Unilateral ovariectomy, flutamide treatment and HCG reverse the anovulatory action of antiprogesterone RU486 in rat.

Administration of antiprogesterone RU486 (4 mg/day) from estrus through proestrus to cyclic rats blocked ovulation. Moreover, RU486 increased basal serum concentrations of LH, PRL, testosterone and estradiol, while it decreased basal serum concentration of FSH. Both unilateral ovariectomy and antiandrogen flutamide treatment, as well as an ovulatory injection of HCG in the proestrus afternoon partially reversed, the ovulatory blockade of RU486. These results indicate that both the decreased FSH concentration and the increased testosterone concentration, as well as the reduced ovulatory LH release are responsible for the anovulatory effects of RU486.     

Control of birth in rats by RU 486, an antiprogesterone compound

Rats, isolated at mating (Day 1 of pregnancy), were submitted to either 8 h (8L:16D, Exp. I) or 14 h (14L:10D, Exp. II) of light daily with lights on from 12:00 h to 20:00 h and from 06:00 to 20:00 h respectively. In Exp. I, a single dose of RU 486 (10 mg in 0.2 ml ethanol) was given cutaneously at 08:00 h (Group A1), 12:00 h (Group B1), 19:00 h (Group C1) on Day 21 and at 08:00 h (Group D1) and 12:00 h (Group E1) on Day 22. In Exp. II, the same dose of RU 486 was given at 08:00 h (Group A2), 12:00 h (Group B2) and 19:00 h (Group C2) on Day 21. The solvent was given once at each of the preceding times to the control groups (T1 and T2) in both experiments. Groups T1 and T2 gave birth at two periods, the first on Day 22, the second on Day 23; the proportion of births during each of these periods depended on the light regimen (66.3% in 8L:16D; 50% in 14L:10D on Day 22). The distribution of births in Groups D1 and E1 treated on Day 22 were similar to their controls (T1). Rats treated on Day 21 (Groups A1, A2, B1, B2, C1, C2) gave birth over single periods on Day 22 after an interval correlated with the time of RU 486 administration. The earlier the treatment was given, the higher was the number of dead young and the lower the weight of live young 1 day after birth. These effects of prematurity did not impair further survival rates or weight at weaning.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of action of an antiprogesterone, RU486, in the rabbit endometrium. Effects of RU486 on the progesterone receptor and on the expression of the uteroglobin gene

RU486 is a recently described antiprogesterone. In order to be able to understand its mechanism of action it is necessary to analyze its effect on a discrete gene product. We show here that the induction of uteroglobin mRNA by progesterone in the rabbit endometrium may be a suitable model for such studies since RU486 totally inhibits this effect without itself exerting any agonistic activity. Moreover, RU486, which does not bind to the estrogen receptor and is devoid of general antiestrogenic activity, partially inhibits the induction by estradiol of uteroglobin mRNA. Studies of the interaction between [3H]RU486 and the progesterone receptor have been undertaken with the aim of understanding the antagonistic effect of this compound. The binding to DNA-cellulose of heat-activated [3H]RU486-receptor complexes was slightly decreased (37%) when compared with that of the agonist [3H]R5020-receptor complexes (47%). Detailed analysis of this difference showed that it was due to both a decreased activation of complexes and to a diminished affinity of activated complexes towards DNA. The change in activation was shown by the fact that at high concentrations of DNA, where all activated complexes are bound, agonist-receptor complexes were bound to DNA in higher proportion than antagonist-receptor complexes. Moreover a difference was also observed when studying the binding of agonist-receptor and antagonist-receptor complexes to charged resins (phosphocellulose, DEAE-cellulose) which are known to discriminate between activated and non-activated complexes. Decreased affinity to DNA of antagonist-receptor complexes was shown by studying their binding at various concentrations of DNA, either in crude cytosol or after isolating a homogenous population of activated-receptor complexes by DNA-cellulose chromatography and by comparing the salt extraction from DNA-cellulose of agonist-receptor and antagonist-receptor complexes. Both effects (decreased activation and diminished affinity towards DNA) were relatively moderate and could account only for a small decrease in the agonistic activity of RU486. Thus, the fact that this compound is a complete antagonist without any agonistic activity can only be explained by a defect in some further step of hormone action as, for instance in the specific interaction with the regulatory regions of the uteroglobin gene. No immunological difference could be detected between [3H]R5020-receptor and [3H]RU486-receptor complexes, both interacted with the five monoclonal antibodies raised against purified R5020-receptor complexes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Normal puberty onset in female rats treated with antiprogesterone RU486 during juvenile period.

