B11
Primate Models for the Study of Antiprogestins in Reproductive Medicine
GARY D. HODGEN, Ph.D.
The Howard and Georgeanna Jones Professor and President, The Jones Institute for Reproductive Medicine
Department of Obstetrics and Gynecology, Eastern Virginia Medical School
PART I: ACTIVITY EXPRESSIONS OF ANTIPROGESTINS
Progesterone Antagonists
The new class of hormonal compounds called antiprogestins are so named because, whether they are steroids or nonsteroids, they bind to the progesterone receptor, thereby competitively inhibiting the binding of progesterone itself (Figure B11.1). In turn, such progesterone antagonists deny the metabolic actions of progesterone within tissues having progesterone receptors. In the virtual absence of progesterone or its potent analogues, the antiprogestins themselves are very weak agonists,
expressing progestin activity (Figure B11.2), albeit less than 1/1,000 that of levo-norgestrel, a synthetic progestin (Gravanis et al., 1985; Koering et al., 1986; Wolf et al., 1989a) (Figure B11.3).
Antiglucocorticoid Activity
In addition, certain antiprogestins are known to exert antiglucocorticoid activity upon the adrenocorticotropic hormone (ACTH)/adrenal cortex axis, therein elevating ACTH and cortisol secretion. The degree of this effect is both dose and compound specific, as well as being more pronounced during nighttime hours, when there are inherent diurnal rises in circulating ACTH and cortisol (Healy et al., 1983; Kettel et al., 1991; Chwalisz et al., 1992).
Noncompetitive Antiestrogenic (Antiproliferative) Activity
In the context of estrogen-induced mitogenesis, as it occurs in proliferative endometrium, antiprogestins can also be antiestrogens (Figure B11.4). However, since such antiproliferative actions do not arise through competitive binding of antiprogestins to the estrogen receptor (apparently postreceptor binding mechanisms intervene), the inhibition of tissue growth is called a noncompetitive antiestrogenic activity. This is very different from that achieved by tamoxifen or clomiphene (van Uem et al., 1989; Wolf et al., 1989b; Chwalisz et al., 1991). Moreover, this antiproliferative action of antiprogestins is not a manifestation of a progestin-like agonist activity. As revealed by the concentration of estrogen receptors in endometrial tissue, antiprogestins elevate the estrogen-receptor concentration by about sixfold (Neulen et al., 1990), yet paradoxically mitogenesis due to estrogen-induced growth is strik-
ingly curtailed (Figure B11.5). In contrast, progestins are known to inhibit endometrial proliferation in association with a marked suppression of estrogen-receptor levels.
Sources of Antiprogestins
It is important to appreciate the limited research and clinical experience to date using antiprogestins. For example, although more than 400 chemical structures of antiprogestins have been devised, and many patented as unique chemical entities, the biological data base from which the above remarks derive largely reflects research on only two antiprogestins. Of the almost 1000 scientific manuscripts and abstracts worldwide, perhaps as much as two-thirds of these reports are on the Roussel-Uclaf (Paris) compound mifepristone (RU 486), with the bulk of the remainder from studies of the Shering AG (Berlin) compound onapristone (ZK 299).
There are substantial additional data on other antiprogestins. Both Organon AKZO (Oss, Netherlands) and Research Triangle Institute
findings will be reported publicly soon. A Chinese version of mifepristone is also being produced for abortifacient use in that country.
PART II: NONCOMPETITIVE ANTIESTROGENIC ACTIVITY OF PROGESTERONE ANTAGONISTS
That some progesterone antagonists express other biological activities, besides being antiprogestins, was revealed by the antiglucocorticoid functions of mifepristone (Healy et al., 1983, 1985). More recently, we reported that mifepristone has a noncompetitive antiestrogenic activity that blocks estrogen-induced endometrial proliferation in primates (van Uem et al., 1989). This action of the antiprogestin was later found to be dose dependent in the presence of physiologic estradiol (Wolf et al., 1989b). Paradoxically, we found that mifepristone elevates the concentration of estrogen receptors in monkey endometrium, yet the mitogenic (proliferative) impact of estrogen on endometrial growth was negated (Neulen et al., 1990).
These observations are consistent with other monkey and human data that may substantiate this antiproliferative activity of mifepristone on primate endometrium (Kettel et al., 1991; Murphy et al., 1991; Batista et al., 1992). Apparently, other progesterone antagonists may also possess this property. For example, Chwalisz and coworkers recently reported that onapristone curtails endometrial growth (Chwalisz et al., 1991). Based on this report, we wonder how general this activity may be among a wider spectrum of antiprogestin compounds.
Below, some basic biological studies are summarized that suggest potential therapeutic uses of the antiproliferative activity of antiprogestins on uterine tissues.
Initial Evidence of Noncompetitive Antiestrogenic Activity of Mifepristone
In previous studies, mifepristone administration arrested spontaneous folliculogenesis (van Uem et al., 1989). To investigate the central versus peripheral effects of mifepristone on the ovarian/menstrual cycle, including endometrial proliferation, mifepristone was administered daily [10 mg/kg per day, intramuscular (IM)] from menstrual cycle day 3 or 7 to day 25 in six normal adult cynomolgus monkeys receiving human menopausal gonadotropin (hMG) treatment [37.5 IU (international units) per day] from days 3 to 8. Mifepristone administration with hMG/human chorionic gonadotropin (hCG) therapy did not inhibit ovarian response, as evidenced by steroidogenesis and ovulation. Nine of 23 oocytes retrieved by lavage or follicular aspiration at laparotomy after ovulation induction were morphologically classified as mature
preovulatory status. Whereas endometrial biopsies performed on cycle day 25 in control monkeys revealed an in-phase mature secretory endometrium, histologic sections from mifepristone plus hMG/hCG-treated females uniformly demonstrated atrophic to weakly proliferative endometrium on cycle day 25. This was despite serum estradiol levels of > 300 pg/ml during hMG/hCG treatment (Figure B11.6). Three months after the initial 25-day study, endometrial biopsies revealed persistent atrophic endometrium even though repeated ovulation induction with hMG/hCG therapy resulted in elevated serum estrogen concentrations. The findings were observed whether mifepristone treatment began on cycle day 3 or 7. The intermenstrual interval was significantly lengthened by mifepristone treatments (28.5 ± 2.0 days for controls versus
131.3 ± 11.5 days for mifepristone treatment; P < .01).
