C.W. Bardin

Clinical pharmacology of RU 486 : an antiprogestin and antiglucocorticoid

Over the last few years, several reviews have focussed on various but limited aspects of antiprogestins in general and RU 486 in particular.lm4 The present review attempts a comprehensive evaluation of the physiology and current clinical uses of RU 486. Progesterone plays a critical role in mammalian reproduction in that it is essential for the initiation and maintenance of pregnancy. Following the discovery of the progesterone receptor,i it was recognized that a progesterone receptor antagonist would be a significant advance in contraceptive technology if it could induce menstruation when used in the luteal phase (once-a-month pill), prevent implantation (morning after pill), and promote abortion in early pregnancy. Furthermore, since progesterone facilitates the action of estradiol in inducing the LH surge prior to ovulation,” a contraceptive action of a progesterone antagonist was also contemplated. The search for such an antiprogesterone extended over more than a decade. The eventual discovery was fortuitous since the scientific team was in fact looking for a glucocorticoid antagonist. (For the interesting story behind the early studies, see reference 1.) In 1981, Philibert and coworkers’ from Roussel-Uclaf reported the first highly effective progesterone antagonist designated RU 38486, subsequently abbreviated to RU 486

Clinical trial with 3-keto-desogestrel subdermal implants.

The study was done to assess the clinical performance and in vivo steroid release rate of 3-keto-desogestrel subdermal implants designed to deliver 5 different doses of the progestin. Volunteers were healthy women of proven fertility who provided blood samples at scheduled intervals during treatment. No pregnancy occurred in 514 woman-months in users of implants delivering 30 and 40 micrograms per day of 3-keto-desogestrel. Three pregnancies, one ectopic, were observed in 109 woman-months recorded with implants delivering 20 micrograms per day or less. Ovulation was inhibited, as judged by depressed progesterone levels, in 57 of 59 (97%) blood samplings in women whose 3-keto-desogestrel plasma levels were greater than 0.28 nmol/L and in 39 of 75 (52%) of cases with lower levels. Users of 4 cm implants manufactured by The Population Council, New York, showed mean levels above 0.28 nmol/L until 18 months of use. Levels achieved with 4.4 cm implants manufactured by Organon, Oss, Holland, were less consistent. No changes were observed in the plasma lipoprotein pattern or clinical chemistry during treatment. The main complaint was the occurrence of bleeding irregularities, particularly with the lower doses. Ovarian cysts found during pelvic examination in 11 (22%) subjects disappeared spontaneously within 7-90 days. 3-keto-desogestrel implants releasing around 40 ug/day and providing plasma levels around 0.28 nmol/L afford efficient contraceptive protection.

Anti-implantation activity of antiestrogens and mifepristone

To develop a better postcoital contraceptive, the following antiestrogens were tested for their anti-implantation activity in the rat: anordrin, anordiol, tamoxifen, ICI 182,780, and RU 39411. The compounds were administered orally or subcutaneously (s.c.) to female rats on days 1, 2, and 3 of pregnancy. All the antiestrogens tested were 100% effective in preventing blastocyst implantation. The lowest effective doses when administered orally were 10, 1.25, 0.062, 6.0 (partially effective), and 0.01 mg/kg/day, respectively. The estimated median effective doses (ED50) were 5.60, 0.40, 0.035, 5.40, and 0.0074 mg/kg/day, respectively. When administered s.c., the minimum effective doses in preventing blastocyst implantation in all animals were 2.0, 0.1, 0.1, 0.1, and 0.01 mg/kg/day, respectively. Anordrin, anordiol, and ICI 182,780 were more potent when administered s.c.; whereas tamoxifen and RU 39411 were effective at similar doses when administered parenterally or orally. RU 39411 was the most potent among the antiestrogens tested and should be evaluated as a potential postcoital contraceptive. The administration of mifepristone, an antiprogestin, at a dose of 8 mg/kg/day blocked blastocyst implantation in all treated animals; whereas at a dose of 4 mg/kg/day or lower, the drug was ineffective. These findings confirm that estradiol and progesterone are essential for blastocyst implantation in the rat. The capacity of mifepristone to potentiate the anti-implantation activity of the antiestrogens was also determined. The combination of a non-effective dose of each of the antiestrogens (anordrin, anordiol, and tamoxifen), and RU 39411, with mifepristone at a non-effective dose, prevented pregnancy, demonstration that an antiprogestin and antiestrogen act synergistically in blocking blastocyst implantation in the rat. The antiestrogen compounds whose anti-implantation activities were potentiated by mifepristone were found to possess significant estrogenic activity, when assayed by measuring the increase in the uterine weights of ovariectomized rats. The only exception was ICI 182,780, which showed no estrogenic activity in the uterine weight bioassay and did not act synergistically with mifepristone in blocking blastocyst implantation. Estradiol was effective in preventing pregnancy at a dose of 1 microgram/kg/day. The combination of non-effective doses of estradiol and mifepristone did not prevent pregnancy. The findings that mifepristone potentiates the anti-implantation activity suggests that the synergistic effect may be a unique property of this class of antiestrogens.