The significance of the increased progesterone plasma levels after day 21 on the onset of puberty in female rats is unclear. To analyze this question, female rats were injected from day 21 to 32 with the potent antiprogestagen RU486 (1 mg/day) or vehicle. Treatment with RU486 did not modify either the age at which vaginal opening or first estrus occurs. Vaginal cycles were regular. Animals sacrificed on day 40 or 50 showed reduced uterus weight, whereas body, pituitary, adrenal and ovaries were not affected. These results evidence that antagonization of progesterone action during the juvenile phase did not affect the onset of puberty.     

Progesterone-like relaxant effect of RU 486 in the rat myometrium

The antihormone RU 486 is characterized by its antiprogesterone and antiglucocorticoid activities. In this work the likelihood of a non-genomic effect for this compound was assessed. Thus, RU 486 was compared with progesterone and the 5β-progestin pregnanolone, for its ability to modify the uterine contractility of the rat. An outstanding relaxant effect elicited by RU 486 was observed, slightly higher than that produced by progesterone but lower than pregnanolone. Moreover, calcium promoted contractions were antagonized by RU 486, in the same way as the endogenous steroids. The data suggest the capability of RU 486 to block the calcium channels. It is concluded that a non-genomic effect of RU 486 is produced before its journey into the cell for its genomic action.     

A possible mechanism for the anti-ketogenic action of alanine in the rat

1. The anti-ketogenic effect of alanine has been studied in normal starved and diabetic rats by infusing l-alanine for 90min in the presence of somatostatin (10μg/kg body wt. per h) to suppress endogenous insulin and glucagon secretion. 2. Infusion of alanine at 3mmol/kg body wt. per h caused a 70±11% decrease in [3-hydroxybutyrate] and a 58±9% decrease in [acetoacetate] in 48h-starved rats. [Glucose] and [lactate] increased, but [non-esterified fatty acid], [glycerol] and [3-hydroxybutyrate]/[acetoacetate] were unchanged. 3. Infusion of alanine at 1mmol/kg body wt. per h caused similar decreases in [ketone body] (3-hydroxybutyrate plus acetoacetate) in 24h-starved normal and diabetic rats, but no change in other blood metabolites. 4. Alanine [3mmol/kg body wt. per h] caused a 72±9% decrease in the rate of production of ketone bodies and a 57±8% decrease in disappearance rate as assessed by [3-¹⁴C]acetoacetate infusion. Metabolic clearance was unchanged, indicating that the primary effect of alanine was inhibition of hepatic ketogenesis. 5. Aspartate infusion at 6mmol/kg body wt. per h had similar effects on blood ketone-body concentrations in 48h-starved rats. 6. Alanine (3mmol/kg body wt. per h) caused marked increases in hepatic glutamate, aspartate, malate, lactate and citrate, phosphoenolpyruvate, 2-phosphoglycerate and glucose concentrations and highly significant decreases in [3-hydroxybutyrate] and [acetoacetate]. Calculated [oxaloacetate] was increased 75%. 7. Similar changes in hepatic [malate], [aspartate] and [ketone bodies] were found after infusion of 6mmol of aspartate/kg body wt. per h. 8. It is suggested that the anti-ketogenic effect of alanine is secondary to an increase in hepatic oxaloacetate and hence citrate formation with decreased availability of acetyl-CoA for ketogenesis. The reciprocal negative-feedback cycle of alanine and ketone bodies forms an important non-hormonal regulatory system.     

