Effect of mifepristone on COX-2 both in eutopic and ectopic endometrium in mouse endometriotic model.

OBJECTIVE: To study the influence of mifepristone on the expression of cyclooxygenase 2 (COX-2) protein and COX-2 mRNA and then to evaluate the mechanism. METHODS: After the establishment of 30 mice endometriosis models, the mice were randomly divided into six groups with 5 mice each group and assigned to experimental and control groups of 1-, 4- and 6-week circle according to whether mifepristone (0.13 mg d(-1)) was taken or not. Small animal optical imaging system was used to detect the fluorescent intensity of the ectopic tissue. Reverse transcript-polymerase chain reaction and western blot was used to examine COX-2 protein and COX-2 mRNA expression. ELISA was used to examine concentration of PGE(2) in serum. RESULT(S): Mifepristone could not affect the fluorescent intensity of the ectopic endometrium after it was taken 1, 4, and 6 (P > 0.05). However, it could decrease the transcription of COX-2 mRNA in the 1 and 4 week groups (P < 0.05), while the difference in the 6 week group was not significant (P > 0.05). It could decrease the expression of COX-2 protein after it was taken 4 and 6 weeks (P < 0.05). The serous PGE(2) in the trial groups was lower than that in the control groups, but the difference was not significant (P > 0.05). CONCLUSION(S): This study showed that mifepristone could not affect the size of the ectopic endometrium, but it could decrease the transcription of COX-2 gene and then reduce the expression of COX-2 protein and its product PGE(2) which is an important factor which mediate pain. This maybe another mechanism that mifepristone takes effect through anti-inflammatory path.

Endometrial shedding effect on conception and live birth in women with polycystic ovary syndrome.

OBJECTIVE: To estimate whether progestin-induced endometrial shedding, before ovulation induction with clomiphene citrate, metformin, or a combination of both, affects ovulation, conception, and live birth rates in women with polycystic ovary syndrome (PCOS). METHODS: A secondary analysis of the data from 626 women with PCOS from the Eunice Kennedy Shriver National Institute of Child Health and Human Development Cooperative Reproductive Medicine Network trial was performed. Women had been randomized to up to six cycles of clomiphene citrate alone, metformin alone, or clomiphene citrate plus metformin. Women were assessed for occurrence of ovulation, conception, and live birth in relation to prior bleeding episodes (after either ovulation or exogenous progestin-induced withdrawal bleed). RESULTS: Although ovulation rates were higher in cycles preceded by spontaneous endometrial shedding than after anovulatory cycles (with or without prior progestin withdrawal), both conception and live birth rates were significantly higher after anovulatory cycles without progestin-induced withdrawal bleeding (live births per cycle: spontaneous menses 2.2%; anovulatory with progestin withdrawal 1.6%; anovulatory without progestin withdrawal 5.3%; P<.001). The difference was more marked when rate was calculated per ovulation (live births per ovulation: spontaneous menses 3.0%; anovulatory with progestin withdrawal 5.4%; anovulatory without progestin withdrawal 19.7%; P<.001). CONCLUSION: Conception and live birth rates are lower in women with PCOS after a spontaneous menses or progestin-induced withdrawal bleeding as compared with anovulatory cycles without progestin withdrawal. The common clinical practice of inducing endometrial shedding with progestin before ovarian stimulation may have an adverse effect on rates of conception and live birth in anovulatory women with PCOS. LEVEL OF EVIDENCE: II.

Induction of overt menstruation in intact mice.

The complex tissue remodeling process of menstruation is experienced by humans and some primates, whereas most placental mammals, including mice, go through an estrous cycle. How menstruation and the underlying mechanisms evolved is still unknown. Here we demonstrate that the process of menstruation is not just species-specific but also depends on factors which can be induced experimentally. In intact female mice endogenous progesterone levels were raised by the induction of pseudopregnancy. Following an intrauterine oil injection, the decidualization of the endometrium was reliably induced as a prerequisite for menstruation. The natural drop of endogenous progesterone led to spontaneous breakdown of endometrial tissue within an average of 3 days post induction of decidualization. Interestingly, morphological changes such as breakdown and repair of the endometrial layer occurred in parallel in the same uterine horn. Most importantly, endometrial breakdown was accompanied by vaginally visible (overt) bleeding and flushing out of shed tissue comparable to human menstruation. Real-time PCR data clearly showed temporal changes in the expression of multiple factors participating in inflammation, angiogenesis, tissue modulation, proliferation, and apoptosis, as has been described for human menstruating endometrium. In conclusion, human menstruation can be mimicked in terms of extravaginally visible bleeding, tissue remodeling, and gene regulation in naturally non-menstruating species such as intact female mice without the need for an exogenous hormone supply.

