protein levels to a similar extent as ICI 182,780 treatment, and, in addition, EM-652 has no effect on ER mRNA levels. In proliferation assays, EM-652 is as effective as raloxifene in inhibiting cell growth. From these studies, we conclude that the reason the pharmacology of EM-652 is similar to that of raloxifene is because they both fit the ER in the same manner, and their biology depends on an interaction of the antiestrogenic side chain with amino acid 351.     

Effect of estrogen and antiestrogens on cell proliferation and synthesis of secreted proteins in the human breast cancer cell line MCF-7 and a tamoxifen resistant variant subline, AL-1.

The human breast cancer cell line MCF-7 contains estrogen receptors and responds to estrogens with an increase in growth rate and to antiestrogens with a decrease in growth rate. Estrogen stimulation of cell proliferation is concomitant with an increase in the synthesis and secretion of three proteins with mol. wt 52 kDa, 61 kDa and 66 kDa and a decrease in the synthesis and secretion of a 42 kDa protein. The antiestrogen ICI 164,384 has a complete estrogen antagonistic effect on the synthesis of these secreted proteins, whereas the antiestrogen tamoxifen has an agonistic effect on the synthesis and secretion of the 52 kDa protein. We believe that the above mentioned estrogen regulated secreted proteins are either directly or indirectly involved in control of cell proliferation, and the less pronounced inhibitory effect of tamoxifen on cell proliferation compared to ICI 164,384 may be due to agonistic effects of tamoxifen. A tamoxifen resistant variant of the MCF-7 cell line, the AL-1 subline, can be growth inhibited by ICI 164,384, although a higher concentration is needed to inhibit the AL-1 cells compared to the parent MCF-7 cells. Tamoxifen has no effect on secreted proteins from the AL-1 cells, whereas ICI 164,384 has a complete estrogen antagonistic effect on secreted proteins, indicating that the mechanisms by which estrogens and antiestrogens influence cell proliferation may be via up and down regulation of secreted proteins with growth regulatory functions.     

Effects of the antiestrogen EM-800 (SCH 57050) and cyclophosphamide alone and in combination on growth of human ZR-75-1 breast cancer xenografts in nude mice.

Human breast cancer proliferates as heterogeneous cell populations that exhibit different sensitivities to therapeutic agents. A logical approach to control these different cancer cell populations is the use of combined treatment with agents that block cell proliferation or induce apoptosis via different mechanisms. We therefore investigated the effect of treatment with the novel pure antiestrogen EM-800, alone or in combination with chemotherapy, on the growth of ZR-75-1 human breast tumors in nude mice, a well-recognized model of human breast cancer. Mice bearing estrone-releasing silastic implants as estrogenic stimulus received EM-800 or cyclophosphamide alone or in combination for 227 days. Cyclophosphamide (256 mg/kg/2 weeks) was administered by i.p. injection in 64 mg/kg fractions over 4 consecutive days with repetition of the cycle every 14 days. EM-800 was administered p.o. once daily at the maximally effective dose of 300 microg/mouse. After 227 days of treatment, average tumor size in mice receiving estrone alone was 192% higher than pretreatment. The average tumor size of mice treated with chemotherapy was reduced by 47%, whereas on the other hand, EM-800 caused a 81% decrease of the value of the same parameter. The combined treatment (EM-800 + cyclophosphamide), on the other hand, resulted in a 95% decrease in tumor size compared with control estrogen alone. In fact, EM-800 alone decreased tumor size to 55% of the value at the start of treatment, whereas the addition of cyclophosphamide to the antiestrogen further decreased tumor size to as low as 15% of the pretreatment value. The combination of EM-800 and cyclophosphamide resulted in 95% of complete or partial responses compared with 61 and 27% with EM-800 and cyclophosphamide alone, respectively. In fact, in the combination therapy group, only one tumor remained stable, while 17 regressed >50% and four disappeared. It is noteworthy that no tumor progressed with EM-800 alone or in combination with cyclophosphamide. The present data show, for the first time, that the addition of cyclophosphamide to a pure antiestrogen used at a maximal dose causes a more potent inhibition of human breast tumor growth, thus suggesting that combined treatment using a maximal dose of a pure antiestrogen and a chemotherapeutic agent(s), two classes of compounds having different mechanisms of action, could further improve breast cancer therapy above the results achieved with a potent and pure antiestrogen alone in estrogen-sensitive breast cancer.     