The antiprogesterone RU486 stabilizes the heterooligomeric, non-DNA-binding, 8S-form of the rabbit uterus cytosol progesterone receptor

The salt-induced (0.3 M KCl) transformation of the non-transformed, heterooligomeric 8S-form of the rabbit uterus cytosol progesterone receptor (PR) was analyzed by density gradient ultracentrifugation (8S----4S conversion) and DNA-cellulose chromatography (non-binding----binding forms). After 1 h treatment at 2 C, greater than 90% of agonist (R5020 or Org2058)-PR complexes were transformed, contrary to antagonist (RU486)-PR complexes, which did not undergo any transformation. Thus, there is stabilization of the non-transformed receptor form by RU486 as compared to the effect of agonist binding. The hydrodynamic parameters of both agonist- and antagonist-bound non-transformed receptors were similar and the calculated Mr were approximately 283,000 and approximately 293,000, respectively. In both cases, purification indicated the presence of a 90-kD non-hormone-binding protein associated with the hormone binding unit(s). Transformation of RU486-PR complexes occurred after exposure to high salt at increased temperature and was correlated to the dissociation of the 90-kD protein from the receptor. Both agonist- and antagonist-bound transformed forms of PR had apparent similar affinities for DNA-cellulose. Molybdate-stabilized and KCl-treated RU486-PR complexes were more stable, as assessed by steroid binding, than the corresponding R5020-PR complexes, arguing in favor of a stabilizing effect of both the 90-kD protein and RU486 against inactivation. These cell-free experiments support the concept that RU486 in the rabbit uterus system stabilizes the 8S non-DNA binding, non-transformed form of the receptor at low temperature. The possibility that impaired dissociation of the heterooligomeric receptor form is involved in the antiprogesterone activity of RU486 is discussed.     

Effect of RU 486 on luteal function in the early pregnant rat

A dose of 30 mg RU 486/kg, an antiprogesterone, was administered to pregnant rats on Day 2 (Group 1) or Day 4 (Group 2) of pregnancy. RU 486 significantly changed serum progesterone and oestradiol concentrations and luteal 3 beta-HSD and 20 alpha-HSD activities in Group 1, and implantation was significantly inhibited. The luteal 3 beta-HSD activity in Group 2 rats on Day 6 was significantly (P less than 0.01) lower than the control value (7.5 +/- 0.6 and 10.1 +/- 0.6 mU/mg protein respectively). This decline in the 3 beta-HSD activity was followed by a marked decrease in the serum progesterone concentration, resulting in a significant decrease of the progesterone/oestradiol ratio and implantation was completely inhibited. The 20 alpha-HSD activity, which could not be detected on Day 6 in the control rats, was twice as great in Group 2 than in Group 1 rats (17.5 +/- 1.2 and 7.4 +/- 3.1 mU/mg protein respectively). Ultrastructural examination of corpora lutea of Group 2 rats confirmed luteolysis. These results suggest that RU 486 has a luteolytic effect and its anti-implantation effect is concomitant with luteolysis of the corpora lutea of pregnancy.     

Differences in the binding mechanism of RU486 and progesterone to the progesterone receptor

The binding mechanism of the antagonist RU486 to the progesterone receptor was compared with that of the agonists progesterone and R5020. Both progesterone and RU486 bound to the receptor with a Hill coefficient of 1.2, indicating the binding of each ligand is positive cooperative. However, when each ligand was used to compete with [3H]progesterone for binding to the receptor at receptor concentrations near 8 nM, at which the receptor is likely a dimer, the competition curve for RU486 was significantly steeper than the curves for progesterone and R5020 (p less than 0.001). This indicated that a difference in the binding mechanism of RU486 and progesterone can be detected when both ligands are present. In contrast, at receptor concentrations near 1 nM, at which the receptor is likely a monomer, the competition curves for all three ligands were indistinguishable (p = 0.915). These results indicate that RU486 and agonists have different binding mechanisms for the receptor and further suggest that this difference may be related to site-site interactions within the receptor.     