Tamoxifen and RU39411 synergize with mifepristone to produce preimplantation pregnancy loss by increasing embryo transport (rat)

Tamoxifen is a non-steroidal antiestrogen that possesses antagonistic as well a agonistic properties, while RU39411 is an antiestrogen that is known to possess only antagonistic properties. These steroid antagonists were administered orally to rats, with or without mifepristone, an antiprogestational agent, prior to implantation on Day 2 of pregnancy. The status of pregnancy was assessed on Day 14. Low doses of tamoxifen reduced litter size and weight and inducing embryonic absorption in some animals; a dose of 0.25 mg/Kg prevented pregnancy in all animals. RU39411 also had a dose-dependent effect on pregnancy, but a higher dose (0.5 mg/Kg) was required to achieve the same degree of pregnancy prevention. Addition of mifepristone to both antiestrogens had a synergistic effect on reducing litter size and weight. To determine the mechanism by which antiestrogens terminate pregnancy in rats, oviducts and uteri of treated rats were examined for the presence of embryos on Day 3 and 4 of pregnancy, times when most embryos would be expected to be in the oviducts. Most of the embryos of the treated animals were found in the oviducts on Day 3 of pregnancy. By Day 4, only a few embryos were still present in oviducts. These observations suggest acceleration of embryo transport by Day 4 of pregnancy. There was no accumulation of embryos in the uterus. Since acceleration of embryo transport through the reproductive tract in rat is induced by low doses of estrogens, it is likely that the agonistic action of tamoxifen is responsible for pregnancy prevention in these experiments. The fact that RU39411 also prevents pregnancy by a similar mechanism suggests that this estrogen antagonist also has some estrogen agonist effects on the oviduct. The fact that both antiestrogens also affected embryo weight could suggest the action of the antihormones on other mechanisms controlling embryo development.

Species differences in the sensitivity to a GnRH antagonist

The effects of the potent GnRH antagonist [Ac-D-NAL(2)1, 4F-D-Phe2, D-Trp3, D-Arg6]-GnRH (GnRH-A), on ovulation in mature rats and rabbits and on serum LH and FSH levels in ovariectomized rats, rabbits and mice were investigated. Dose-response studies showed that 1 microgram (4 micrograms/kg) of GnRH-A was sufficient to inhibit ovulation completely in cycling rats, while 500 micrograms (135 micrograms/kg) were required to inhibit mating induced ovulation in 8 of 11 rabbits. Two of the 3 rabbits which ovulated in spite of the antagonist treatment had delayed LH surges. The mean LH peak of these 3 rabbits was significantly (p less than 0.001) lower than that of controls. Pituitary response to GnRH-A, as measured by plasma gonadotropin levels following GnRH-A treatment in ovariectomized rabbits, rats and mice showed highest sensitivity of the rat to the inhibitory effects of the antagonist. Serum FSH levels were slightly suppressed in rats, but remained undiminished in rabbits and mice. The difference in the response of the three species to the antipituitary effects of GnRH-A is most likely due to differences in the affinity of the pituitary GnRH receptor to the antagonist.

