Yves Merand

EM-652 (SCH 57068), a third generation SERM acting as pure antiestrogen in the mammary gland and endometrium

Breast cancer is the most frequent cancer in women while it is the second cause of cancer death. Estrogens are well recognized to play the predominant role in breast cancer development and growth and much efforts have been devoted to the blockade of estrogen formation and action. The most widely used therapy of breast cancer which has shown benefits at all stages of the disease is the use of the antiestrogen Tamoxifen. This compound, however, possesses mixed agonist and antagonist activity and major efforts have been devoted to the development of compounds having pure antiestrogenic activity in the mammary gland and endometrium. Such a compound would avoid the problem of stimulation of the endometrium and the risk of endometrial carcinoma. We have thus synthesized an orally active non-steroidal antiestrogen, EM-652 (SCH 57068) and the prodrug EM-800 (SCH57050) which are the most potent of the known antiestrogens. EM-652 is the compound having the highest affinity for the estrogen receptor, including estradiol. It has higher affinity for the ER than ICI 182780, hydroxytamoxifen, raloxifene, droloxifene and hydroxytoremifene. EM-652 has the most potent inhibitory activity on both ER alpha and ER beta compared to any of the other antiestrogens tested. An important aspect of EM-652 is that it inhibits both the AF1 and AF2 functions of both ER alpha and ER beta while the inhibitory action of hydroxytamoxifen is limited to AF2, the ligand-dependent function of the estrogen receptors. AF1 activity is constitutive, ligand-independent and is responsible for mediation of the activity of growth factors and of the ras oncogene and MAP-kinase pathway. EM-652 inhibits Ras-induced transcriptional activity of ER alpha and ER beta and blocks SRC-1-stimulated activity of the two receptors. EM-652 was also found to block the recruitment of SRC-1 at AF1 of ER beta, this ligand-independent activation of AF1 being closely related to phosphorylation of the steroid receptors by protein kinase. Most importantly, the antiestrogen hydroxytamoxifen has no inhibitory effect on the SRC-1-induced ER beta activity while the pure antiestrogen EM-652 completely abolishes this effect, thus strengthening the need to use pure antiestrogens in breast cancer therapy in order to control all known aspects of ER-regulated gene expression. In fact, the absence of blockade of AF2 by hydroxytamoxifen could explain why the benefits of tamoxifen observed up to 5 years become negative at longer time intervals and why resistance develops to tamoxifen. EM-800, the prodrug of EM-652, has been shown to prevent the development of dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma in the rat, a well-recognized model of human breast cancer. It is of interest that the addition of dehydroepiandrosterone, a precursor of androgens, to EM-800, led to complete inhibition of tumor development in this model. Not only the development, but also the growth of established DMBA-induced mammary carcinoma was inhibited by treatment with EM-800. An inhibitory effect was also observed when medroxyprogesterone was added to treatment with EM-800. Uterine size was reduced to castration levels in the groups of animals treated with EM-800. An almost complete disappearance of estrogen receptors was observed in the uterus, vaginum and tumors in nude mice treated with EM-800. EM-652 was the most potent antiestrogen to inhibit the growth of human breast cancer ZR-75-1, MCF-7 and T-47D cells in vitro when compared with ICI 182780, ICI 164384, hydroxytamoxifen, and droloxifene. Moreover, EM-652 and EM-800 have no stimulatory effect on the basal levels of cell proliferation in the absence of E2 while hydroxytamoxifen and droloxifene had a stimulatory effect on the basal growth of T-47D and ZR-75-1 cells. EM-652 was also the most potent inhibitor of the percentage of cycling cancer cells. (ABSTRACT TRUNCATED)

Age-related changes in central nervous system enkephalins and substance P

Abstract Concentrations of substance P and met- and leu-enkephalins were measured by radioimmunoassay in discrete rat brain nuclei of young (4–5 months) and old (24–26 months) rats. The substance P content of n. anterior (hypothalami), n. ventromedialis, n. premamillaris ventralis, n. interstitialis striae terminalis, n. entopeduncularis and n. dorsalis raphes is reduced in old rats. The met-enkephalin content is decreased in n. suprachiasmaticus, n. arcuatus and n. premamillaris ventralis while the leu-enkephalin content of n. preopticus medialis, n. suprachiasmaticus, n. paraventricularis, n. ventromedialis and n. premamillaris ventralis is decreased in old rats.

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 7β-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.

Changes in the β-endorphin content of discrete hypothalamic nuclei during the estrous cycle of the rat

Abstract Concentrations of β-endorphin were measured in discrete brain nuclei of 4-day cycling rats on each day of the estrous cycle. On the afternoon of proestrus β-endorphin levels were significantly higher in median eminence and suprachiasmatic nucleus, and lower in arcuate nucleus, when compared to levels found on other days of the cycle. These changes coincided with the peak of plasma prolactin, which was blocked by prior administration of naloxone.