Progesterone, but not 17-alpha-hydroxyprogesterone caproate, inhibits human myometrial contractions.

OBJECTIVE: The aim was to determine whether progesterone (P4) or 17-alpha-hydroxyprogesterone caproate (17P) directly inhibit human uterine contractility in vitro and thereby clarify their mechanisms of action. STUDY DESIGN: Myometrial tissues were suspended in organ chambers and exposed for 2 to 20 hours to varying concentrations of P4 or 17P or solvent. Contractile activity was registered, stored, and analyzed. Dose response curves were then generated for P4 or 17P at various times. RESULTS: P4 significantly inhibited spontaneous contractility dose dependently. The inhibition was not blocked by RU486 but was reversible after washing. Surprisingly, 17P dose dependently stimulated contractility. HPLC and GC-MS methods were used to determine the detectable concentrations of progestins in the baths. CONCLUSION: P4, at concentrations equivalent to those present in the placenta and uterus, inhibit spontaneous myometrial contractility in vitro by nongenomic mechanisms.

Menstrual-like changes in mice are provoked through the pharmacologic withdrawal of progesterone using mifepristone following induction of decidualization.

BACKGROUND: Cyclic shedding of the endometrium is unique to menstruating species, and mouse menstruation models by physiologic progesterone withdrawal have been previously reported. Since progesterone action ablated pharmacologically may provide more insight into the mechanism of action, a mouse menstruation model using mifepristone was established. METHODS: Mifepristone was administered following oil-induced decidualization in ovarectomized mice primed with hormones. Morphology, hormone levels, leukocytes and apoptosis were evaluated over a period of 48 h after treatment. Vaginal smears were used to monitor bleedings. RESULTS: Mifepristone induced menstrual-like changes. Tissue breakdown was drastic by 16 h, and the decidual zone was shed by 24 h while the mice bled. The endometrium regenerated from 24 h onwards and became completely restored by 48 h. These results are consistent with previous reports. However, although progesterone levels remained constant, estradiol levels increased after the treatment. The CD45(+) cells showed two peaks of increase at 16 h (breakdown phase) and 32 h (regeneration phase) (Leukocyte levels also increased in the unstimulated horns, but no breakdown changes occurred there). Moreover, apoptosis drastically increased by 16 h concurrent with tissue destruction. These results differed from those of the physiologic withdrawal models. CONCLUSIONS: The pharmacologic withdrawal of progesterone by mifepristone successfully provoked a menstrual-like process in mice after artificial decidualization.

Uterine stretch regulates temporal and spatial expression of fibronectin protein and its alpha 5 integrin receptor in myometrium of unilaterally pregnant rats.

The adaptive growth of the uterus during pregnancy is a critical event that involves increased synthesis of extracellular matrix (ECM) proteins and dynamic remodeling of smooth muscle cell (SMC)-ECM interactions. We have previously found a dramatic increase in the expression of the mRNAs that encode fibronectin (FN) and its alpha5-integrin receptor (ITGA5) in pregnant rat myometrium near to term. Since the myometrium at term is exposed to considerable mechanical stretching of the uterine wall by the growing fetus(es), the objective of the present study was to examine its role in the regulation of FN and ITGA5 expression at late gestation and during labor. Using myometrial tissues from unilaterally pregnant rats, we investigated the temporal changes in Itga5 gene expression in gravid and empty uterine horns by Northern blotting and real-time PCR, in combination with immunoblotting and immunofluorescence analyses of the temporal/spatial distributions of the FN and ITGA5 proteins. In addition, we studied the effects of early progesterone (P4) withdrawal on Itga5 mRNA levels and ITGA5 protein detection. At all time-points examined, the Itga5 mRNA levels were increased in the gravid uterine horn, compared to the empty horn (P < 0.05). Immunoblot analysis confirmed higher ITGA5 and FN protein levels in the myometrium, associated with gravidity (P < 0.05). Immunodetection of ITGA5 was consistently high in the longitudinal muscle layer, increased with gestational age in the circular muscle layer of the gravid horn, and remained low in the empty horn. ITGA5 and FN immunostaining in the gravid horn exhibited a continuous layer of variable thickness associated directly with the surfaces of individual SMCs. In contrast to the effects of stretch, P4 does not appear to regulate ITGA5 expression. We speculate that the reinforcement of the FN-ITGA5 interaction: 1) contributes to myometrial hypertrophy and remodeling during late pregnancy; and 2) facilitates force transduction during the contractions of labor by anchoring hypertrophied SMCs to the uterine ECM.