Effects of anordiol, an antiestrogen, on the reproductive organs of the male rat

Anordiol, an antiestrogen, was administered at doses of 2.5 and 10 mg/kg day-1 for 30 and 60 days to adult male rats. A significant decrease in serum LH, FSH, and testosterone levels occurred in the treated animals as compared to controls. Serum testosterone and LH levels decreased to about 15-35% of control value on day 15 of treatment and reached undetected levels thereafter. Serum FSH level also decreased to about 25-50% of control value by day 15 of treatment and remained at this level. Significant decrease in the weights of the tests and accessory reproductive organs occurred following anordiol treatment, while the body weights remained at pretreatment level. Histological examination of the tests revealed significant decrease in the diameter of the seminiferous tubules and in the number of germ cell elements. Spermatogenesis was arrested at the secondary spermatocyte stage. Leydig cells appeared atrophic and contained pyknotic nuclei. The epididymis, prostate, and seminal vesicle showed degenerative changes. The secretory activity of the glandular epithelium of the prostate gland appeared to be markedly reduced. In conclusion, anordiol acts by blocking gonadotropin production and/or release by the pituitary; thereby testosterone production by Leydig cells is not stimulated, causing spermatogenesis arrest.

Comparative effectiveness of three antiprogestins alone and in combination with anordiol in terminating pregnancy in the rat.

The effectiveness of mifepristone, onapristone, and ORG 31806 alone or in combination with anordiol to terminate pregnancy in the rat was evaluated. ORG 31806 at a dose of 2 mg/kg/day, mifepristone at 4 mg/kg/day, and onapristone at 8 mg/kg/day, terminated pregnancy in all treated animals. Anordiol, an antiestrogen, at a dose of 5 mg/kg/day, terminated pregnancy in all treated animals. Anordiol acted synergistically with all three antiprogestins terminating pregnancy in the rat. The antiprogestins at doses that were either partially effective or non-effective became 100% effective when administered with a non-effective dose of anordiol. Thus, combination of ORG 31806 (1 mg/kg/day) plus anordiol (0.31 mg/kg/ day), mifepristone (1 mg/kg/day) plus anordiol (0.62 mg/ kg/day), and onapristone (2 mg/kg/day) plus anordiol (2.5 mg/kg/day) terminated pregnancy in all treated animals. These combinations of the antiprogestins and anordiol decreased significantly the serum progesterone levels but not serum 17 beta-estradiol levels. The present results indicate that the most potent combination was ORG 31806 plus anordiol.

Potentiating effect of epostane on pregnancy terminating activity of RU 486 in the rat

The effectiveness of a combination of RU 486, an antiprogestin, and epostane, a 3 beta-hydroxysteroid dehydrogenase inhibitor, for termination of pregnancy in female rats was determined. Epostane administered at doses ranging from 8 to 48 mg/Kg/day on days 7, 8, and 9 of pregnancy did not influence the progress of the pregnancy. RU 486 in doses of 1, 2, and 4 mg/Kg/day terminated pregnancy in 0, 40, and 100% of rats, respectively. The combination of epostane (48 mg/Kg/day) plus RU 486 (1 and 2 mg/Kg/day) terminated pregnancy with complete resorption of fetuses in all treated animals. Unexpectedly, the drug combination that terminated pregnancy used a dose of epostane that was not highly effective in lowering serum progesterone levels when epostane was administered alone. Thus, in the rat, the synergistic action of epostane plus RU 486 may not be dependent upon reduced progesterone secretion plus receptor blockage as anticipated. The present results suggest that the combination of epostane plus RU 486 is effective for pregnancy termination.