The molecular biology of RU486. Is there a role for antiprogestins in the treatment of breast cancer?

PIP: Considerable animal research and clinical trials demonstrate that progesterone antagonists could treat hormone-dependent breast cancers. Since endogenous progesterone does include mitosis in epithelial cells of the breast, in theory, exogenous progesterone can cause breast cancer. Thus administration of progesterone antagonists could block endogenous progesterone. Yet we do not know the mitosis pattern in breast cancer cells during the menstrual cycle, so research obtaining such data is needed. Ethical problems arise, however, since researchers need multiple breast tumor samples to analyze proliferative activity at various times during the cycle. A possible solution is using an aspirated tumor sample for initial mitotic analysis immediately followed by RU-486 treatment then tumor removal 24 hours later for reanalysis. Ideally well controlled studies using organ-cultured human breast tumors, human breast cancer lines, and human tumors implanted into nude mice are needed to understand the mechanisms of the mitogenic actions of progestins and progestin antagonists. Progestin antagonist may be used to treat locally advanced or metastatic cancers either as an adjuvant endocrine therapy alone or with tamoxifen. An obstacle to longterm use of RU-486 as a treatment for breast cancer is its antiglucocorticoid side effects. But the molecules of newer progesterone antagonists appear to produce maximal antiprogestin activity and minimal antiglucocorticoid activity. In addition, if RU-486 is administered with drugs that prevent adrenal steroidogenesis or peripheral aromatization of adrenal steroids to estrogens, women may take it for longterm treatment. Researchers must have the opportunity to continue basic tumor biological and molecular research to gain an understanding of the exact molecular targets and mechanisms of antagonist action. The current political climate in the US hinders such research, however.^ieng

Differences in the association of the progesterone receptor ligated by antiprogestin RU38486 or progestin ORG 2058 to chromatin components.

We assessed the hypothesis that due to variations in the conformation of the progesterone receptor induced by the antiprogestin RU38486 compared to the progestin ORG 2058, differences may result in the association of the receptor with some of the chromatin components. The physical properties of the receptor-bound chromatin fragments released by micrococcal nuclease digestion were characterized by sucrose gradient sedimentation and by gel filtration on Agarose A-1.5m or Agarose A-5m columns. The nuclear fraction was isolated from T47D cells previously exposed to 0.1 microM [3H]RU38486 or 0.1 microM [3H]ORG 2058. Micrococcal nuclease digestion solubilized two receptor forms sedimenting at 4.4 S and 6.3 S for the antiprogestin bound receptor and only one receptor at 4.4 S for the progestin ligated receptor. High-salt buffer dissociated either the antiprogestin or the progestin-bound receptor to smaller receptor forms sedimenting at 3.5 S. Chemical cross-linking with the cross-linker 2-iminothiolane of the micrococcal nuclease solubilized receptor forms resulted in 6.7-S and 4.4-S forms sedimenting on 0.4 M KCl gradients for the antiprogestin and progestin ligated receptors, respectively. Stokes radii of 7.3 nm and 6.4 nm were determined by gel filtration in 0.4 M KCl for the 6.7-S and the 4.4-S receptor forms, respectively. Using the sedimentation coefficient and the Stokes radius, molecular weights of 202,000 and 116,000 were calculated for the antiprogestin and progestin ligated receptors. We conclude that the micrococcal nuclease solubilized antiprogestin ligated receptor is associated with additional or different chromatin components compared to the progestin bound receptor.

PIP: The hypothesis that variations in the conformation of the progesterone receptor induced by the antiprogesterone RU38486 compared to the progestin ORG2058 may cause differences in the association of the receptor with some of the chromatin components was investigated. The approach to analyzing the functional organization of the receptor in the chromatin was to study the receptor-bound fragments released by nuclear digestion. The physical properties of these receptor-bound chromatin fragments were characterized by sucrose gradient sedimentation and gel filtration. Micrococcal nuclease digestion solubilized 2 receptor forms sedimenting at 4.4 S and 6.3 S for the antiprogestin bound receptor and only 1 receptor at 4.4 S for the progestin ligated receptor. High-salt buffer dissociated either the antiprogestin or the progestin-bound receptor to smaller receptor forms sedimenting at 3.5 S. Chemical cross-linking resulted in 6.7 S forms for the antiprogestin receptors and 4.4 S forms for the progestin ligated receptors. Use of the sedimentation coefficient and the Stokes radius yielded molecular weights of 202,000 and 116,000 for the antiprogestin and progestin ligated receptors, respectively. Overall, these kinetic studies were unable to explain the variation in responses of the target cell to agonist or antagonist ligands. It is postulated that the antagonist exerts a subtle, but important, conformational change in the receptor, which alters the receptor's ability to associate with some chromatin components and thus affects the normal events in gene expression.

Disruption of follicular maturation and delay of ovulation after administration of the antiprogesterone RU486.