The effects of aromatase inhibitors and antiestrogens in the nude mouse model

The effects of antiestrogens, tamoxifen and ICI 182,780, and aromatase inhibitors, arimidex (anastrozole ZD1033) and letrozole (CGS 20,267), on the growth of tumors were studied in nude mice. In this model, estrogen dependent MCF-7 human breast cancer cells stably transfected with the aromatase gene were inoculated in four sites per mouse. Sufficient estrogen is produced from aromatization of androstenedione supplement (0.1 mg/mouse/day) by the cells to stimulate their proliferation, tumor formation, and maintain the uterus similar to that of the intact mouse. Once the tumors reached a measurable size, the mice were injected with antiestrogen or inhibitor for 35–56 days. Tumor volumes were measured weekly. At autopsy, the tumors were removed, cleaned, and weighed. Statistical data was determined from tumor weights. Both antiestrogens were effective in reducing tumor growth in these mice. Tamoxifen appears to be more effective than ICI 182,780, although the former stimulated the uterine weight whereas the pure antiestrogen did not. However, both aromatase inhibitors were more effective than the antiestrogens. Tumor regression was observed with letrozole. Thus, after-treatment tumor weights were less than those of a group of mice at the start of treatment. The aromatase inhibitors also reduced the weight of the uterus, suggesting that these compounds, as well as the pure antiestrogen, may not cause endometrial proliferation, unlike tamoxifen. These aromatase inhibitors may not only benefit patients who have relapsed from tamoxifen, but may be more effective in patients as first line agents for suppressing the effects of estrogen.     

Antiestrogenic properties of keoxifene,trans-4-hydroxytamoxifen,and ICI 164384, a new steroidal antiestrogen,in ZR-75-1 human breast cancer cells

The agonistic/antagonistic properties of two non-steroidal antiestrogens, namelytrans-4-monohydroxy-tamoxifen (OH-TAM) and keoxifene (LY156758), and the new steroidal antiestrogen ICI164384, a 7-alkylamide derivative of estradiol (E₂), were assessed by measuring their effect on the proliferation of ZR·75-1 cells, an estrogen-responsive human breast cancer cell line. While subnanomolar concentrations of both OH-TAM and LY156758 had significant estrogenic stimulatory activity on cell growth in the absence of estrogens and higher concentrations were inhibitory, ICI164384 behaved exclusively as a growth inhibitor and more potently so than the two other compounds. The three antiestrogens had similar potency to inhibit the mitogenic effect of E₂ and at 300 nM, all antiproliferative effects were completely reversible by the estrogen. ICI164384 was a weaker competitor of³H-labeled E₂ or R2858 (moxestrol) uptake in intact ZR-75-1 cells in a 1-hour assay, partly because of a slower intracellular access to estrogen specific binding sites. Moreover, ICI164384 interacted in a rapidly (~ 6 h) reversible manner with estrogen-specific binding sites, while the non-steroidal antiestrogens induced a longer-acting (> 24 h) down-regulation of specific [³H]R2858 uptake. The present data indicate that, among the antiestrogens studied, ICI164384 is the only compound acting as a pure antiestrogen in ZR-75-1 breast cancer cells, while LY156758 and OH-TAM behave as antiestrogens endowed with partial agonistic activity in this system.     

Effect of combined treatment with the pure antiestrogen EM-800 and radiotherapy on the growth of human ZR-75-1 breast cancer xenografts in nude mice.