In summary, mifepristone consistently blocked ovulation unless hMG/hCG was provided. It elicited a persistent retardation of early proliferative endometrium when administered daily beginning in early or midfollicular phase. The normal mitogenic effects of elevated ovarian estrogen secretion on endometrial tissue were quelled, which uniformly resulted in amenorrhea (Figure B11.3). The long-lasting action of mifepristone in these studies—causing ovulation inhibition and atrophic endometrium—may be due to the depot effect of IM injection. In addition, mifepristone did not prevent ovarian steroidogenesis, ovulation, or oocyte maturation when an ovulation induction regimen of hMG/hCG was given. These findings show that mifepristone alone prevents ovulation by diminishing pituitary gonadotropin secretion, rather than by direct effects on ovarian folliculogenesis. It induces amenorrhea by inhibiting estrogen-induced endometrial proliferation.
Dose-Dependent Blockade of the Proliferative Action of Estradiol Endometrium by Mifepristone
The noncompetitive antiestrogenic effects of mifepristone were examined using estradiol (E2)-treated ovariectomized monkeys given mifepristone, progesterone (P), or both. The E2-induced luteinizing hormone surge of control animals was abrogated by P, mifepristone, or both. Secretory transformation by P was inhibited by mifepristone coadministration. Mifepristone alone at a dose of 1 mg/kg induced endometrial secretory transformation, but higher doses (5 mg/kg) inhibited proliferation and secretory activity (Table B11.1). Thus, in the presence of P, mifepristone is antagonistic, but in the absence of P, it exhibits endometrial progestational effects at low doses and an antiproliferative (antiestrogenic) effect at higher doses (Figure B11.4). These data are encouraging and suggest that mifepristone should continue to be evaluated as a potential contraceptive agent acting at the pituitary or endometrial level or both.
Mifepristone-Induced Elevations of Estrogen Receptor in Primate Endometrium
We have conducted a study to investigate the effect of the antiprogestin mifepristone on estradiol-receptor concentrations in the endometrium of monkeys given physiologic estrogen replacement therapy. Estradiol-17ß (E2) silastic implants were inserted infrascapularly into 12 long-term ovariectomized cynomolgus monkeys (Macaca fascicularis), resulting in an average peripheral serum level of approximately 100
TABLE B11.1 Effect of RU 486 and Progesterone on Ovariectomized Monkeys
pg/ml E2. On day 6 of E2 treatment, four treatment groups were initiated:
Group I: E2 implants only;
Group II: E2 implants plus 11 µmol progesterone/kg body weight in sesame oil, IM, on days 6, 7, and 8;
Group III: E2 implants plus 2.2 µmol mifepristone/kg in sesame oil, IM, on days 6, 7, and 8;
Group IV: E2 implants plus 11 µmol mifepristone/kg, IM, on days 6, 7, and 8.
On treatment day 9, endometrial biopsies were removed by hysterotomies. Cytosolic and nuclear E2-receptor contents of tissues were estimated by the charcoal method (Figure B11.5). In group I, the tissue contained 376 ± 123 pmol bound [3H]E2 per gram of protein; the nuclear portion of binding was about 16 percent. In group II, the tissue contained 216 ± 64 pmol bound [3H]E2 per gram of protein; the nuclear binding portion was only 8 percent. In group III, tissue contained 654 ± 47 pmol bound [3H]E2 per gram of protein; the nuclear binding portion was about 22 percent. In group IV, the tissue contained 1198 ± 172 pmol bound [3H]E2 per gram of protein; the nuclear binding portion was about 17 percent. Scatchard plot analysis indicated that the Kd app of the estrogen receptor (1.04 × 10-9 M) was not altered by mifepristone. This study demonstrates that after physiologic E2-replacement, antiprogestin treatment will cause E2 receptor concentrations to rise dramatically; this effect was dose dependent.
Whether this noncompetitive antiestrogenic (antiproliferative) property of certain antiprogestins extends to breast cancers that are estrogen or estrogen-progestin dependent is not known. In addition, the effects of mifepristone, onapristone, and other progesterone antagonists on estrogen-dependent physiological functions, such as bone density and lipid-related cardiovascular health, remain to be evaluated, especially in the context of long-term regimens.
REFERENCES
Batista, M.C., Cartledge, T.P., Zellmer, A.W., et al. Delayed endometrial maturation induced by daily administration of the anti-progestin RU 486: A potential new contraceptive strategy. American Journal of Obstetrics and Gynecology 167:60–65, 1992
Chwalisz, K., Hegele-Hartung, C., Fritzemeier, K.H., et al. Inhibition of the estradiol
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Chwalisz, K., Hsiu, J.G., Williams, R.F., et al. Evaluation of the anti-proliferative actions of the progesterone antagonists mifepristone (RU 486) and onapristone (ZK98299) on primate endometrium. [Abstract] Presented at the 39th Annual Meeting of the Society for Gynecologic Investigation, San Antonio, Texas, March 18–23, 1992.
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