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.

Regional distributions of somatostatin and cholecystokinin-like immunoreactivities in rat and bovine brain.

The distribution of somatostatin- and cholecystokinin octapeptide (CCK-8)-like immunoreactivities in the central nervous system of bovine and rat has been studied using a sensitive and specific radioimmunoassay. The most interesting information is the high concentration of CCK-8 in the various cortical areas in rat and bovine. The nuclei of amygdala contain the highest amount of octapeptide in rat while in bovine brain, this structure contains the second highest concentration after polus frontalis.

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.

Binding Characteristics of Novel Nonsteroidal Antiestrogens to the Rat Uterine Estrogen Receptors

Tamoxifen (TAM), the only antiestrogen currently available for the endocrine therapy of breast cancer behaves as a mixed agonist/antagonist of estrogen action, thus limiting its therapeutic potential. We report the binding characteristics of a novel series of nonsteroidal antiestrogens to the rat uterine estrogen receptor. As measured by competition studies, the affinity of EM-652, the active metabolite of the prodrug EM-800, for the estrogen receptor is 7-11 times higher than that of 17beta-estradiol (E2), ICI 182780, and hydroxy-tamoxifen (OH-TAM), the active metabolite of Tamoxifen. EM-652 is 20x more potent than ICI 164384 and Droloxifene while it is 400 times more potent than Toremifene in displacing [3H]E2 from the rat uterine estrogen receptor. On the other hand, the prodrug EM-800 and Tamoxifen have respectively 150-fold and 410-fold less affinity for the estrogen receptor than the pure antiestrogen EM-652. No significant binding of EM-652, EM-800, TAM or OH-TAM was observed to the rat uterine progesterone receptor at concentrations up to 10,000 nM except for TAM that caused a 50% displacement of labeled R5020 at 4000 nM. No significant binding of EM-652 or EM-800 was observed on the rat ventral prostate androgen receptor or the rat uterine progesterone receptor. The present data demonstrate the high affinity and specificity of the new antiestrogen, EM-652, for the rat uterine estrogen receptor. The antiestrogen EM-652 thus becomes the compound having the highest known affinity for the estrogen receptor. Due to its unique potency and its pure antiestrogenic activity already demonstrated in many systems, this antiestrogen could well offer an important advance for the endocrine therapy of breast cancer, uterine cancer, and other estrogen-sensitive diseases in women.

Comparison of the Effects of the New Orally Active Antiestrogen EM-800 with ICI 182 780 and Toremifene on Estrogen-Sensitive Parameters in the Ovariectomized Mouse

The nonsteroidal antiestrogen EM-800 is approximately 10-fold more potent than ICI 182 780, the most potent known steroidal antiestrogen, at inhibiting estrone-stimulated uterine weight in ovariectomized mice (half-maximal inhibitory daily sc doses of 0.2 and 2.0 μg, respectively). At maximal doses, however, both compounds lead to a similar maximal 90% inhibition of estrone-stimulated uterine weight. A 10-fold higher activity of EM-800 compared with ICI 182 780 was also observed on estrone-stimulated vaginal weight, with maximal inhibitions of 96% and 90%, respectively, achieved by the two compounds. In addition, EM-800 injected sc or administered orally led to a marked loss of uterine and vaginal estrogen receptor levels measured by binding assay, whereas ICI 182 780 exerted no inhibitory effect on this parameter under the experimental conditions used. Comparable effects were observed when estrogen receptor protein levels were measured by enzyme immunoassay. After oral administration, EM-800 exerted maxi...

Effect of neonatal hypothyroidism on the serotonin system of the rat brain

The effects of neonatal thyroidectomy and thyroid hormone replacement therapy on the development of serotonin-containing neurons in discrete rat brain nuclei were studied. Newborn male rats were rendered hypothyroid by the injection of 125 mu Ci 131I, and, after 45 days, were compared with normal littermate controls and 131I-injected animals subsequently maintained by daily T4 injections. The serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) contents of discrete brain nuclei removed by punches of frozen brain slices were measured by HPLC with electrochemical detection. 5-HT and 5-HIAA contents were significantly increased in many nuclei of the hypothyroid rat brain. By blocking the biosynthesis of 5-HT with p-chlorophenylalanine we found that the activity of tryptophan hydroxylase is an important step in the stimulatory effect of hypothyroidism on the 5-HT and 5-HIAA contents. Furthermore, we demonstrated after blockage of monoamine oxidase activity with pargyline, a less pronounced decline of 5-HIAA in neonatal hypothyroid animals, thus causing a relative accumulation of this metabolite. These results demonstrate that there are important modifications of the 5-HT system in the brain of neonatal hypothyroid rats. This may have an important role in the development of hypothyroid-induced impairments of central nervous system function.