To investigate the role of progesterone in the follicular phase, we examined the effects of RU486 in eight normal cycling women studied with daily and frequent blood sampling (every 10 min for 10 h) during three menstrual cycles (control-treatment-recovery). RU486 (3 mg/kg, orally) was administered for 3 consecutive days after ultrasound documentation of a dominant follicle. In six of the eight women, RU486 was given after emergence of the dominant follicle, while in two women, RU486 was initiated during the preovulatory period when estradiol levels had exceeded 917 pmol/L. In the six women given RU486 after emergence of the dominant follicle, RU486 significantly prolonged the follicular phase duration from 15.6 +/- 1.9 (+/- SD) to 28.6 +/- 9.3 days (P less than 0.01) and extended the treatment cycle length to 42.3 +/- 9.1 (+/- SD) days (P less than 0.01). During RU486 treatment, mean serum estradiol levels decreased from 385 +/- 43 to 228 +/- 28 pmol/L (P less than 0.01), while LH, FSH, ACTH, cortisol, and progesterone levels changed little. LH pulse frequency and amplitude on the last day of RU486 administration did not differ from control values. Collapse of the dominant follicle was evident on ultrasound after RU486 administration and was not accompanied by uterine bleeding. In the two women treated during the preovulatory period, the follicular phase was not prolonged, and RU486 failed to delay the onset of the LH surge. Our findings indicate that RU486 treatment during the follicular phase interrupts normal follicular development, resulting in a delay of ovulation and a reinitiation of follicular recruitment.

PIP: The effect of the antiprogesterone RU486 on the hypothalamic-pituitary-ovarian axis during the follicular phase of the menstrual cycle was investigated in 8 normally cycling women. Frequent, daily blood sampling was conducted during 3 cycles--control, treatment, and recovery. In 6 subjects, RU486 administration was initiated after emergence of the putative dominant follicle (documented by ultrasound). The 2 remaining subjects received RU486 during the preovulatory period when estradiol levels had already exceeded 917 pmol/L. When RU486 was administered after emergence of the dominant follicle, the duration of the follicular phase significantly increased from 15.6 + or - 1.9 days to 28.6 + or - 9.3 days, thus prolonging the intermenstrual length of the RU486 cycle from 28.9 + or - 2.7 days to 42.3 + or - 9.1 days. Endometrial breakdown did not occur during RU486 administration. RU486 further induced a significant decline in estradiol levels from 385 + or - 43 pmol/L to 228 + or - 28 pmol/L, but there was no significant change in luteinizing hormone (LH), follicle-stimulating hormone (FSH), or progesterone levels. RU486 produced a decrease in size or collapse of the putative dominant follicle in 5 of the 6 subjects who received this agent after emergence of the follicle. In the 2 women treated during the preovulatory period, the follicular phase was nor prolonged and RU486 failed to delay the onset of the LH surge. Overall, this study demonstrates that administration of RU486 in the follicular phase disrupts normal follicular development, resulting in functional demise of the dominant follicle, reinitiation of folliculogenesis, and extended menstrual cycle length. These events are not accompanied by uterine bleeding or significant alterations in LH pulsatile frequency and amplitude.

Effects of RU486 on progesterone secretion by human preovulatory granulosa cells in culture.

The effects of RU486 on progesterone synthesis were studied in human preovulatory granulosa cells in culture. No effect was observed at 1 and 10 micrograms/mL, but at 100 micrograms/mL, RU486 inhibited the simulation of progesterone secretion induced by LH and cAMP. It is suggested that the main target of RU486 is the cytochrome P450scc function [catalyzing the formation of pregnenolone (D5P) from cholesterol], since no accumulation of D5P or hydroxy derivatives of progesterone was observed. As RU486 is an antiglucocorticosteroid and antiprogesterone agent, the effects of dexamethasone and progesterone were also investigated. Dexamethasone did not modify progesterone secretion, but progesterone inhibited its own synthesis in both the presence and absence of LH. Thus, under these experimental conditions RU486 displayed a progesterone-like effect. However, since the effect of RU486 was observed only at a concentration around 10(-4) M, the mechanism of action may not involve a receptor pathway and may not apply to most clinical circumstances.

The steroid hormone antagonist RU486. Mechanism at the cellular level and clinical applications.

The cellular and molecular mechanism of RU486, a steroid hormone antagonist, is discussed in detail. Principally, RU486 opposes the action of two types of hormones: progesterone and glucocorticosteroids. The clinical applications are also described, as well as the future outlook.

