11-Deoxy,16,16-dimethyl prostaglandin E2 induces specific proteins in association with its ability to protect against oxidative stress.

Prostaglandins (PGs) act locally to maintain cellular homeostasis and stimulate stress response signaling pathways. These cellular effects are diverse and are tissue-dependent. PGE(2), and the synthetic analogue, 11-deoxy,16,16-dimethyl PGE(2) (DDM-PGE(2)), protect renal proximal tubular epithelial (LLC-PK1) cells against cellular injury induced by the potent nephrotoxic and nephrocarcinogenic metabolite of hydroquinone, 2,3,5-tris-(glutathion-S-yl)hydroquinone. Although this cytoprotective response (in LLC-PK1 cells) is mediated through a thromboxane or thromboxane-like receptor coupled to AP-1 signaling pathways, the mechanism of cytoprotection is unknown. In this study, we utilized HPLC-electrospray ionization tandem mass spectrometric (ESI MS/MS) and matrix-assisted laser desorption ionization time-of-flight mass spectrometric (MALDI TOF) analysis of proteins isolated from DDM-PGE(2)-stimulated LLC-PK1 cells to identify candidate cytoprotective proteins. DDM-PGE(2) selectively stimulated the synthesis of several proteins in LLC-PK1 cells. Peptide sequencing by ESI-MS/MS of in-gel tryptic protein digests revealed the identity of eight proteins: endothelial actin binding protein, myosin, elongation factor 2 (EF-2), elongation factor 1alpha-1 (EF-1alpha), heat shock protein 90beta (HSP90beta), glucose-regulated protein 78 (GRP 78), membrane-organizing extension spike protein, and actin. Both ESI-MS/MS and MALDI-MS analysis resulted in the same protein identification. Western analysis confirmed the temporal induction of the majority of these proteins, including EF-2, EF-1alpha, HSP90beta, GRP78, and actin. The collective expression of these proteins suggests that DDM-PGE(2)-mediated cytoprotection may involve alterations in cytoskeletal organization and/or stimulation of an endoplasmic reticulum (ER) stress response. The present studies provide insights into potential downstream targets of PG signaling.

A multicentre randomized comparative clinical trial of 200 mg RU486 (mifepristone) single dose followed by either 5 mg 9-methylene PGE(2) gel (meteneprost) or 600 microg oral PGE(1) (misoprostol) for termination of early pregnancy within 28 days of missed menstrual period. ICMR Task Force Study. Indian Council of Medical Research.

A multicentre, randomized, comparative clinical trial of 200 mg RU486 (Mifepristone) followed 48 h later by either 5 mg 9-methylene PGE(2) vaginal gel (meteneprost) or 600 microg oral PGE(1) (misoprostol) for termination of pregnancy within 28 days of the missed period, was carried out through the Indian Council of Medical Research's (ICMR) network of Human Reproduction Research Centres (HRRCs). A total of 893 subjects were assessed regarding their therapeutic responses to the two different treatment groups. The results indicated a success rate of 84.6% among 453 women treated with RU486 followed by 9 methylene PGE(2) vaginal gel, that was not significantly different from the success rate of 87.7% observed in 440 women treated with RU486 followed by oral PGE(1). The majority of study subjects (90%) started bleeding within 72 h. About 26% of the subjects had started bleeding before the administration of any prostaglandin. The average duration of bleeding in all the subjects was about 7 days. No life threatening side effects were observed among the subjects in two treatment groups. Gastro-intestinal complaints were reported more often by women treated with oral PGE(1) as compared to those treated with 9-methylene vaginal PGE(2) gel; nausea occurred in 25.7% and 19.2%, vomiting in 6.8% and 4.6%, and diarrhoea in 4.8% and 0.9% of the subjects in the 2 treatment groups, respectively. Fever higher than 38 degrees C and severe abdominal pain were reported by 4.2% and 5.0% of all subjects treated, respectively. Intravenous infusion of glucose and saline was required by 6 subjects in each treatment of the prostaglandin treated groups. Blood transfusion was required in 2 subjects, one in each treatment group, for profuse bleeding.

Early abortion by mifepristone (RU 486) followed by vaginal gel (meteneprost) versus oral (misoprostol) prostaglandin.

