The distinct roles of the two estrogen receptor (ER) isotypes, ERalpha and ERbeta, in mediating the physiological responses to estrogens are not completely understood. Although knockout animal experiments have been aiding to gain insight into estrogen signaling, additional information on the function of ERalpha and ERbeta will be provided by the application of isotype-selective ER agonists. Based on the crystal structure of the ERalpha ligand binding domain and a homology model of the ERbeta-ligand binding domain, we have designed steroidal ligands that exploit the differences in size and flexibility of the two ligand binding cavities. Compounds predicted to bind preferentially to either ERalpha or ERbeta were synthesized and tested in vitro using radio-ligand competition and transactivation assays. This approach directly led to highly ER isotype-selective (approximately 200-fold) and potent ligands. To unravel physiological roles of the two receptors, in vivo experiments with rats were conducted using the ERalpha- and ERbeta-selective agonists in comparison to 17beta-estradiol. The ERalpha agonist induced uterine growth, caused bone-protective effects, reduced LH and FSH plasma levels, and increased angiotensin I, whereas the ERbeta agonist did not at all or only at high doses lead to such effects, despite high plasma levels. It can thus be concluded that estrogen effects on the uterus, pituitary, bone, and liver are primarily mediated via ERalpha. Simultaneous administration of the ERalpha and ERbeta ligand did not lead to an attenuation of ERalpha-mediated effects on the uterus, pituitary, and liver parameters.

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Asoprisnil (J 867; benzaldehyde, 4-[(11beta, 17beta)-17-methoxy-17-(methoxymethyl)-3-oxoestra-4, 9-dien-11beta-yl]-, 1-oxime) is the prototype of a novel class 11beta-benzaldoxime-substituted selective progesterone receptor modulators (SPRMs) and the first-in-class SPRM to reach an advanced stage of clinical development for the treatment of uterine fibroids and endometriosis. This compound was selected in a drug discovery program aimed to identify progesterone receptor (PR) ligands with predominant agonist but also some antagonist activities. The screening program included a range of receptor binding studies and a hierarchy of in vivo tests. A series of 11beta-benzaldoxime-substituted steroidal compounds exhibiting mixed PR agonist/antagonist effects were synthesized and characterized. For inclusion in this class of compounds, two methods of synthesis were developed and optimized. The 11beta-benzaldoxime-substituted SPRMs showed high PR binding affinities, reduced glucocorticoid receptor affinities compared with the antiprogestin mifepristone, marginal androgen receptor binding affinities, and no binding to estrogen receptors. Animal tests in guinea pigs (luteolysis inhibition assay) and rabbits (McPhail test) constituted the secondary screening tests. A mosaic of progesterone agonist and antagonist effects were found in various models. The most agonistic compounds were selected for further evaluation in animal models with respect to labor induction and endometrial effects. Unlike progesterone antagonists, asoprisnil and related compounds showed marginal effects on labor and parturition in guinea pigs. Proof-of-concept studies in nonhuman primates revealed endometrial antiproliferative effects of selected compounds, including asoprisnil and J 1042, in the presence of amenorrhea and follicular phase estradiol concentrations. Asoprisnil was selected for further clinical development. It shows promising results in the treatment of uterine leiomyomata and endometriosis.

Sulfamate substitution (-O-SO2-NH2) at carbon atom 3 of the steroid skeleton leads to orally active prodrugs of estrogens with much higher systemic, but lower hepatic, estrogenic activity than their parent steroids. This dissociation is achieved by first passage through the liver in erythrocytes, followed by systemic hydrolysis which releases the 'parent' estrogen. In the rat, orally administered tritiated estradiol sulfamate, unlike estradiol, appears in the circulation at high concentrations. At Cmax, approximately one third of the administered dose forms a depot in the circulation (98% in erythrocytes, 2% in plasma). Significant estradiol, estrone and estrone sulfate concentrations were recorded in plasma during depletion of the red blood cell pool. Estradiol sulfamate (J995) has no estrogen receptor affinity per se or estrogenic activity in vitro ( i.e. without hydrolysis). Its oral uterotropic activity in rats is approximately 100 times greater than that of estradiol, however, its hepatotropic activity is only marginally elevated. These functions include bile secretion, the secretion of angiotensinogen, lipoproteins (total and high-density lipoprotein cholesterol) and insulin-like growth factor I (IGF-I). Orally administred estradiol sulfamate led to systemic estrogenic effects without significant hepatic responses, whereas estradiol and other 'conventional' estrogens exerted parallel systemic and hepatic estrogenic effects. Sulfamate technology represents an approach to the use of natural estrogens for fertility control and hormone replacement therapy in both genders. In this context, reduced effects on hemostatic factors, angiotensinogen, bile and IGF-I secretion seem the most important aspects. In addition, blood concentrations of estrogens are less variable than with conventional estrogens.

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