PIP: This review of the mechanism of action of the anti-progestational agent RU-486 concentrates on its interactions with the sub-molecular regions of the progesterone receptor, and implications for clinical applications. The progesterone receptor, like other steroid hormone receptors, has 4 distinct regions, the ligand-binding domain (LBD), the DNA-binding domain (DBD), a highly charged hinge region, and an N-terminal section thought to be involved in regulation of transactivation. The progesterone receptor occurs in 2 forms, A and B, and these are linked in dimers. The dimer binds to DNA at locations called hormone response elements (HREs). The receptor remains in the nucleus, prevented from binding by heat shock protein 90 (hsp90). DNA binding, which can occur separately from hormone in experimental conditions, first causes release of hsp90, and a shape change in the receptor, and then elicits an effect on the DNA chromatin, modification of transcription factors, and other effects such as phosphorylation or related proteins. When RU-486 is substituted for progesterone, DNA binding is tight, but the hsp-90-receptor complex is stabilized, and the receptor shape is abnormal, incompatible with subsequent hormone effects in the cell. The implications for research on the mechanism of action of the progesterone receptor were discussed. Examples include the suggestion that the 1% of women who do not respond to RU-486 with early abortion may be genetically distinct; that research of progesterone receptors in several types of cancer such an meningiomas and breast cancer are justified; and alto that use of RU-486 for local or systemic hypercorticism may be applicable. On the other hand, while new users of RU-486 for contraception are theoretically of interest, it is unlikely that any company will invest in another type of oral contraceptive.

Contraceptive efficacy of low doses of mifepristone.

OBJECTIVE: To determine whether a 5-mg dose of mifepristone is sufficient to prevent pregnancy. DESIGN: Clinical study. SETTING: Academic research center. SUBJECT(S): Healthy, fertile, sexually active female volunteers. INTERVENTION: Volunteers received a 5-mg dose of mifepristone once weekly, starting on cycle day 2, for up to 6 months. This was their only contraceptive method. MAIN OUTCOME MEASURE(S): Number of pregnancies. RESULT(S): The treatment resulted in a significant decrease in pregnancy rate without affecting the menstrual cycle or causing disturbing side effects. CONCLUSION(S): A low dose of mifepristone, which does not inhibit ovulation, reduces fertility significantly by affecting the endometrium. However, the contraceptive effect needs to be improved for the drug to compete with other contraceptive methods.

PIP: Clinical research has demonstrated that the effect of mifepristone on the endometrium is sufficient to prevent pregnancy. The efficacy of a low dose of mifepristone in preventing implantation was investigated in 18 healthy, fertile women with normal menstrual cycles from Stockholm, Sweden. Study participants received 5 mg of mifepristone once/week, starting on cycle day 2, and were followed for 1-6 months. Three pregnancies occurred in the 63 treatment cycles observed. The mean frequency of sexual intercourse was 2.2 times/week. If ovulation occurred 14 days before the onset of menstruation, at least 1 coital act took place during the period 3 days before and 1 day after ovulation in at least 45 cycles, resulting in a probability of pregnancy of 0.067. Without treatment, 14 pregnancies would have been expected. Although ovulation was not measured objectively, the regular bleeding pattern recorded in all cycles but one makes it unlikely that the contraceptive effect of mifepristone was due to ovulation inhibition. Although the failure rate of 5 mg of mifepristone is unacceptably high for a contraceptive regimen, other treatment schedules merit investigation.

Sequential regimen of the antiprogesterone RU486 and synthetic progestin for contraception.

OBJECTIVE: To examine the effect of sequential use of the antiprogesterone RU486 and synthetic progestin on ovarian function of healthy women. DESIGN: Healthy women were given a sequential antiprogesterone-progestin treatment. Blood samples were taken twice a week during one control cycle and one to three treatment cycles; prospective analysis. SETTING: The outpatient clinic of the Helsinki City Maternity Hospital, Helsinki, Finland, and Steroid Research Laboratory, Department of Medical Chemistry, University of Helsinki, Helsinki, Finland. PATIENTS: Eleven healthy women, volunteers, 20 to 34 years of age. INTERVENTIONS: A dose of 25 mg/d of RU486 was given during cycle days 1 to 21, and synthetic progestin (5 mg of norethisterone to six and 5 mg of medroxyprogesterone acetate to five women) during cycle days 22 to 31. MAIN OUTCOME MEASURES: Serum P, E2, FSH, and LH were measured from serum samples. RESULTS: In 20 of the 24 treatment cycles analyzed the serum concentrations of P were anovulatory. In the remaining 4 cycles, P levels rose above 3 ng/mL, suggestive of ovulation. Folliculogenesis was not completely inhibited, but serum E2 profiles were subnormal and delayed. Bleeding control was satisfactory. CONCLUSIONS: Antiprogesterone RU486 hampers or delays follicular development, suggesting a possible use as an estrogen-free oral contraceptive. However, the synthetic progestins used in this regimen induced serum P rises in some cycles. The synthetic progestin provides the cycle control, but its possible effect on the reliability of the method remains to be evaluated.