The present study was conducted to compare the therapeutic regimens of low-dose mifepristone (200 mg) plus vaginal meteneprost versus oral misoprostol in terms of efficacy and safety for medical termination of early pregnancy. A randomized clinical trial was conducted by the Department of Obstetrics and Gynecology at the All India Institute of Medical Sciences, New Delhi. A total of 101 subjects were enrolled within 56 days of amenorrhea. A single dose of 200 mg of mifepristone (RU 486) was given and, 48 hr later, prostaglandin was administered as either 5 mg of 9 methylene PGE2 vaginal gel, meteneprost (classified as group I) or 600 microg of oral PGE1 derivative misoprostol (classified as group II). In group I, 50 subjects and in group II, 51 subjects were treated with the respective schedule. The success rate with mifepristone + misoprostol (group II) was 88.63% which was significantly higher than that with mifepristone + meteneprost (group I) which was 82% (p < 0.05). The average duration of bleeding in cases with complete abortion was 8.95+/-5.67 and 9.77+/-6.51 in group I and II, respectively. There were no serious side-effects. Only one subject in group I (2%) required blood transfusion for heavy bleeding. This study indicated that oral prostaglandin after a low dose of mifepristone (200 mg) could be developed into an effective method to terminate early pregnancy. Oral administration of both drugs would be a more convenient, feasible, private and acceptable regimen.

Prostanoid receptor with a novel pharmacological profile in human erythroleukemia cells.

The purpose of this study was to characterize the prostanoid receptors coupled to intracellular calcium in human erythroleukemia (HEL) cells, a cell line with platelet/megakaryocytic characteristics. Both prostaglandin E1 (PGE1) and iloprost increased cyclic AMP (cAMP) in HEL cells, but modulated [Ca2+]i by different mechanisms. Iloprost (10(-9) to 10(-6) M) had no effect on basal [Ca2+]i, but greatly potentiated the increase in [Ca2+]i produced by thrombin. This effect was mimicked by cholera toxin and other Gs-coupled receptors, and involved calcium influx since iloprost had no effect on [Ca2+]i in cells incubated in Ca2+-free buffer. Furthermore, iloprost did not increase the generation of baseline or thrombin-induced inositol phosphates at these concentrations. In contrast, PGE1 (10(-7) to 10(-5) M), but not iloprost, increased basal [Ca2+]i through a pertussis toxin-sensitive mechanism that involved stimulation of inositol phosphate generation and mobilization of intracellular calcium. The order of potencies of other prostaglandins that increased [Ca2+]i was not consistent with known IP, EP, DP, FP, or TP receptors. 11-Deoxy-16,16-dimethyl PGE2 was the most potent of the analogs tested (EC50 = 28 nM). In summary, at least two prostaglandin receptors are functionally coupled to intracellular calcium in HEL cells: a putative IP receptor coupled to Gs proteins that increases cAMP and enhances calcium influx, and a novel prostanoid receptor that evokes calcium mobilization through stimulation of phospholipase C by a pertussis toxin-sensitive pathway.

PGE2-mediated cytoprotection in renal epithelial cells: evidence for a pharmacologically distinct receptor.

Although the exact mechanism of prostaglandin E2 (PGE2)-mediated cytoprotection has not been elucidated, its ability to induce cytoprotection in cell culture suggests this action occurs at the cellular level. The present studies were conducted to determine whether PGE2 induces protection against 2,3,5-(trisglutathion-S-yl)-hydroquinone [2,3,5-(trisglutathion-S-yl)-HQ]-mediated cytotoxicity in a renal proximal tubule epithelial cell line (LLC-PK1) and to delineate the cellular and molecular mechanisms associated with this response. Pretreatment of LLC-PK1 cells with 0.01-40 microM PGE2 for 24 h fully protects against a moderately toxic concentration of 2,3,5-(trisglutathion-S-yl)-HQ. PGE2-mediated cytoprotection is observed in cells pretreated at pH 7.4 but not at pH 7.8. However, cytoprotection is observed in LLC-PK1 cells pretreated with the PGE2 analog, 11-deoxy-16,16-dimethyl PGE2 (DDM-PGE2) but not with the PGE2 receptor [E-prostanoid (EP)] agonists 17-phenyltrinor PGE2 (EP1), 11-deoxy PGE1 (EP2/EP4), sulprostone (EP1/EP3), PGE1, or PGA2. 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent activator of protein kinase C (PKC), also induces cytoprotection, supporting a role for this pathway in the cytoprotective response. PGE2, DDM-PGE2, and TPA all induce the binding of nuclear proteins to a TPA responsive element (TRE), whereas analogs that did not induce cytoprotection (PGE1, 17-phenyltrinor PGE2, sulprostone) were without effect. DDM-PGE2- and TPA-mediated cytoprotection and TRE binding activity are inhibited by N-(2[[3-(4-bromophenyl)-2-propenyl]-amino]-ethyl)-5-isoquinolinesulfonam ide (H-89), a PKC inhibitor. These data suggest that cytoprotection by PGE2 and DDM-PGE2 in LLC-PK1 cells is mediated by a PKC-coupled receptor, which is pharmacologically distinct from the presently classified EP receptor subtypes.