Hormonal regulation and localization of estrogen, progestin and androgen receptors in the endometrium of nonhuman primates: effects of progesterone receptor antagonists.

This article reviews the effects of estradiol (E(2)), progesterone (P) and P receptor antagonists (PA) on the rhesus macaque endometrium. Ovariectomized macaques can be treated with implants of estradiol (E(2)) and P to induce precisely controlled, artificial menstrual cycles. During these cycles, treatment with E(2) alone induces an artificial proliferative phase marked by extensive endometrial epithelial cell proliferation and increased expression of stromal and epithelial estrogen receptor (ER) and P receptor (PR). Androgen receptor (AR) is also upregulated by E(2) but is expressed only by the endometrial stroma. Progesterone acts on the E(2) primed endometrium to induce secretory differentiation and causes suppression of epithelial and stromal ER, epithelial PR, and stromal AR in the functionalis zone. However, epithelial ER and PR are retained in the basalis zone during the secretory phase. When potent P antagonists (PA) are administered acutely at the end of an E(2) + P induced cycle, menses typically ensues similar to P withdrawal at the end of the menstrual cycle. When PAs are administered chronically there is significant blockage of all P- dependent effects including upregulation of ER, PR and AR and suppression of glandular secretory function. However, chronic PA administration also inhibits estrogen-dependent endometrial cell proliferation and growth. This endometrial antiproliferative effect is the basis of the clinical use of PA to control various diseases such as endometriosis.

Menstrual induction with mifepristone and misoprostol.

Menstrual induction refers to early uterine evacuation without laboratory confirmation of pregnancy in women with delayed menses. Mechanical aspiration is the method used in many countries but, as suggested by a pilot study, mifepristone followed by a prostaglandin analogue could also be effective. We launched the present study to evaluate the efficacy and side effects of 150 mg of mifepristone, followed 2 days later by 0.4 mg misoprostol vaginally, for menstrual induction among women with a menstrual delay of up to 7 days. The outcome of treatment was uterine evacuation, which could mean menstruation or termination of early pregnancy. A total of 720 women were recruited. The mean delay of menstruation at recruitment was 4.9 (SD = 1.7) days. Retrospective analysis of human chorionic gonadotropin from serum samples taken at admission showed that 492 (68.3%) women were pregnant at admission, and 228 (31.7%) women had delayed menstruation without pregnancy. One nonpregnant woman was lost to follow-up. Bleeding was induced in 479 (97.4%) pregnant women and in 222 (97.8%) nonpregnant women. Among the pregnant women, 455 (92.5%) had complete abortion, 12 (2.4%) had incomplete abortion and pregnancy continued in 25 (5.1%) women, including one ectopic pregnancy. Side effects were mild and uncommon. We conclude that 95.8% of the women treated had the expected outcome. Further research is needed to compare the efficacy, safety and acceptability of the medical regimen to vacuum aspiration. A rather high continuing pregnancy rate in this study is a concern.

The possibility of using mifepristone for menstrual induction.

Menstrual induction is a variant of suction aspiration that was originally defined to be performed in women with a menstrual delay of up to 2-3 weeks without knowing if the women is pregnant or not. Prerequisites for a pharmacological method for menstrual induction are a high efficacy to induce a bleeding in nonpregnant women and an expulsion of the pregnancy in pregnant women. Treatment with prostaglandins, specifically intramuscular sulprostone, can be as effective as suction aspiration for menstrual induction. However, the administration of prostaglandin in therapeutically effective doses was associated with a high frequency of gastrointestinal side effects and, to a lesser extent, of strong abdominal pain, which limited their routine use. More recent studies indicate that mifepristone in combination with either misoprostol or gemeprost is a more promising alternative. Further studies to identify the best treatment schedule are, however, needed, as is a randomized comparison with suction aspiration before a pharmaceutical method can be recommended.