Human breast tumors are usually composed of heterogeneous cell populations that exhibit different sensitivities to therapeutic agents. We therefore investigated the effect of treatment with various regimens of the novel pure antiestrogen EM-800, alone or in combination with external beam radiation therapy, on the growth of human ZR-75-1 xenografts in athymic mice. The animals received a maximal dose of EM-800 (300 microg, p.o.) and/or radiotherapy at the dose of 10 Gy. 2.5 Gy fractions were administered over a 9-day period in four sessions of 13.7 min each (250-kilovolt Siemens with 2-mm aluminum filtration at 90 cm from the source origin). EM-800 was administered p.o. once daily, whereas radiotherapy was repeated every 35 days. Tumor size was expressed as a percentage of the initial tumor size, which was assigned a value of 100%. Average tumor size increased by 514% in ovariectomized mice supplemented with estrone alone for 259 days compared with the pretreatment value. Treatment with radiotherapy or EM-800 alone resulted in 11 and 73% decreases in mean tumor size, respectively, whereas combined treatment given simultaneously at the beginning caused a dramatic 98% decrease in tumor size. The start of radiotherapy on day 35 in EM-800-treated mice, or conversely, the start of EM-800 in irradiated mice at the 35-day time interval, resulted in somewhat lower, 88% and 95%, decreases in tumor size, respectively. In animals receiving EM-800 alone, 40% of tumors disappeared, thus indicating a cytotoxic effect caused by the estrogen blockade achieved with the pure antiestrogen. Eighty-six % of the original tumors disappeared under continuous combined treatment. Most importantly, no tumor reappeared under estrogenic stimulation after stopping treatment, thus indicating cure of 86% of the tumors in the group of animals who received the combination therapy. The present data indicate that combined treatment with EM-800 and radiotherapy yields a faster response, a greater decrease in tumor size, and a higher percentage of complete responses or tumor disappearance (cure) than either treatment used alone. The present data also suggest that maximal benefits are achieved when the pure antiestrogen is administered continuously, starting at the same time as radiation therapy and continued without interruption as adjuvant therapy. The present data also clearly show that efficient blockade of estrogens with a potent and pure antiestrogen is not only cytostatic but is cytotoxic and can lead to the disappearance of an important proportion of tumors or cure.     

Characterization of the effects of the novel non-steroidal antiestrogen EM-800 on basal and estrogen-induced proliferation of T-47D,ZR-75-1 and MCF-7 human breast cancer cells in vitro

Since estrogens play a predominant role in the development and growth of human breast cancer, antiestrogens represent a logical approach to the treatment of this disease. The present study compares the effects of the novel non-steroidal anti-estrogen EM-800 and related compounds with those of a series of anti-estrogens on basal and 17β-estradiol (E₂)-induced cell proliferation in human breast cancer cell lines. In the absence of added E₂, EM-800 and related compounds failed to change basal cell proliferation, thus showing the absence of intrinsic estrogenic activity in the ER-positive T-47D, ZR-75-1 and MCF-7 cell lines. The stimulation of T-47D cell proliferation induced by 0.1 nM E₂ was competitively blocked by a simultaneous incubation with EM-652, EM-800, OH-tamoxifen, OH-toremifene, ICI 182780, ICI 164384, droloxifene, tamoxifen and toremifene at apparent Ki values of 0.015, 0.011–0.017, 0.040–0.054, 0.043, 0.044, 0.243 and 0.735 nM, approx. 10 nM and >10 nM, respectively. Similar data were obtained in ZR-75-1 and/or MCF-7 cells. Moreover, EM-652 was 6-fold more potent than OH-Tamoxifen in inhibiting the proportion of cycling MCF-7 cells. Our data show that EM-800 and EM-652 are the most potent known antiestrogens in human breast cancer cells in vitro and that they are devoid of the estrogenic activity of OH-tamoxifen and droloxifene suggested by stimulation of cell growth in the absence of estrogens in ZR-75-1 and MCF-7 cells. Int. J. Cancer 73:104–112, 1997. © 1997 Wiley-Liss, Inc.     

Effects of a new clinically relevant antiestrogen (GW5638) related to tamoxifen on breast and endometrial cancer growth in vivo.