In vitro studies on the regulation of rainbow trout (Oncorhynchus mykiss) macrophage respiratory burst activity.

Modulation of the respiratory burst activity of head kidney macrophages isolated from rainbow trout (Oncorhynchus mykiss) was observed following treatment with several biologically active substances. Macrophage-activating factor (MAF) induced the highest increment if respiratory burst activity relative to treatment with lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF alpha) or beta-glucans from Saccharomyces cerevisiae. Increased responses were more evident when these molecules were combined in pairs. Negative regulation of respiratory burst activity was observed when diMePGE2 was added to the macrophages, with maximal inhibition seen using a concentration of 2.6 microM. Inhibition was also seen using stimulated macrophages, either by co-incubation of stimuli and diMePGE2 or by adding diMePGE2 to previously stimulated cells. The inhibitory effect on macrophages was detectable with 3 h of incubation with diMePGE2 and by 24 h the level of the response was even lower than that from unstimulated (control) macrophages. Of significance was the finding that the inhibitory effect of prostaglandin on macrophage function could be overcome by co-incubation with stimulatory molecules or by pre-treatment with MAF and LPS or MAF and TNF alpha Thus, the regulation of macrophage activation in fish is likely to be as complex as in mammals.

A multicentre clinical trial with RU 486 followed by 9-methylene-PGE2 vaginal gel for termination of early pregnancy: a dose-finding study. Indian Council of Medical Research Task Force on Hormonal Contraception.

A dose-finding study was carried out with two different doses of RU 486 (200mg or 600mg) each in combination with two doses of 9-methylene-PGE2 gel (3mg or 5mg) for termination of pregnancy between 7 to 28 days after missed menstrual period. It was observed that the success rates with 200mg RU 486 followed by 5mg 9-methylene-PGE2 gel were 94.5% and 89.6% in women with 7-14 days and 15-28 days of missed menstrual period, respectively. These rates were similar to those observed with 600mg RU 486 given along with 3mg or 5mg 9- methylene-PGE2 gel and were significantly higher than those observed with 200mg RU 486 given along with 3mg 9-methylene-PGE2 gel. All subjects except four started bleeding following the treatment. The average duration of bleeding in subjects with successful outcome (complete abortion) ranged between 7.0 to 11.8 days in the four different treatment schedules. There were no serious side effects with any of the treatment schedules; only one subject required transfusion of one unit of blood for heavy bleeding. The immediate and delayed complication rates were similar with the four treatment schedules.

The use of antiprogestin (RU 486) for termination of second trimester pregnancy.

Pretreatment with laminaria tent is often used in prostaglandin-induced second-trimester abortion to increase efficacy and shorten induction-to-abortion time. In the present study, two alternatives to soften the cervix and dilate the cervical canal, the antiprogestin RU 486 and intra-cervical application of PGE2, were studied. The study included 71 women requesting legal abortion in the 15th to 23rd week of pregnancy who were treated with repeated vaginal applications of 9-methylene PGE2 in a hydrophilic gel (5 mg every 4th hour) following pretreatment with 200 mg of RU 486 and/or intracervical administration of 0.5 mg of PGE2 gel. The mean interval from start of vaginal prostaglandin treatment to abortion was 13.2 h after intracervical PG-treatment, 10.0 h after antiprogestin and 6.6 h after the combined pretreatment. Patients who received pretreatment with RU 486 alone or in combination with intracervical PGE2 experienced the lowest frequency of episodes of vomiting. Of these two pretreatment alternatives, RU 486 alone has the advantage of a shorter hospital stay. It can be concluded that vaginal administration of 9-methylene PGE2 after pretreatment with RU 486 was a highly effective, safe and rapid procedure for termination of mid-trimester pregnancy, was well tolerated by the patients and was associated with few side effects.

Vacuum aspiration for termination of early second trimester pregnancy after treatment with vaginal prostaglandin.