Pharmacological characterization of pre- and postsynaptic prostanoid receptors in pig gastric fundus.

This study characterized the subtype of prostanoid receptors on the cholinergic neurones and smooth muscle cells in circularly oriented muscle strips of the pig gastric fundus. Tissues were electrically stimulated (40 V, 4 Hz, 0.25 ms, 2 min) to induce tritium outflow after incubation with [3H]-choline. Indomethacin increased the electrically induced tritium outflow, suggesting an inhibitory effect of endogenous prostanoids. In the presence of indomethacin, PGE2 > PGF2alpha >PGI2 inhibited tritium release while the TP-receptor agonist U-46619 and PGD2 had no effect. The EP2-receptor agonist butaprost had no effect while the EP1- and EP3-receptor agonist sulprostone mimicked the effect of PGE2. The effect of sulprostone was not affected by AH 6809, that antagonizes EP1- and EP2-receptors, suggesting the presence of presynaptic EP3-receptors on the cholinergic nerve endings. All prostanoid receptor agonists, except butaprost, contracted the tissues concentration-dependently; the rank order of potency (U-46619 > sulprostone > PGE2 > PGF2alpha > PGD2 = PGI2) suggests the presence of TP- and EP1- and EP3-receptors on the circular smooth muscle cells.

Preclinical experience with two selective progesterone receptor modulators on breast and endometrium.

Org 31710 and Org 33628 are two highly selective progesterone receptor modulators (PRMs) with respect to their anti-progestational and anti-glucocorticoid activity. The compounds have been studied both in vitro and in vivo. Org 33628 has approximately four times stronger anti-progestational activity in vitro than does Org 31710, and in rats it is about 15 times more potent in the pregnancy interruption test. Two main indications for the use of PRMs are breast cancer and fertility regulation. The effects of both Org 31710 and Org 33628 were tested in relevant models for these indications. The effects of the two compounds on breast tumor development were assessed and in rats using the DMBA model. Their potency in menses induction was tested in monkeys on a 4-day regimen in the luteal phase, and after a single dose at day 21 of the normal cycle, and under a continuous progestin treatment using desogestrel. The compounds were also tested alone in a continuous low-dose regimen. The effects on follicular development and ovulation were determined by measuring estradiol and progesterone levels. Cycle control was monitored by daily vaginal swabs. In the DMBA model, Org 31710 at oral doses of 0.8, 2.0, and 5.0 mg/kg showed a clear dose-related reduction in tumor load. With the two highest doses, an even lower tumor load was seen after a 3-week treatment period compared to the tumor load at the start of treatment. Org 33628 showed a similar efficacy as Org 31710 at a dose of 2.0 mg/kg. RU 486 after oral treatment was two times less potent in this model than Org 31710 and Org 33628. The efficacy of menses induction using the 4-day regimen is dependent on the time of administration relative to the progesterone peak in the luteal phase. The highest efficacy is achieved in the descending part of the peak, at which a 100% success rate is found with a dose of 1 mg/kg of either Org 31710 or Org 33628. In Cynomolgus monkeys, at a single dose of 15 mg/kg of Org 31710 or Org 33628 in the luteal phase, menses induction was achieved only in 60% of the treatment cycles. Surprisingly menses induction can be achieved with a single dose that is about a ten-times lower when the monkeys are treated continuously with desogestrel. Cycle control is better at low than at high doses of antiprogestin in combination with daily dosing of 4 microg/kg desogestrel. Despite the difference in receptor affinity, no difference between Org 31710 and Org 33628 was found in menses induction. In the continuous low-dose (1 mg/kg) regimen with the PRMs, follicular development occurs normally while ovulation is inhibited. Ovulation is resumed shortly after stopping treatment, and a normal menses occurs after the first progesterone peak. Both compounds may be interesting options for the prevention and treatment of breast cancer and for fertility control.

Nonsteroidal progestins and antiprogestins related to flutamide.