PURPOSE: Cross-resistance is an important issue for the evaluation of new antiestrogens to treat advanced breast cancer patients who have failed tamoxifen therapy. In addition, postmenopausal patients treated with long-term adjuvant tamoxifen show a 3-4-fold increase in the risk of developing endometrial cancer. Consequently, a new second line agent should be more antiestrogenic and less estrogen-like on the uterus, and be effective at controlling the growth of breast cancer after exposure to tamoxifen. The purpose was to evaluate the effects of the new tamoxifen analogue GW5638 on breast and endometrial cancer growth. EXPERIMENTAL DESIGN: Athymic mice were transplanted with an endometrial tumor model (ECC-1 E2) that is responsive to estrogen and has never been exposed to antiestrogen. In addition, we used three breast tumor models: a tamoxifen-na?ve tumor (T47D-E2) and two tamoxifen-stimulated tumors (MT2 TAM and MCF-7 TAM LT). The antiestrogen GW5638 (1.5 mg daily), tamoxifen (0.5 mg or 1.5 mg daily), and raloxifene (1.5 mg daily) were given p.o. The pure antiestrogen ICI182,780 (5 mg once a week) was given s.c. Western blots from MCF-7 TAM breast tumors were performed to demonstrate the regulation of estrogen receptor alpha expression by different ligands. RESULTS: Estradiol and GW5638 down-regulated the receptor compared with control. ICI182,780 completely degraded the receptor but tamoxifen had no effect. GW5638 did not promote tumor growth, and was effective in blocking the effects of postmenopausal estradiol on the growth of tamoxifen-na?ve breast and endometrial tumors. However, raloxifene did not completely block the effects of postmenopausal estradiol on the growth of tamoxifen-na?ve endometrial tumor after 14 weeks. GW5638 and ICI182,780 but not raloxifene were also effective in blocking the tamoxifen-stimulated breast tumor growth in athymic mice. CONCLUSIONS: GW5638 is more effective than raloxifene in blocking the effect of estrogen on tamoxifen-na?ve endometrial cancer. More importantly, GW5638, like the pure antiestrogen ICI182,780, is able to block the growth of breast cancer stimulated by tamoxifen differently from raloxifene. GW5638 down-regulates estrogen receptor but does not completely destroy the receptor. Therefore, based on our findings, GW5638 could be developed as a second line agent for advanced breast cancer patients and an important first line agent to evaluate as an adjuvant treatment or chemopreventive.     

Development of antiestrogens and their use in breast cancer: eighth Cain memorial award lecture

This paper describes the laboratory discovery and clinical testing of the first nonsteroidal antiestrogen, MER-25 (ethamoxytriphetol). The compound blocks estrogen action in all species tested and has only slight but transient estrogenic effects. No other antisteroidal actions are noted. MER-25 is antiestrogenic in primates and was investigated in the clinics in a wide range of gynecological conditions, including breast and endometrial cancer. Unfortunately toxic side effects (hallucinations, etc.) precluded further investigation. A derivative of triphenylethylene, clomiphene, has some partial agonist (estrogen-like) actions in laboratory animals and following clinical evaluation is now an established agent for the induction of ovulation in subfertile women. Although clomiphene is active in advanced breast cancer, it was not developed further. In the late 1960s a related compound, tamoxifen, was evaluated to treat a number of estrogen-responsive disorders but was successfully introduced in the 1970s for the treatment of advanced breast cancer. Although there was only modest initial interest in the palliative use of tamoxifen, an enormous increase in basic and applied studies with antiestrogens resulted in a definition of the target site-specific and tumoristatic actions of tamoxifen. Close cooperation between laboratory and clinical evaluation has guided the subsequent development of tamoxifen which is now available to treat all stages of breast cancer. Long-term adjuvant tamoxifen therapy, a concept developed in the laboratory, is currently the treatment strategy of choice. The considerable success of tamoxifen has focused attention on new antiestrogens with different pharmacological properties for other potential clinical applications.     

“Studies on the estrogen receptor in breast cancer” — 20 years as a target for the treatment and prevention of cancer

Summary In 1973, McGuire and Chamness (In: O'Malley BW and Means AR (eds) Receptors for Reproductive Hormones, Plenum Press) summarized their work on the estrogen receptor in animal and human breast tumors, and in so doing described a target for therapeutic intervention. At that time there were no clinically useful antiestrogens, but the subsequent development of tamoxifen for breast cancer therapy has revolutionized the approach to treatment. Long-term adjuvant tamoxifen adjuvant therapy (i.e. greater than one year) has proven efficacy to enhance the survival of breast cancer patients. In addition, because there is an associated 40% decrease in contralateral breast cancer during adjuvant tamoxifen therapy and tamoxifen maintains bone density and reduces fatal myocardial infarction, clinical trials to test the worth of tamoxifen as a preventive for breast cancer in high risk women have started in the United States, United Kingdom, and Italy. Initial concerns that long-term tamoxifen causes endometrial cancer have been placed in perspective and analyzed by a review of the literature. Tamoxifen only doubles the normal risk of detecting endometrial cancer, (i.e. to 2 per 1,000 tamoxifen-treated women per year), and 80% of these cases are early stage, good prognosis disease. Annual gynecological examinations and education are essential to provide reassurance for patients.The success of tamoxifen has encouraged the development of new antiestrogens to exploit the estrogen receptor as a therapeutic target. Droloxifene and TAT-59 mimic the metabolite 4-hydroxytamoxifen in having a high affinity for the estrogen receptor (Jordan et al, J Endocrinol 75:305, 1977). These drugs appear to have a pharmacological profile similar to tamoxifen. In contrast, the new pure antiestrogens have a distinct mechanism of action and will be valuable either as a first line therapy for advanced breast cancer or as a second line endocrine therapy after the failure of long-term adjuvant tamoxifen therapy.Finally, a new strategy is being developed to exploit the target site specific action of antiestrogens. Raloxifene, an antiestrogen with high affinity for the estrogen receptor but only weak estrogenicity for the uterus, prevents rat mammary tumorigenesis and maintains bone density. The drug is to be evaluated as a treatment for osteoporosis, but may also prevent the development of breast and endometrial cancer in a broad group of treated subjects.The identification of the estrogen receptor as a target for therapeutic opportunities has proved to be extremely beneficial for the control of breast cancer and has the added potential to control osteoporosis and coronary heart disease in women.Presented at the 17th Annual San Antonio Breast Cancer Symposium. The William L. McGuire Memorial Lectures are sponsored by an educational grant from Wellcome Oncology.     