RU 486 completely inhibits the action of progesterone on cell proliferation in the mouse uterus

The antiprogestagen RU 486 completely inhibited the progesterone-induced switch in cell proliferation from the luminal and glandular epithelia to the stroma in response to oestradiol-17 beta. It also inhibited the progesterone-induced differentiation of the uterine epithelium. Since the proliferative switch of the uterus and the differentiation of the epithelium are prerequisites for implantation, these inhibitory actions may, in part, explain the ability of RU 486 to prevent implantation. Furthermore, it also suggests that the proliferative response to oestradiol in the presence of progesterone may be a sensitive assay for compounds with anti-progestational activity.     

Effect of RU486 on development and implantation of rat embryos

This study evaluated the effects of postcoital treatment with the antiprogestin RU486 on transport, development and implantation of rat embryos. Doses of 0.1, 0.5, 1.0, 2.0, or 3.0 mg/rat of RU486 were injected subcutaneously on days 1, 1 + 2, or 4 of pregnancy. Autopsies were carried out on days 5 or 12 of pregnancy. RU486 provoked a significant dose-related reduction in the number of recovered embryos and inhibited their development (day 5) and decreased the number and size of implanted embryos (day 12). Treatment on day 4 was the least effective. Blastocysts recovered from RU486-treated rats exhibited comparable rate of trophoblastic outgrowth in vitro as the controls. Blastocysts transferred from RU486-treated rats to synchronous untreated pseudopregnant recipients yielded implanted embryos 12 days later in all recipients, although at a significantly lower rate than the controls. Blastocysts transferred from control pregnant rats to RU486-treated pseudopregnant recipients failed to implant completely when the dose was greater than or equal to 1.0 mg. The interceptive mechanism of postcoital treatment with RU486 in the rat involves loss of embryos from the reproductive tract and altered development prior to implantation. Endometrial receptivity or the ability of the uterus to retain the embryos until the time of implantation are also impaired by RU486. The embryos that survive these effects may experience delayed implantation in their mothers.     

Pharmacokinetics and metabolism of RU 486

The effects of dose on the initial pharmacokinetics and metabolism of an antiprogesterone steroid RU 486 (mifepristone) were studied in healthy female volunteers after administration of RU 486 as a single dose of 50-800 mg. The concentrations of RU 486 and its monodemethylated, dimethylated and hydroxylated non-demethylated metabolites were measured specifically after Chromosorb-column chromatography by HPLC. Their relative binding affinities to the human uterine progesterone receptor were also determined. Micromolar concentrations of the parent compound in blood were reached within the first hour after oral administration. The pharmacokinetics of RU 486 followed two distinct patterns in a dose-dependent fashion. With a low dose of 50 mg the pharmacokinetics followed an open two-compartment model with a half-life of over 27 h. With the doses of 100-800 mg the initial redistribution phase of 6-10 h was followed by zero-order kinetics up to 24 h or more. Importantly, after ingestion of doses higher than 100 mg of RU 486 there were no significant differences in plasma concentrations of RU 486 within the first 48 h, with the exception of plasma RU 486 concentrations at 2 h. After single oral administration of 200 mg unchanged RU 486 was found 10 days later in two subjects. The elimination phase half-life with this dose, calculated between day 5 and 6, was 24 h. Micromolar concentrations of monodemethylated, didemethylated and non-demethylated hydroxylated metabolites were measured within 1 h after oral administration of RU 486. In contrast to plasma RU 486 concentrations, circulating plasma concentrations of metabolites increased in a dose-dependent fashion. With higher doses the metabolite concentrations were close to, or even in excess to the parent compound. The relative binding affinities of RU 486, monodemethylated, didemethylated and hydroxylated metabolites (progesterone = 100%) to the human progesterone receptor were 232, 50, 21, and 36, respectively. The existence of a high affinity-limited capacity serum binding protein would explain the long half-life and the observed diverging dose-dependent pharmacokinetics. The extravasation of RU 486 after the saturation of serum binding sites would explain the blunted serum peak concentrations of RU 486 with higher doses. The return of the drug back to circulation thereafter explains the zero-order kinetics. High concentrations of circulating metabolites capable of binding to the progesterone receptor suggest a significant contribution of these steroids in the overall antiprogestational action.     