From the dual progestin/antiandrogenic properties of certain synthetic steroids (e.g. cyproterone acetate), it was apparent that the progesterone (P) and androgen (A) receptors must have some common ligand binding features. The nonsteroidal antiandrogen (aA) hydroxyflutamide was therefore considered a possible starting point for medicinal chemistry aimed at antiprogestin (aP) activity. Various modifications to the side chain and aryl ring substituents of flutamide yielded both P and aP activity, but always coupled with varying degrees of A or aA activity. Mineralocorticoid activity was present in some structures, but glucocorticoid and antiglucorticoid activities were not detected. Species (rat, rabbit and monkey) and chiral differences presented formidable difficulties in developing simple structure activity patterns, and low ( < 1%) in vitro uterine receptor binding belied in vivo potency of some aPs. One of the most active aPs, ZM172406, the R enantiomer of ZM150271, N-(3-chloro-4-cyanophenyl)-3,3, 3-trifluoro-2-hydroxy-2-methylpropanamide, had comparable oral potency to mifepristone in rats and monkeys. The racemate ZM150271 was an effective abortifacient during early pregnancy in pigtailed monkeys (3 x 10 mg/kg) but less effective in cynomolgus monkeys. One of the most active progestins (Pn), ZM182345, N-(4-nitro-3-trifluoromethylphenyl)-4-phenyl-2-hydroxy-2-trifluoromet hyl-pentanamide, was at least as potent as P in rats and rabbits but also possessed A activity.

Biological mechanisms underlying the clinical effects of mifepristone (RU 486) on the endometrium.

The abortifacient and menstrual effects of the potent antiprogestin, RU 486 (mifepriston) are associated with both endometrial hemorrhage and extracellular matrix (ECM) degradation. Such processes reflect reduced perivascular decidual cell hemostatic and increased ECM-degrading protease activity. In this review, we summarize the effects of RU 486 on different proteases involved in these processes and expressed by in vitro decidualized endometrium stromal cells. The expression of tissue factor (TF), the primary initiator of hemostasis; urokinase-type plasminogen activator (uPA); tissue-type plasminogen activator (tPA); plasminogen activator inhibitor-1 (PAI-1) as well as the potent matrix metalloprotease, MMP-3 was assessed. These endpoints of decidualization are regulated by progesterone. It was observed, that RU 486 blocks and reverses progestin-enhanced stromal cell TF protein and mRNA expression and PAI-1 protein and mRNA expression, whereas blocks and reverses progestin-inhibited stromal cell uPA, tPA and MMP-3 protein and mRNA expression. These coordinate enhancement of plasminogen activator and MMP-3 expression promotes proteolysis of the decidual ECM, which leads to endometrial sloughing during menstruation. Moreover, destabilization of endometrial microvessels resulting from degradation of their surrounding ECM is consistent with the heavy bleeding after RU 486 administration. On the other hand, with blocking the expression of TF and PAI-1, RU 486 creates a haemorrhagic and fibrinolytic milieu around the endometrial vessels, suggesting a mechanism for RU 486-induced endometrial hemorrhage. The steroid antagonist RU 486 (mifepristone) causes menstrual bleeding when given during the luteal phase of the menstrual cycle (1) and induces abortion in 64-85% of pregnant patients when administered before the 50. postmenstrual days (2, 3). These clinical actions are thought to reflect the antiprogestational effects of RU 486. Pathologic studies showed, that the effects of RU 486 on primate and human luteal phase endometrium include reduced stromal edema, increased venular diameter. Erythrocyte and leukocyte diapedesis, focal hemorrhage, degeneration of the stromal extracellular matrix, and eventual disruption of the superficial layer of the endometrium (4, 5). This antihormone acts at the receptor level and possibly also at the postreceptor level(s) (6). The most important mechanism of action is to compete with progesterone at the level of their respective binding site in the ligand binding domain of the progesterone receptors. The binding of RU 486 to the receptor leads to conformational changes in the DNA-binding site of the progesterone-receptor (7). As a consequnce of these changes, the interaction between the receptor and the progesterone-response elements in the promoter region of progesterone-responsive genes is altered (7). The menorrhagic and abortifacient properties of RU 486 are associated with the induction of endometrial hemorrhage. The physiological mechanisms by which human endometrium permits menstrual hemorrhage in the absence of pregnancy yet maintains hemostasis during endovascular trophoblast invasion (avoiding early abortion) has been investigated in our laboratory by evaluating endometrial expression of different proteins that play role in the process of hemostasis. Besides the endometrial haemostasis, we also examined the proteolytic processes leading extracellular matrix (ECM) degradation, which is also an integral part of menstruation. In this review we sought to summarize the biological mechanisms underlying the clinical effects of RU 486 on endometrial haemorrhage/haemostasis and on ECM degradation.