Metabolites of tamoxifen in animals and man: Identification,pharmacology, and significance

Summary Over the past decade, the non-steroidal antiestrogen tamoxifen has gained general acceptance for the palliative treatment of breast cancer. Although there has been much interest in the pharmacology of tamoxifen, our knowledge of its metabolism in laboratory animals and patients is incomplete and the precise mechanism of action within target tissue and breast tumor cells is unknown. This review briefly describes the pharmacology of tamoxifen in various laboratory species and patients. Several metabolites of tamoxifen are known and their relative potencies as estrogens and antiestrogens are compared with the parent compound. Apart from monohydroxytamoxifen, none of tamoxifen's metabolites are more potent antiestrogens, but a metabolite in the dog, Metabolite E, is fully estrogenic. Routine assays (tlc, HPLC, glc/ms) are available to detect tamoxifen, N-desmethyltamoxifen, monohydroxytamoxifen, and a newly identified metabolite, designated Metabolite Y, in biological fluids. Continuous therapy with tamoxifen (10 mg bid) produces steady-state levels (100–200 ng/ml serum) within 4 weeks. Levels of N-desmethyltamoxifen are often up to twice the levels achieved with tamoxifen, while levels of monohydroxytamoxifen and Metabolite Y are below 10 ng/ml. Although monohydroxytamoxifen has a high binding affinity for the estrogen receptor, the metabolic activation of tamoxifen is an advantage rather than a requirement for antiestrogenic activity. The action of tamoxifen in vivo is the net result of the individual actions of the parent compound and its metabolites competing for the occupation of receptors within target tissues and tumors.     

Effect of the nonsteroidal antiestrogen ZK 119.010 on growth and metastasis of the EnDa endometrial carcinoma

For patients with disseminated endometrial cancer the prognosis is poor. Radiotherapy, chemotherapy or high-dose progestins have been of limited value in the clinic, with low response rates and a usually short duration. Because of the role of estrogen in the etiology of this disease, a rationale exists for therapies using estrogen antagonists. In order to test this strategy, we used the EnDA endometrial carcinoma of the rat recently described by us. The nonsteroidal antiestrogen ZK 119.010 inhibited the primary-tumor growth of the s.c. implanted EnDA endometrial carcinoma by 50%, being superior to high-dose progestin and tamoxifen (TAM). Moreover, in intact as well as in castrated estrogen (E₂)-substituted rats, ZK 119.010 substantially reduced metastatick-tumor growth in the lymph nodes and lungs. With TAM, however, the number of lung metastases in intact and in castrated E₂-substituted rats either rose or remained stable and the weight of lymph nodes in intact rats increased. After TAM treatment, almost no low-salt-extractable (cytosolic) estrogen receptor (ER) was measurable in the tumor, whereas ZK 119.010 did not alter ER concentrations. The stimulation of metastatic tumor growth, as well as the loss of cytosolic ER under TAM therapy, may reflect the well-known agonist activity of this compound in uterine tissues. ZK 119.010, however, not only lacks this agonist activity, but it exerts a strong antagonistic one. In conclusion, pure antiestrogens may help to improve treatment of endometrial cancer.     

Activity and safety of the antiestrogen EM-800, the orally active precursor of acolbifene, in tamoxifen-resistant breast cancer.