PIP: To assess the effectiveness of an estrogen-free oral contraceptive (OC) involving the sequential administration of an antiprogesterone and synthetic progestin, 11 healthy Finnish women 20-34 years of age were enrolled in a prospective investigation for 1-3 cycles. 25 mg of RU-486 was administered for the first 21 days of the cycle, followed by 5 mg of norethisterone or medroxyprogesterone acetate for the next 10 days. It was hypothesized that prolonged RU-486 administration would prevent the follicle from surviving for ovulation. During the RU-486 phase, P increases above 3 ng/ml (suggestive of ovulation) were recorded in only four of the 24 treatment cycles analyzed. Mean serum estradiol concentrations were 147 pg/ml in ovulatory women and 72 pg/ml in anovulatory women, indicating that follicle development was subnormal and delayed, but not completely inhibited. Baseline secretion of luteinizing hormone (LH) increased during RU-486 administration, while an LH surge greater than 100% over baseline was recorded in the majority of cycles during progestin administration. No significant changes occurred in follicle-stimulating hormone levels. Menstrual bleeding began 1-5 days after progestin discontinuation; women who took norethisterone experienced no breakthrough bleeding or spotting during treatment. It is concluded that the main antiovulatory effect of RU-486 is attributable to its antiprogestational effect on the preovulatory P rise.

Heterosteroids and drug research.

PIP: This updated literature review on heterosteroids and drug research has information on chemical structure, pharmacology, and effects. It first discusses the anti-inflammatory heterosteroids, such as mometasone furoate and cortivazol. It also covers heterosteroidal antimineralocorticoids and anabolic hetero derivatives. The review discusses at length the 19-norsteroid, mifepristone (RU-486), which exhibits antiprogestational activity and is being used for fertility control in women. It also has antiglucocorticoid activity and shows promise as a treatment of diseases characterized by muscle atrophy. In vitro studies indicate that mifepristone inhibits growth of breast cancer cell lines and of endometrial cancer cell lines. It has already exhibited growth inhibitory effects in some breast cancer patients. Discussions of mifepristone's pharmacokinetics and structural modifications of mifepristone follow. Danazol is an antigonadotropin and is used to treat endometriosis, benign breast disease, precocious puberty, hereditary angioneurotic edema, menorrhagia, some types of infertility, and gynecomastia. Danazol effects considerable changes in lipid metabolism. Other hormonal, antihormonal, and/or antifertility heterosteroids and/or aspects include androgen antagonists (e.g., cyproterone acetate), estrogen activity, antiestrogens, STS-557, and oximinosteroids. Heterosteroidal inhibitors of steroid hormone biosynthesis discussed are aromatase inhibitors, 5 alpha-reductase inhibitors, and 3 beta-hydroxysteroid dehydrogenase inhibitors (trilostane, epostane, and azastene). Heterosteroids affect the cardiovascular system, including the cardiac glycosides, antiarrhythmic agents, and antilipemic agents. Some heterosteroids affect central nervous system activity (e.g., RU-5135 causes convulsions in rodents). Pancuronium analogues and chandonium and analogues are neuromuscular blocking azasteroids. In addition to danazol and RU-486, several other antineoplastic heterosteroids exist (e.g., estramustine phosphate sodium, a prostate cancer drug).^ieng

Pharmacokinetic study of orally administered RU 486 in non-pregnant women.

A method based on HPLC was devised for the estimation of RU 486 in blood and utilised to study the pharmacokinetics of a single dose of 50 mg RU 486 administered orally to 12 women on day 7 of the cycle. The dose was rapidly absorbed with peak plasma concentration between 1 and 2 hours. Distribution was also rapid (mean t1/2 alpha: 1.4h), whereas elimination was slow (mean t1/2 beta: 28.3 h). RU 486 was still detectable in some women at 72 h after administration. The plasma concentrations fitted the equation for a two-compartment open model from which the pharmacokinetic parameters were calculated. The mean total plasma clearance was 3.0 l/h, and the comparison of our data with those published studies suggests that the pharmacokinetics of RU 486 in Chinese women are similar to those of other populations.

PIP: A method based on high performance liquid chromatography (HPLC) was developed for the estimation of RU-486 in blood and used to analyze the pharmacokinetics of a single dose of 50 mg of RU-486 administered orally to 12 Chinese women on day 7 of the menstrual cycle. In the 1st 6 subjects, blood samples were taken immediately before and 1, 2, 4, 8, 24, and 48 hours after RU-486 administration; in the 2nd group of 6 women, additional samples were taken at 0.33, 0.5, 0.75, and 72 hours. Since there were no significant differences between the 2 groups in values, the data were combined. RU-486 was found to be rapidly absorbed, with significant amounts present in the 20 minute samples and peak concentrations at 1-2 hours. In contrast, elimination was slow and significant levels of RU-486 were still detectable in most subjects at 72 hours. The mean value for the half-life of absorption was less than 1 hour, while the mean half-life of distribution was 1.4 hours. Mean total plasma clearance was 3.0 liters/hour. The plasma concentrations fitted the equation for a 2-compartment open model from which the pharmacokinetic parameters were calculated. Comparison of these findings with those from other studies suggest that the pharmacokinetics of RU-486 in Chinese women are similar to those in non-Asian populations.

