Sylvain Gauthier

Androgen Receptor Antagonists (Antiandrogens) Structure-Activity Relationships

Prostate cancer, acne, seborrhea, hirsutism, and androgenic alopecia are well recognized to depend upon an excess or increased sensitivity to androgens or to be at least sensitive to androgens. It thus seems logical to use antiandrogens as therapeutic agents to prevent androgens from binding to the androgen receptor. The two predominant naturally occurring androgens are testosterone (T) and dihydrotestosterone (DHT). DHT is the more potent androgen in vivo and in vitro. All androgen-responsive genes are activated by androgen receptor (AR) bound to either T or DHT and it is believed that AR is more transcriptionally active when bound to DHT than T. The two classes of antiandrogens, presently available, are the steroidal derivatives, all of which possess mixed agonistic and antagonistic activities, and the pure non-steroidal antiandrogens of the class of flutamide and its derivatives. The intrinsic androgenic, estrogenic and glucocorticoid activities of steroidal derivatives have limited their use in the treatment of prostate cancer. The non-steroidal flutamide and its derivatives display pure antiandrogenic activity, without exerting agonistic or any other hormonal activity. Flutamide (89) and its derivatives, Casodex (108) and Anandron (114), are highly effective in the treatment of prostate cancer. The combination of flutamide and Anandron with castration has shown prolongation of life in prostate cancer. Furthermore, combined androgen blockade in association with radical prostatectomy or radiotherapy are very effective in the treatment of localized prostate cancer. Such an approach certainly raises the hope of a further improvement in prostate cancer therapy. However, all antiandrogens, developed so-far display moderate affinity for the androgen receptor, and thus moderate efficacy in vitro and in vivo. There is thus a need for next-generation antiandrogens, which could display an equal or even higher affinity for AR compared to the natural androgens, and at the same time maintain its pure antiandrogenic activity, and thus providing improved androgen blockade using possibly antiandrogens alone.

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)

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.

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...

Pure selective estrogen receptor modulators, new molecules having absolute cell specificity ranging from pure antiestrogenic to complete estrogen-like activities

Publisher Summary The new chemically provided antiestrogens induce three-dimensional structural changes of the estrogen receptor (ER), which lead to a multitude of different activities of the ER–antiestrogen complex that are specific for each target cell type and gene. Such ligand-induced modifications of the three-dimensional structure of the ER, which are unique to each antiestrogen, lead at one extreme to a complete blockade of the normal action of estrogens in some tissues while in other tissues, the same ER complex completely mimics or even surpasses this natural action. While of major importance for preventive and therapeutic medicine, these unexpected cell-specific properties of the new antiestrogens offer unique tools to investigate and further understand the detailed mechanisms of action of estrogens and antiestrogens, including the structural domains of ERs responsible for their delicate interactions with the numerous other intracellular regulatory proteins (coactivators and corepressors) that culminate in cell-specific stimulation or inhibition of the expression of each estrogen-sensitive gene. The present findings suggest that the progression of breast cancer that occurs with tamoxifen treatment can be due to the intrinsic estrogenic stimulatory activity of tamoxifen on breast cancer proliferation. Raloxifene, on the other hand, represents an important improvement in terms of decrease of the stimulatory activity in the endometrium, although some small estrogenic activity persists in the endometrium as well as on breast cancer cell proliferation.

Long-term inhibitory effects of a novel anti-estrogen on the growth of ZR-75-1 and MCF-7 human breast cancer tumors in nude mice

The effects of the novel anti‐estrogen EM‐343 on the growth of 2 hormone‐responsive human breast cancer tumors have been examined in athymic nude mice. At the low daily dose of 5 μg, EM‐343 administered subcutaneously for 6 months completely blocked the stimulatory effect of endogenous estrogens on the growth of ZR‐75‐1 and MCF‐7 tumors implanted in nude mice. In addition, uterine weight decreased by 60% while ovarian weight increased by 37%. Estrogen receptor (ER) levels measured by [3H]‐labeled estrogen binding were markedly reduced (by 96%, 96% and 92%) in ZR‐75‐1 and MCF‐7 tumors, and in the mouse uterus, respectively. Accompanying the decrease in ER, progesterone receptor levels were reduced by 79%, 87% and 76%, respectively, in the above‐mentioned tissues following EM‐343 treatment. Our data show the pure anti‐estrogenic properties of EM‐343 and its high potency as an inhibitor of growth of human ZR‐75‐1 and MCF‐7 breast tumors in nude mice. Int. J. Cancer 73:735–739, 1997.

Steroid derivatives as pure antagonists of the androgen receptor.

BACKGROUND While the androgens of testicular origin (representing about 50% of total androgens in men over 50 years) can be completely eliminated by surgical or medical castration with GnRH (gonadotropin-releasing hormone) agonists or antagonists, the antiandrogens currently available as blockers of androgen binding to the androgen receptor (AR), namely bicalutamide (BICA), flutamide (FLU) and nilutamide have too weak affinity to completely neutralize the other 50% of androgens made locally from dehydroepiandrosterone (DHEA) in the prostate cancer tissue by the mechanisms of intracrinology. MATERIALS AND METHODS Series of steroid derivatives having pure and potent antagonistic activity on the human and rodent AR were synthesized. Assays of AR binding and activity in carcinoma mouse Shionogi and human LNCaP cells as well as in vivo bioavailability measurements and in vivo prostate weight assays in the rat were used. RESULTS The chosen lead steroidal compound, namely EM-5854, has a 3.7-fold higher affinity than BICA for the human AR while EM-5855, an important metabolite of EM-5854, has a 94-fold higher affinity for the human AR compared to BICA. EM-5854 and EM-5855 are 14 times more potent than BICA in inhibiting androgen (R1881)-stimulated prostatic specific antigen (PSA) secretion in human prostatic carcinoma LNCaP cells in vitro. MDV3100 has a potency comparable to bicalutamide in these assays. Depending upon the oral formulation, EM-5854 is 5- to 10-times more potent than BICA to inhibit dihydrotestosterone (DHT)-stimulated ventral prostatic weight in vivo in the rat while MDV3100 has lower activity than BICA in this in vivo model. These data are supported by respective 40-fold and 105-fold higher potencies of EM-5854 and EM-5855 compared to BICA to inhibit cell proliferation in the androgen-sensitive Shionogi carcinoma cell model. CONCLUSIONS Although the present preclinical results data need evaluation in clinical trials in men, combination of the data obtained in vitro in human LNCaP cells as indicator of potency in the human prostate and the data on metabolism evaluated in vivo on ventral prostate weight in the rat, could suggest the possibility of a 70- to 140-fold higher potency of EM-5854 compared to bicalutamide (Casodex) for the treatment of prostate cancer in men.

Synthesis and structure-activity relationships of analogs of EM-652 (acolbifene), a pure selective estrogen receptor modulator. Study of nitrogen substitution.

EM-652 (acolbifene) analogs have been synthesized as selective estrogen receptor modulators. Substitution on the nitrogen atom of these 2H-1-benzopyran derivatives has been studied for its influence on antiestrogenic activity. Binding to the rat estrogen receptor, inhibition of estradiol-stimulated proliferation of T-47D breast cancer cells, as well as antiuterotrophic and uterotrophic activities in ovariectomized mice have been evaluated. 2H-1-Benzopyran 1b (EM-343, racemic form of EM-652), which contains a piperidine ring, shows the best pharmacological profile; RBA=380, IC50value=0.110 nM (in T-47D cells), as well as 63% and 84% antiuterotrophic inhibitions at the 7.5 and 75 nmol doses, respectively.