PURPOSE: To determine the efficacy and safety of EM-800 (SCH-57050), the precursor of acolbifene, a new, highly potent, orally active, pure antiestrogen in the mammary gland and endometrium, for the treatment of tamoxifen-resistant breast cancer. PATIENTS AND METHODS: Forty-three post menopausal/ovariectomized women with breast cancer who had received tamoxifen, either for metastatic disease or as adjuvant to surgery for > or = 1 year, and had relapsed were treated in a prospective, multicenter, phase II study with EM-800 (20 mg/d [n = 21] or 40 mg/d [n = 22] orally). Results Thirty-seven patients had estrogen receptor (ER)-positive tumors (>10 fmol/mg; mean, 146 fmol/mg cytosolic protein), three patients had ER-negative/progesterone receptor-positive tumors, and three patients had undetermined ER status. The objective response rate to EM-800 was 12%, with one complete response and four partial responses. Ten patients (23%) had stable disease for > or = 3 months, and 7 patients (16%) had stable disease for > or = 6 months. With a median follow-up of 29 months, median duration of response was 8 months (range, 7 to 71+ months). Treatment with EM-800 was well tolerated. No significant adverse events related to the study drug were observed clinically or biochemically. CONCLUSION: EM-800 produced responses in a significant proportion of patients with tamoxifen-resistant breast cancer, thus showing that this highly potent, selective estrogen receptor modulator, which lacks estrogenic activity in the mammary gland and endometrium, has incomplete cross-resistance with tamoxifen, thus suggesting additional benefits in the treatment of breast cancer.     

Antagonism of chemotherapy-induced cytotoxicity for human breast cancer cells by antiestrogens

In a prior National Surgical Adjuvant Breast and Bowel Project (NSABP) adjuvant study, the addition of the antiestrogen tamoxifen to chemotherapy with melphalan and fluorouracil adversely affected survival in several patient subsets, suggesting an antagonistic drug interaction. To investigate this possibility, we studied the interaction of tamoxifen and other antiestrogens with several cytotoxic drugs in cultured human breast cancer cell lines. Clinically relevant concentrations of tamoxifen and melphalan reduced colony survival of estrogen receptor (ER)-positive breast cancer cells when used alone in a colony-forming assay. However, pretreatment of cells with tamoxifen followed by exposure to melphalan resulted in antagonism, with more colonies surviving treatment with the combination than with melphalan alone. Identical effects were seen using several other triphenylethelene antiestrogens. An antagonistic interaction was observed even with a brief preincubation with tamoxifen that had no effect on cell proliferation, indicating that antagonism was not due to tamoxifen's known cell kinetic effects. Tamoxifen even antagonized melphalan cytotoxicity in ER-negative breast cancer cells and in cultured liver cells. An additive drug interaction occurred when melphalan was combined with pharmacologic concentrations of estradiol or medroxyprogesterone acetate, but antagonism was also observed with dexamethasone. Tamoxifen also antagonized the cytotoxicity of fluorouracil in these cells. However, an additive interaction occurred when the antiestrogen was combined with doxorubicin or 4-hydroxy-cyclophosphamide, an alkylating agent that is transported into the cell by a different carrier-mediated mechanism than melphalan. To avoid potential antagonism in the clinic, combinations of tamoxifen with melphalan and/or fluorouracil should be avoided.     

Endometrial Carcinoma in Tamoxifen-Treated Breast Cancer Patients

Summary

Tamoxifen is an important agent for the treatment of breast cancer. Occasionally the drug, which is an antiestrogen, has agonistic estrogenic activity. The authors describe three new cases of endometrial carcinoma developing in breast cancer patients taking tamoxifen and stress the necessity of carefully monitoring the uterine cavity under tamoxifen treatment.     