Roles of prostaglandin E receptors in mesangial cells under high-glucose conditions.

BACKGROUND: High glucose reportedly stimulates prostaglandin (PG) E2 production and DNA synthesis in mesangial cells (MCs). However, the pathophysiological significance of PGE2 in MCs has remained unclear. METHODS: The effects of prostanoids on [3H]-thymidine uptake and cAMP production in rat MCs cultured with 5.6 mM glucose, 25 mM glucose, or 5.6 mM glucose supplemented with 19.4 mM mannitol were examined. The gene expression of PGE2 receptor (EP) subtypes in MCs was analyzed with Northern blotting techniques. RESULTS: Northern blotting indicated EP1 and EP4 gene expression in MCs. EP1 agonists and PGE2 stimulated [3H]-thymidine uptake in MCs. EP1 antagonists dose dependently attenuated high-glucose-induced [3H]-thymidine uptake, which suggests EP1 involvement, by an increase in intracellular Ca2+, in DNA synthesis of MCs. On the other hand, forskolin, db-cAMP, and 11-deoxy-PGE1, an EP4/EP3/EP2 agonist, significantly decreased DNA synthesis in MCs. These inhibitory effects are thought to be mediated via EP4 as a result of an increase in cAMP synthesis. The effects via EP4 seem to be particularly important because PGE2-induced cAMP synthesis was significantly attenuated in the high-glucose group compared with the mannitol group, in which [3H]-thymidine uptake did not increase in spite of augmented PGE2 production. CONCLUSION: The increase in DNA synthesis in MCs under high-glucose conditions can be explained, at least in part, by the high-glucose-induced inhibition of cAMP production via EP4, which augments EP1 function in conjunction with the overproduction of PGE2.

Prostaglandin E receptor subtypes in cultured rat microglia and their role in reducing lipopolysaccharide-induced interleukin-1beta production.

Prostaglandins (PGs) are potent modulators of brain function under normal and pathological conditions. The diverse effects of PGs are due to the various actions of specific receptor subtypes for these prostanoids. Recent work has shown that PGE2, while generally considered a proinflammatory molecule, reduces microglial activation and thus has an antiinflammatory effect on these cells. To gain further insight to the mechanisms by which PGE2 influences the activation of microglia, we investigated PGE receptor subtype, i.e., EP1, EP2, EP3, and EP4, expression and function in cultured rat microglia. RT-PCR showed the presence of the EP1 and EP2 but not EP3 and EP4 receptor subtypes. Sequencing confirmed their identity with previously published receptor subtypes. PGE2 and the EP1 agonist 17-phenyl trinor PGE2 but not the EP3 agonist sulprostone elicited reversible intracellular [Ca2+] increases in microglia as measured by fura-2. PGE2 and the EP2/EP4-specific agonists 11-deoxy-PGE1 and 19-hydroxy-PGE2 but not the EP4-selective agonist 1-hydroxy-PGE1 induced dose-dependent production of cyclic AMP (cAMP). Interleukin (IL)-1beta production, a marker of activated microglia, was also measured following lipopolysaccharide exposure in the presence or absence of the receptor subtype agonists. PGE2 and the EP2 agonists reduced IL-1beta production. IL-1beta production was unchanged by EP1, EP3, and EP4 agonists. The adenylyl cyclase activator forskolin and the cAMP analogue dibutyryl cAMP also reduced IL-1beta production. Thus, the inhibitory effects of PGE2 on microglia are mediated by the EP2 receptor subtype, and the signaling mechanism of this effect is likely via cAMP. These results show that the effects of PGE2 on microglia are receptor subtype-specific. Furthermore, they suggest that specific and selective manipulation of the effects of PGs on microglia and, as a result, brain function may be possible.

RU 486 (mifepristone). A short overview of its mechanisms of action and clinical uses at the end of 1996.