Effect of follicular phase administration of mifepristone (RU486) on blastocyst implantation in the rhesus monkey.

In the present study our aim was to test two hypotheses: 1) inhibition of preovulatory phase progesterone action can inhibit or delay ovulation, and 2) inhibition of preovulatory phase progesterone action can inhibit postovulatory phase endometrial receptivity for blastocyst implantation. Female rhesus monkeys showing normal cycle lengths were randomly assigned to two groups: group 1 (n = 5) and group 2 (n = 7). The pretreatment cycles were monitored for ovulatory pattern and, in treatment cycles, females were allowed to cohabit with males from cycle days 6 to 28; group 1 animals received vehicle alone, and group 2 animals received mifepristone (RU486, subcutaneously), 1 mg/animal 3 consecutive cycle days (days 7, 8, and 9 for 26-day pretreatment cycle length; and days 8, 9, and 10 for 28-day pretreatment cycle length). Follicular phase mifepristone resulted in a delay of ovulation (p < 0.01) when compared with pooled data of pretreatment and treatment cycles of group 1 and pretreatment cycles of group 2. Despite delay of ovulation, there was only a 20% decrease in the incidence of pregnancy in group 2 as compared with that in group 1. However, a delay (p < 0.05) in the appearance of CG was noted in follicular phase mifepristone-treated cycles as compared with control treatment cycles. On the other hand, ovulation could not be detected in three monkeys in group 2; and, of these, two cycles were extended, but all three cycles were negative for CG. These results support earlier reports that follicular phase mifepristone can inhibit or disrupt follicular maturation, and delay ovulation. However, follicular phase mifepristone failed to inhibit implantation, because gonadal hormones, including progesterone, resume normal functions once ovulation takes place.

PIP: Available research evidence suggests that, when administered during the follicular phase, a high affinity antiprogestin such as mifepristone can act at different levels of the hypothalamus-pituitary-ovary-uterus axis, resulting in contraceptive action. The administration of mifepristone during the late follicular phase of naturally mated cycles of rhesus monkeys was used to test the hypotheses that inhibition of preovulatory phase progesterone action can: 1) inhibit or delay ovulation, and 2) inhibit postovulatory phase endometrial receptivity for blastocyst implantation. During the preovulatory period, group 1 monkeys (n = 5) were treated with 1 mL vehicle and group 2 animals (n = 7) received mifepristone for 3 consecutive days. Peripheral hormone profiles suggested no ovulation occurred in 3 of 8 mifepristone-treated cycles. Follicular phase mifepristone treatment was associated with a delay of ovulation in 4 of 8 treated cycles. Despite the delay of ovulation, there was only a 20% decrease in the incidence of pregnancy in group 2 compared to group 1. A delay in the appearance of chorionic gonadotropin was noted in follicular phase mifepristone-treated cycles. These findings confirm that mifepristone treatment during the late follicular phase can inhibit or delay ovulation without affecting uterine function during the luteal phase. Further studies are required using this primate model to investigate the role of progesterone and other physiological modulators on ovulation and implantation.

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.

PIP: The pregnancy termination potency of varying doses of mifepristone, onapristone, and ORG 3806--alone and in combination with the estrogenic/antiestrogenic compound anordiol--was evaluated in adult rats. The antiprogestins and anordiol alone were administered to pregnant female rats on days 7, 8, and 9 of pregnancy and the presence or absence of embryos in utero was determined on day 16. 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 100% of animals; 5 mg/kg/day of anordiol was required. Anordiol acted synergistically with all three antiprogestins. Antiprogestin doses that were either partially effective or ineffective became 100% effective when administered with a noneffective dose of anordiol. The combination of ORG 31806 (1 mg/kg/day) and anordiol (0.31 mg/kg/day) had the most potent pregnancy termination activity. The administration of antiprogestins in combination with anordiol at doses that effectively terminate pregnancy was associated with a significant, persistent reduction in serum progesterone, but no change in serum estradiol levels. The effectiveness of ORG 31806 and anordiol in terminating pregnancy should be evaluated in a non-human primate model to determine its potential clinical use.

Pharmacokinetics of mifepristone after low oral doses.