Activity of tamoxifen and its metabolites on endocrine-dependent and endocrine-independent breast cancer cells

To better understand the mechanism of action of antiestrogens, the growth-inhibitory effect of tamoxifen and its main metabolites N-desmethyltamoxifen and 4-hydroxytamoxifen, was studied in 6 breast cancer cell lines characterized by different steroid receptor contents. On the basis of the results, our cell lines could be classified into three groups: a first group, including 734B and ZR-75.1 cell lines, characterized by a clear endocrine-dependent behavior, in which cells were sensitive to antiestrogens although to different degrees; a second group, including MDA-MB 231 and BT20 cell lines, characterized by a clear endocrine-insensitive behavior, in which cells were affected only by the highest (10(-6) M) antiestrogen concentration; a third group, including MCF7 and T47D cell lines, characterized by a peculiar behavior. The T47D cell line displayed an increased growth rate after treatment with all three antiestrogens considered. Despite the positive receptor content in the MCF7 cell line, only 4-hydroxytamoxifen showed a clear antiestrogen dose-dependent effect, whereas tamoxifen decreased the cell growth rate only at lower concentrations (10(-8) and 10(-7) M). These results and the well-known heterogeneity of human breast tumors explain the failure of antiestrogen treatment in a certain percentage of patients with breast cancer with a positive estrogen receptor status.     

Comparative effects of 28-day treatment with the new anti-estrogen EM-800 and tamoxifen on estrogen-sensitive parameters in intact mice

Following 28 days of oral administration, in intact mice, the novel non-steroidal anti-estrogen EM-800 was at least 30-fold more potent than tamoxifen in inhibiting uterine weight. Moreover, the maximal inhibitory effect achieved with tamoxifen on uterine weight was only 40% that with EM-800. The pure anti-estrogenic activity of EM-800 on the hypothalamo–pituitary-gonadal axis is illustrated by the increase in ovarian weight, while tamoxifen, due to its estrogenic activity, decreased ovarian weight. EM-800 is 10- to 30-fold more potent than tamoxifen in inhibiting uterine and vaginal estrogen receptors. Since 17β-hydroxysteroid dehydrogenase (17β-HSD) is the key enzyme in estradiol formation, the potent inhibitory effect of EM-800 on uterine 17β-HSD could play an additional role by decreasing the availability of estradiol in the uterine tissue, while tamoxifen, on the contrary, stimulates activity of the enzyme. The atrophic changes in both the endometrial and myometrial layers achieved with EM-800 almost reached those observed 28 days after ovariectomy. EM-800 also resulted in a marked decrease in the number of ovarian developing follicles and corpora lutea, while the number of atretic follicles was increased. Tamoxifen treatment, on the other hand, produced an increase in both the number and crowding of the endometrial glands and a mild atrophy of the myometrial layer. Tamoxifen caused atrophic changes of the vaginal epithelium, especially at the highest doses, though the atrophy was much less pronounced than that following EM-800 treatment or ovariectomy. In addition to being at least 30-fold more potent than tamoxifen in inhibiting uterine weight, the novel anti-estrogen causes atrophy of the endometrium, stimulates the hypothalamo–pituitary–gonadal axis and inhibits uterine 17β-HSD activity, while tamoxifen exerts opposite and estrogen-like effects on these parameters. Int. J. Cancer 73:381–391, 1997. © 1997 Wiley-Liss, Inc.     

Molecular mechanisms of antiestrogen action in breast cancer

The success of antiestrogen therapy to treat all stages of breast cancer, and the evaluation of tamoxifen as a preventive for breast cancer in normal women, have focused attention on the molecular mechanisms of antiestrogen action and mechanisms of drug resistance. The overall goal of research is to enhance current therapies and to develop new approaches for breast cancer treatment and prevention. Recent studies show that tamoxifen and the new pure antiestrogens appear to have different mechanisms of action: tamoxifen and related compounds cause a change in the folding of the steroid binding domain that prevents gene activation whereas the pure antiestrogens cause a reduced interaction at response elements and cause a rapid loss of receptor complexes. Tamoxifen treatment produces changes in the cellular and circulating levels of growth factors that could influence both receptor negative or receptor positive tumor growth and the metastatic potential of a tumor. These events may explain the survival advantage observed with tamoxifen therapy. However, the current therapeutic challenge is to avoid drug resistance during long-term tamoxifen therapy. Numerous explanations for drug resistance to tamoxifen have been suggested, including elevated estrogen levels, increased tumor antiestrogen binding sites, receptor mutations, and impaired signal transduction. However, it is probable that multiple mechanisms evolve to facilitate tumor survival. Most importantly, current research is examining mechanisms responsible for the beneficial actions of tamoxifen on bones and lipids as well as the potentially deleterious effects of tamoxifen on liver and endometrial carcinogenesis and retinopathy. The urgent need to understand antiestrogenic drug mechanisms and toxicity is being facilitated by the application of the technology developed for basic molecular biology.