RU 486 (mifepristone) has proved to be a remarkably active antiprogesterone and antiglucocorticosteroid agent in humans. The mechanism of action of RU 486 involves the intracellular receptors of the antagonized hormones progesterone and glucocorticosteroids. At the molecular level, the most important features are high binding affinity to the receptor, interaction of the phenyl-aminodimethyl group in the 11 beta-position with a specific region of the receptor binding pocket, and RU 486-induced transconformation in the ligand binding domain. These properties have consequences at different steps of the receptor function as compared with agonists. However, this cannot be limited to the RU 486-receptor interaction; for instance, a switch from an antagonistic property to an agonist activity is possible, depending on the intervention of other signaling pathways. Derivatives with only one of the two antagonistic properties (antiprogestin, antiglucocorticosteroid) would be desirable in spite of similarities between the steroid structures, receptors involved, and responsive machineries in target cells. Clinically, the RU 486 plus prostaglandin method is ready to be used on a large scale, and is close to being as practical and safe as any medical method of abortion may be. The early use of RU 486, as a contragestive--that is, for use by a woman as soon as she fears a pregnancy she does not want--will help to defuse the abortion issue. Research should now be conducted to define an efficient and convenient contraceptive method with RU 486 or other antiprogestins. The usefulness of RU 486 for obstetrical indications, including facilitation of difficult delivery, has to be assessed rapidly. Gynecological trials, particularly in leiomyomata, should also be systematically continued. The very preliminary results obtained with tumors, including breast cancers, do indicate that further studies are necessary.

Inducible nitric oxide synthase expression in activated rat microglial cultures is downregulated by exogenous prostaglandin E2 and by cyclooxygenase inhibitors.

Prostaglandins and nitric oxide (NO) are among the numerous substances released by activated microglial cells, the brain resident macrophages, and they mediate several important microglial functions. We have previously shown that cyclooxygenase-2 (COX-2) and inducible NO synthase (iNOS), the two key enzymes in prostaglandin and NO synthesis, respectively, are rapidly co-induced in rat neonatal microglial cultures activated by bacterial endotoxin (lipopolysaccharide [LPS]) and that COX-2 expression appears to be under the negative control of endogenous as well as exogenous NO. In this study we show that exogenous prostaglandin E2 (PGE2), which is known to increase cyclic adenosine monophosphate (cAMP) levels in microglial cells, downregulates LPS-induced iNOS expression in a dose-dependent manner. The involvement of cAMP in the PGE2-dependent inhibition of iNOS is supported by several pieces of evidence. First, iNOS expression was also inhibited by agents such as isoproterenol and forskolin, which cause an elevation of cAMP levels, and by dibutyryl cAMP (dbcAMP), a cAMP stable analogue. Second, the inhibitory effect of PGE2 was mimicked by 11-deoxy-16,16-dm PGE2, a selective agonist at the PGE2 receptor subtype EP2, coupled to the activation of adenylyl cyclase, but not by sulprostone, a potent agonist at receptor subtypes EP3 and EP1, associated with an inhibition of adenylyl cyclase activity and intracellular Ca2+ elevation, respectively. Third, the inhibitory effect of PGE2 on NO synthesis was blocked by SQ 22,536, a specific inhibitor of adenylyl cyclase. Interestingly, the abrogation of endogenous prostanoid production by several COX inhibitors caused a reduction of iNOS expression, suggesting a positive modulatory effect of endogenous prostanoids of iNOS expression, as opposed to the inhibitory effect of exogenous PGE2.

Anti-implantation activity of luteal phase mifepristone administration is not mimicked by prostaglandin synthesis inhibitor or prostaglandin analogue in the rhesus monkey.