Relatively low doses of the antiprogestin mifepristone (RU 486) have recently proven to be efficient for a variety of possible clinical uses of the drug. However, the pharmacokinetics after low single oral doses have not been characterized. We evaluated the pharmacokinetics of mifepristone following single ingestion of 2 and 25 mg in five women as well as repeated ingestion of 8 mg in two women. Maximal serum concentrations were reached rapidly (within 0.5-2 h) with all doses used. Serum mifepristone concentrations were proportional to the oral doses taken. The mean (+/- SD) areas under the concentration curves (AUCs) (0-24 h) were 1134 (+/- 144), 4846 (+/- 64), and 17,015 (+/- 4,421) h x ng/mL following 2, 8, and 25 mg doses, respectively. No cumulative increases in serum concentrations were detected with prolonged daily administration of 8 mg of mifepristone. The study subjects appeared to vary in their ability to metabolize mifepristone, as two different half-lives (t1/2) emerged after both 2 and 25 mg single doses (24.2 +/- 0.6 [SD] h for three subjects; and 44.4 +/- 1.8 [SD] h for two subjects). We conclude that within the dose range of 2-25 mg/day, the pharmacokinetics of mifepristone are linear, unlike those seen following ingestion of higher daily doses. Keeping in mind previously published data on the biological effects of low dose mifepristone administration, these data infer that certain effects of the drug, such as inhibition of ovulation, might be achieved at serum concentrations of approximately 100 ng/mL.

PIP: At the outpatient clinic of Lohja District Hospital in Lohja, Finland, clinical researchers examined the pharmacokinetics of mifepristone in 5 healthy women, 29-37 years old, receiving a single dose of 2 mg mifepristone and of 25 mg mifepristone and in 2 other women receiving 8 mg mifepristone each day for 30 days. Regardless of the dose, serum concentrations of mifepristone peaked within 1.2-1.4 hours. These concentrations were proportional to the oral doses: 104-227 ng/ml after 2 mg dose; 474-561 ng/ml after 8 mg dose; and 1285-4851 ng/ml after 25 mg dose. The areas under the concentration curves (0-24 hours) were also proportional to the oral doses: 1134.4, 4846, and 17,015.2, respectively. Daily doses of 8 mg mifepristone over 30 days did not effect cumulative increases in serum concentrations. The women taking the 2 mg and 25 mg single oral doses exhibited different half-lives (24.2 [3 women] vs. 44.4 [2 women] hours; p = 0.001), suggesting that they varied in their ability to metabolize mifepristone. These findings show that, at daily ingestion of 2-25 mg mifepristone, the pharmacokinetics of mifepristone are linear. Based on these findings, the authors think that inhibition of ovulation might be achieved at serum concentrations of about 100 ng/ml.

Pharmacokinetic study of RU 486 and its metabolites after oral administration of single doses to pregnant and non-pregnant women.

RU 486 and three of its metabolites (RU 42633-monodemethyl, RU 42848-didemethyl, and RU 42698-hydroxymetabolite) were determined by HPLC in plasma from nine non-pregnant and 36 pregnant women. Each non-pregnant subject took an oral dose of RU 486 (25, 100, 400 and 600 mg consecutively) once per menstrual cycle. Six of the nine women also received a dose of 200 mg. The 36 pregnant women were randomized into four groups which were given a single dose of 25, 100, 400 or 600 mg RU 486. Blood samples were taken up to 120 h after dosing. Peak concentrations of RU 486 occurred on most occasions within 2 h. Plasma concentrations at 1 h and at 24 h increased in proportion to log dose. There was a wide variability (up to ten-fold) in the pharmacokinetic parameters within each dose group. Plasma concentrations of RU 42633 were similar to those of RU 486 but concentrations of RU 42848 and RU 42698 were much lower. As with RU 486, the plasma concentrations of the metabolites were maintained at high levels for up to 48-72 h after dosing. The findings were consistent with a rapid metabolism of RU 486 to RU 42633; removal of the second methyl group leading to RU 42698 occurred much more slowly and to a much less extent than removal of the first. There appeared to be no significant differences between the non-pregnant and pregnant women in either the plasma concentrations or pharmacokinetic parameters of RU 486 and its metabolites.

Fertility impairment after mifepristone treatment to rats at proestrus. Actions on the hypothalamic-hypophyseal-ovarian axis.

Accumulated evidence indicates that the antigestagen mifepristone affects the reproductive axis acting on hypothalamic, pituitary, ovarian, and uterine tissues. The purpose of this study was to further investigate which reproductive functions are impaired by the antagonist, critically compromising the reproductive process, leading to unsuccessful pregnancy. Circulating pituitary and ovarian hormones, sexual receptivity, ovulation, and implantation rates were studied in cycling rats receiving a single dose of mifepristone (1 or 10 mg/kg subcutaneously) at 12:00 proestrus, before luteinizing hormone (LH) stimulation of the ovulatory process. Mifepristone-treated rats had decreased preovulatory surges of LH and prolactin (PRL), and hypersecretion of LH, PRL, and progesterone at estrus. The sexual receptivity was dramatically affected by the antagonist as indicated by the profound decrease in the lordosis response evaluated on the night of proestrus. The number of ovulating animals and the number of oocytes recovered from the oviduct on the morning of estrus were not affected by mifepristone. The low number of rats that succeeded in mating with potent males became pregnant. However, they delivered an average of only two pups at parturition, indicating a failure in the implantation of the fertilized ova, as ovulation was not affected by the antagonist at the dose used. We conclude that a dramatic inhibition of the sexual receptivity and unsuccessful implantation, preceded by a reduction on LH and PRL secretion, are the major components leading to fertility impairment after a single dose of mifepristone administered before the preovulatory surge of LH.