Antiprogestagen RU486 prevents the LH-dependent decrease in the serum concentrations of inhibin in the rat

Summary 1. In the rat, the LH-dependent ovarian progesterone rise mediates several actions of the primary surge of LH on the ovary. This experiment was aimed at elucidating the effects of the antiprogestagen RU486 on the LH-dependent decrease in both the serum concentrations and the ovarian content of inhibin.2. All rats in this experiment were treated with an antagonist of LHRH (1 mg/200 µl saline at 0800 h in proestrus) to supress the endogenous release of LH. One group of rats received 32 µg LH/250 µl saline at 1200 h in proestrus. Other group was given 4 mg RU486/200 µl oil at 0800 h in proestrus. The third group was injected with both RU486 and LH. Rats from the control group were injected with 250 µl saline and 200 µl oil. Animals were decapitated at 1700 h in proestrus and trunk blood and ovaries collected to determine the serum concentrations of LH, FSH, progesterone, 17ß-estradiol and inhibin as well as the ovarian content of inhibin.3. The ovulatory dose of LH in LHRHa-treated rats decreased both the serum concentrations and the ovarian content of inhibin and increased the serum concentrations of FSH. The administration of RU486 blocked the effect of LH on the serum concentrations of inhibin but not that on the ovarian content of inhibin.4. Since the antiprogestagen RU486 blocked the effect of LH on the serum concentrations of inhibin, we conclude that ovarian progesterone, besides mediating the effects of the primary LH surge on the ovulatory process and luteinization, participates in the LH-dependent drop in the serum concentrations of inhibin in proestrous afternoon.     

Mechanism of the stimulatory action of antisteroid RU486 on follicle-stimulating hormone secretion in the cyclic Rat

These experiments explored the mechanism underlying FSH hypersecretion on estrous afternoon in rats injected with RU486 (RU) on proestrus. Four-day cyclic rats were injected with RU at 12:00 h on proestrus (1 or 4 mg/0.2 ml oil; s.c.), and its effects on LH and FSH secretion at 18:30 h on estrus were compared with those of antiprogestagens ZK299 (ZK) (1 or 4 mg/0.2 ml oil; s.c.) and Org31806 (OR) (2 or 8 mg/0.2 ml oil; s.c.). Additionally, rats treated with RU or nembutal (PB) (60 mg/kg; i.p. at 13:00 h on proestrus) were injected with an LHRH antagonist (LHRHa) at 10:00 h on estrus (1 mg/0.2 ml saline; s.c.) or progesterone (P) (7.7, 15.5 or 30.9 mg/0.2 ml oil; s.c.) on proestrus at 10:00 h in RU-injected rats and at 14:00 h in PB-injected rats. Animals were killed by decapitation at 18:30 h on estrus and serum LH and FSH concentrations were determined. Rats treated with 1 or 4 mg of RU or Org or 4 mg of ZK recorded increased serum FSH on estrous afternoon, while 1 mg ZK had no effect. PB increased mainly serum LH levels and, to a lesser extent, FSH levels. P decreased serum FSH concentrations in both RU- and PB-injected rats. LHRHa reversed the effects of PB on FSH secretions, but reduced FSH hypersecretion induced by RU only. These results are interpreted to mean that, in the absence of proestrous afternoon P-inhibitory action of the neural stimulus controlling LHRH release, FSH secretion on estrous afternoon involves two components: one is LHRH dependent while, in contrast to LH secretion, the other is LHRH independent, and only expressed in a low estrogen background.     

RU486 is not an antiprogestin in the hamster

The biological activity and progestin receptor binding activity of the synthetic steroid RU486 (RU38486; 17-beta-hydroxy-11-beta-(4-dimethylaminophenyl)-17-alpha-(1-propynl++ +)- estra-4,9-diene-3-one) were investigated in the hamster. RU486 demonstrated no antiprogestational activity in the female hamster in that it was ineffective in blocking decidualization or interrupting early pregnancy. Competitive binding assays showed RU486 did not compete from hamster uterine progestin receptor. It is concluded that hamster uterine progestin receptor has unique steroid binding specificity.