The use of mifepristone as an anti-implantation agent in the primate has been explored in the rhesus monkey with two specific aims: (i) to determine the contraceptive efficacy of very low-dose mifepristone administered on mated cycle days 16, 17, and 18; and (ii) to test the hypothesis that alteration in endometrial prostaglandin milieu by using either prostaglandin analogue or prostaglandin synthesis inhibitor can intervene the antifertility effect induced by mifepristone. Thirty female monkeys were randomly assigned to one of the six treatment groups. Five monkeys in the control group (group 1) were subjected to mating during cycle days 8-22. Four out of five monkeys became pregnant in the first mated cycle (80%) with detection of serum mCG by 12.7 +/- 1.5 days after ovulation. In group 2, 12 mated cycles were studied in five monkeys, mifepristone [RU486, 2 mg/day/animal, s.c. in 1 ml vehicle (1:4, benzyl benzoate:olive oil, v/v)] was given on cycle days 16, 17, and 18. In this group, no pregnancy was observed, thus providing complete pregnancy protection. Though there was an apparent extension of treatment cycle lengths in five cases with no incidence of inter-menstrual bleeding or spotting, there were no significant changes in serum estradiol (E) and progesterone (P). In group 3, four monkeys received prostaglandin (PG) synthesis inhibitor, diclofenac sodium (D, 25 mg/day/animal, i.m.) on cycle days 16, 17, and 18 in seven ovulatory menstrual cycles. Four of these cycles (57%) resulted in normal pregnancies; however, mCG detection (16.8 +/- 1.2 days after ovulation) was significantly (p < 0.05) delayed as compared to group 1. In group 4, four monkeys received 100 micrograms misoprostol (M), a PGE1 analogue, by gavage on mated cycle days 16, 17, and 18. Four pregnancies occurred in five treatment cycles (80%) with normal profiles of serum E and Pi mCG was first detected 13.2 +/- 1.7 days after ovulation. In group 5, seven monkeys received same dosages of RU486 and D on mated cycle days 16, 17, and 18. One hundred percent pregnancy protection was observed with luteal phase lengthening in eight treatment cycles but with unaltered E and P profiles. In group 6, five monkeys in nine treatment cycles received same dosages of RU486 and M on mated cycle days 16, 17, and 18. One pregnancy occurred; evaluation of E and P levels showed that the drug was given in the preovulatory period, which delayed ovulation and implantation, as mCG was detected 19 days post-ovulation. A delay in vaginal bleeding was observed in four treatment cycles with unaltered E and P profiles. Low-dose mifepristone appears to be a potential candidate for luteal phase and post-coital emergency contraception. However, the hypothesis that altered endometrial prostaglandin milieu may be responsible for mediating the anti-implantation effect of RU486 does not appear to be tenable based on our results in the rhesus monkey.

Mifepristone: a potential contraceptive.

Use of standard estrogen-progestin contraception remains problematic in several subgroups of patients (e.g., those in whom exogenous estrogens are contraindicated, such as survivors of hormone-dependent cancer, or patients with endometriosis or uterine fibroids). In addition, the postcoital contraception, even if already available, remains at least underused. Additionally, continuous intake of contraceptive steroids is under increasing scrutiny. Would antiprogestins, and specifically the applications of mifepristone such as the ones reviewed in this article offer significant improvements at such problematic occasions? The most attractive novel contraceptive application of mifepristone is that of emergency postcoital contraception after unprotected intercourse. In addition, various options in the endometrial category, specifically those requiring drug administration only during a limited time, might be used in the future. Mifepristone might offer contraceptive and therapeutic relief to patients suffering from endometriosis or uterine fibroids, both conditions that have been shown to benefit from mifepristone therapy. However, the use of mifepristone in contraceptive preparations that are widely available would pose an additional problem of possible misuse of the compound, the reason for currently limiting access to mifepristone. However, such risk should be easily avoidable in the case of postcoital contraception in which only one dose of mifepristone is needed. Regarding the future of mifepristone, and more broadly that of antiprogestins in contraception, the authors believe that they will have a place in the future contraceptive armament. As already emphasized, the strongest clinical areas are those of immediate postcoital and endometrial contraception. Additional studies evaluating parenteral modes of administration, the long-term endometrial effects, and safety and metabolic effects of prolonged antiprogestin administration are needed before mifepristone can be considered a part of the contraceptive arena.

PIP: The evaluation of mifepristone (RU-486) as a potential contraceptive has entailed three strategies: endocrine, endometrial, and postcoital. Both animal and human studies have indicated that continuous preovulatory administration of RU-486 (at least 2 mg/day) inhibits ovulation, presumably through its perturbation of the hypothalamo-pituitary-ovarian axis and disruption of normal folliculogenesis. However, the return of menses after RU-486 termination is unpredictable. Early luteal phase or continuous RU-486 administration eliminates this side effect and delays endometrial maturation. On the other hand, late luteal phase administration of a high single dose is associated with failure rates of 3-16%. The use of RU-486 for immediate postcoital contraception lengthens the subsequent menstrual cycle, but is highly effective and well tolerated. At present, the immediate postcoital and endometrial strategies appear most promising. Urged is further research evaluating parenteral modes of administration, the long-term endometrial effects, and the safety and metabolic effects of prolonged antiprogestin use.