PIP: Mifepristone has been demonstrated to act on hypothalamic, pituitary, ovarian, and uterine tissue. To further investigate impairments in reproductive function triggered by this antagonist, circulating pituitary and ovarian hormones, sexual receptivity, ovulation, and implantation rates were studied in cycling Wistar rats receiving a single dose (1 or 10 mg/kg subcutaneously) of mifepristone at 12:00 proestrus, before luteinizing hormone (LH) stimulation of the ovulatory process. Treated rats had decreased preovulatory LH and prolactin (PRL) surges and hypersecretion of LH, PRL, and progesterone as estrus. A profound decrease in the lordosis response on the night of proestrus indicated a dramatic effect on sexual receptivity. There was no affect on the number of ovulating animals and the number of oocytes recovered from the oviduct on the morning of estrus. The few rats who succeeded in mating with potent males became pregnant, but they delivered an average of only two pups, indicating a failure in the implantation of the fertilized ova. These findings suggest that the dramatic inhibition of sexual receptivity and unsuccessful implantation, preceded by a reduction in LH and PRL secretion, are the major factors producing fertility impairment after a single dose of mifepristone before the preovulatory LH surge. factors such as smoking and parity.

Interaction of RU 486, a glucocorticoid and progestin antagonist with human circulating mononuclear leukocytes.

The glucocorticoid and progestin antagonist, RU 486 (17 beta-hydroxy-11 beta-[4-dimethylaminophenyl]-17 alpha-[1-propynyl]-estra-4, 9-dien-3-one), antagonizes the suppressive effect of dexamethasone on [14C]2-deoxyglucose uptake by intact human mononuclear leukocytes in a concentration-dependent fashion. The authors found at least two types of specific-binding sites for this compound in the mononuclear leukocytes. The first type of sites (receptor content [Ro], 10.8 +/- 1.6 fmoles/10(6) cells [mean +/- SD of 4 experiments]; equilibrium dissociation constant (Kd), 0.3 +/- 0.1 nM) have a capacity similar to that of the dexamethasone binding site (Ro, 11.2 fmoles/10(6) cells; Kd, 1.2 nM). The second type of sites (Ro, 56 +/- 27 fmoles/10(6) cells: Kd, 19 +/- 5 nM) have a higher capacity and lower affinity for RU 486 than the first type of sites and do not interact with dexamethasone. The authors were unable to demonstrate the presence of the second type of binding sites in subcellular fractions. This finding suggests that this site is unstable and lost in the fractionation process. The role of the second type of low-affinity, high-capacity RU 486 specific-binding site in intact human mononuclear leukocytes, as well as its possible occurrence in other tissue, requires further investigation.

Mifepristone (RU 486).

PIP: Clinical trials in Europe have found that a single oral dose of RU-486, followed by prostaglandin administration, effectively and safely terminates early pregnancy in about 95% of women. In a multicenter study carried out in France, 1 600-mg dose of RU-486, followed 36-48 hours later by intramuscular sulprostone or intravaginal gemeprost, induced complete abortion in 96% of 2000 women with up to 49 days of amenorrhea. In a British multicenter study, the combination of RU-486 and intravaginal gemeprost induced abortion in 95% of 600 women with up to 63 days of amenorrhea. RU-486 is not effective in aborting ectopic pregnancies. Bleeding usually starts 1-2 days after treatment and lasts for 1-2 weeks. About 1% of patients have required curettage since the amount of blood loss exceeds that in a normal menstrual period. The other side effects recorded with any frequency--abdominal cramping, nausea, vomiting, diarrhea, and headache--are all transitory and mild. Only 1 case of myocardial infarction has been reported in the more than 20,000 RU-486 acceptors studied. Endometritis and salpingitis are extremely rare complications. There have been no maternal deaths, and no fetal abnormalities have been observed in women who took RU-486 alone early in pregnancy and failed to abort.^ieng

[RU-486: clinical application in gynecology].

PIP: Antiprogesterone preparations such as gestrinone, anordin, and ORF 3971 were ineffective and dangerous in preventing the implantation of fertilized egg to prevent pregnancy. Trilostane and epostane, inhibitors of 3-beta-hydroxysteroid dehydrogenase, have not been approved in clinical practice. RU-486 is a synthetic 19-norsteroid 1st reported in 1982 with antiprogesterone and antiglucocorticoid activity. Its use in 7-8 weeks pre gnancy caused detachment of the placenta by competitive inhibition of progesterone receptors in the decidua. The resultant increased prostaglandin levels induced uterine contractions expulsing the conceptus. Administration during 22-25 days of menstruation caused the inhibition of the luteal body. RU-486 decreased the gonadotropin level in 3 healthy women on the 22nd day of menstruation irrespective of dosage. The reduction of luteinizing hormone (LH) secretion can be prevented by treatment with RU-486. The administration of 10 ngm/kg of RU-486 to monkeys resulted in significant elevation of ACTH (adrenocorticotropic hormone) and cortisol blood levels for 4 hours. The human dosage is 200 mg/day. 100 mg/day in women treated did not induce elevated ACTH, cortisol, or aldosterone blood levels. An 1982 experiment indicated a dose of 200 mg/day for 4 days for terminating a pregnancy of 6-10 weeks or a single dose of 400-600 mg. In 2nd trimester pregnancy RU-486 can be used for termination of fetal abnormality. It could also aid in term delivery by its ability to dilate the cervix. Early treatment in tubal (ectopic) pregnancy allowed removal of the conceptus without endangering a later pregnancy. As a post-coital agent administration of 5 mg/kg of RU-486 was effective in destroying the ovulated follicle. In breast cancer treatment RU-486 administration induced a positive response in 20% of patients.^ieng

Intravaginal administration of RU 486 in humans and rats: inadequate absorption in humans.

Vaginal absorption of the antiprogesterone steroid RU 486 was studied in humans and rats. In rats, RU 486 was sufficiently absorbed to terminate early pregnancy following vaginal, oral or intramuscular administration. The quantitation of RU 486 in serum showed that the ratios of the areas under the concentration curves per mg administered were 1:2.8:3.4 for the vaginal, intramuscular and oral routes, respectively. Female volunteers received RU 486 vaginally in polyethylene glycol (PEG) suppositories, in tampons and in oil solution. Following vaginal (PEG-suppository) and oral administration of 100 mg of RU 486, the ratio of the areas under the serum concentration curves was 1:56, respectively. From tampons and oil, RU 486 was absorbed in a similar manner to that of the PEG-suppositories, resulting in nanomolar serum concentrations. In humans no biological effects were noted following vaginal administration of RU 486. These data suggest that vaginal release of RU 486 is not a successful route of administration in humans.

[Application of anti-progesterone agents for contraception].

PIP: RU-486, powerful anti-progesterone agent, has proven to be effective as a contraceptive and as an abortifacient. It is more appropriately labeled as a contragestational pill. It functions both as anti-progesterone and anti-glucocorticoid and also as anti-androgen to some extent. RU-486 inhibits formation of deciduoma in rats, blocks uteroglobulin gene expression (mRNA) in rabbits, induces menstruation and induces miscarriages in rats, mice, monkey and humans. RU-486 inhibits endometria production of glycogen by progesterone and antagonistically blocks progesterone's growth inhibitor of endometria cells. It is effective for the treatment of adrenal cortical hyperplasia and Cushing's syndrome related to hypernephroma but not Cushing's syndrome related to hypophysis. RU-486 does not impair gonadotropin. 1 oral dose of RU-486 10 mg/kg in midluteal phase induced in 18 women menses-like bleeding which lasted 4 days according to Nieman, who regards RU-486 as a potential contraceptive agent.^ieng

The antiprogesterone steroid, RU 486 (mifepristone).

RU 486 (Mifepristone) represents a major development in the field of hormone antagonists as the first effective antiprogestogen. A number of therapeutic roles for the drug are envisaged. It is already being used extensively around the world for the procurement of first trimester abortion--particularly in combination with prostaglandins. It also has been shown to be effective as a cervical ripening agent and for the induction of labour. Initial human studies have involved nonviable pregnancies and more work is needed in animal models before the drug can be deemed safe for use in viable human pregnancies in the third trimester.

PIP: RU 486, developed from norethisterone by Roussel Manufacturers, represents the first effective antiprogestogen. Although RU 486 binds strongly to the progesterone receptor, it inhibits progestogenic effects, making this a receptor blocking drug. When administered in the late follicular phase of the menstrual cycle, RU 486 has been shown to delay ovulation. In the first half of the luteal phase, RU 486 destroys the corpus luteum. If luteolysis occurs, RU 486 administration is followed by a single bleeding episode; in the absence of luteolysis, a second bleeding episode occurs several days later in conjunction with the disappearance of the corpus luteum. No clinical use has been found for RU 486's ability to delay ovulation, and luteal phase administration of RU 486 is not yet an effective means of menstrual induction. RU 486 has been most successfully used to induce early abortion, either alone or in combination with a prostaglandin. In most recent study of RU 486, involving 75 women, complete abortion was achieved in 95% of cases. Animal studies have found RU 486 to be effective as a cervical ripening agent and in the induction of labor. They further suggest that the drug can accelerate lactation, prevent breast cancer proliferation, minimize the need for salpingectomy in ectopic pregnancy, and treat Cushing syndrome and glaucoma. To date, there has been no evidence of any teratogenic or antiglucocorticoid side effects. However, more research is needed to confirm the safety of RU 486 for use in viable human pregnancies.