Vaginal and intramuscular administration of prostaglandin analogues are routinely used for dilatation of the cervical canal prior to vacuum aspiration in first trimester abortion. Whether the same procedure is also useful during the first weeks of the second trimester has been much less investigated. In the present study, 127 women in the 13th and 14th week of pregnancy were pretreated with 3 mg 9-deoxo-16,16-dimethyl-9-methylene PGE2 administered vaginally 12 hours before surgery. At surgery the cervical canal was dilated to 9.8 mm +/- 2.5 mm (mean +/- SD) and the evacuation of the uterus was uneventful. In 21% of the patients vaginal bleeding occurred prior to the operation. The mean blood loss at surgery was 49 ml and exceeded 100 ml in only 6 patients. Gastrointestinal side effects were rare but analgesic injections were demanded by 29% of the patients during the pretreatment period. No subsequent curettage was performed during the follow-up period but 2 patients (1.6%) were readmitted because of post-abortion endometritis. It can be concluded that after pretreatment with PG, vacuum aspiration can be safely performed during the first weeks of the second trimester.

Termination of early pregnancy through a combination of the antiprogestin and prostaglandin analogue.

Termination of early pregnancy (less than 49 days of amenorrhea) was induced in 75 patients with the combination of the antigestagen mifepristone and a prostaglandin analogue, meteneprost. After 48 h a single oral dose of 600 mg of mifepristone was followed by a 10 mg meteneprost vaginal pessary. Pregnancy was confirmed by clinical and ultrasound examinations and plasma HCG assessment. Complete abortion occurred in 72 patients (96%) and the three others required a surgical uterine aspiration. Bleeding continued for 4 to 12 days (mean = 8). Uterine pain and side effects occurred during the 3 h following the use of prostaglandin. Only minor analgesic were required in 30 patients. The combination of mifepristone and meteneprost is a safe and effective method to terminate an early pregnancy.

PIP: Termination of early pregnancy (49 days of amenorrhea) was induced in 75 patients with the combination of the antigestagen mifepristone and a prostaglandin (PG) analogue, meteneprost. After 48 hours, a single oral dose of 600 mg mifepristone was followed by a 10 mg meteneprost vaginal pessary. Pregnancy was confirmed by clinical and ultrasound examinations and plasma hCG assessment. Complete abortion occurred in 72 patients (96%) and the 3 others required a surgical uterine aspiration. Bleeding continued for 4-12 days (mean=8). Uterine pain and side effects occurred during the 3 hours following the use of PG; only minor analgesics were required in 30 patients. The combination of mifepristone and meteneprost is a safe and effective method of terminating and early pregnancy.

Oral administration of RU 486 and 9-methylene PGE2 for termination of early pregnancy.

It has been shown that the antiprogestin RU 486 increases the sensitivity of the early pregnant human uterus to the stimulatory action of prostaglandin E analogues administered vaginally or intramuscularly. To examine if RU 486 also increases uterine sensitivity to a PGE analogue given orally, two investigative approaches were used in the present study: 1) direct registration of uterine contractions before and after administration of 9-methylene PGE2 in untreated and RU-486-treated early pregnant women; and 2) an efficacy trial involving treatment of pregnant women (amenorrhea of 49 days or less) with 25 mg RU 486 twice daily for three or four days followed by 2.5, 5.0 or 10 mg 9-methylene PGE2, or 600 mg RU486 followed by 10 mg 9-methylene PGE2 administered on day 3 and 4. The results showed that oral 9-methylene PGE2 had a clear stimulatory effect on uterine contractility which was further increased by pretreatment with RU 486. Following 2.5, 5.0 or 10.0 mg 9-methylene PGE2, the frequency of complete abortion was the same, or approximately 80%. The success rate is higher than that generally reported for RU 486 treatment alone. If 600 mg RU 486 was complemented with 10 mg 9-methylene PGE2 administered on both days 3 and 4, the frequency of complete abortion increased to 95%. Side effects were of a mild nature and generally occurred following administration of 9-methylene PGE2. The results of the present study indicate that a combined treatment based on oral administration of both the antiprogestin and the prostaglandin analogue can be developed into a highly effective and simple method to terminate early pregnancy.

The effects of 16,16-dimethyl PGE2 and phosphodiesterase inhibitors on Con A blastogenic responses and NK cytotoxic activity of mouse spleen cells.

Prostaglandins function as down regulators of immune responses probably by increasing the concentration of intracellular cAMP. Phosphodiesterase inhibitors, which prevent the breakdown of cAMP, also increase the intracellular levels of cAMP. Prostaglandins and phosphodiesterase inhibitors have both been shown to suppress immune responses in vitro. In this study 16,16-dimethyl PGE2 (dm-PGE2), added in vitro, suppressed the mouse spleen cell concanavalin A (Con A) response by 38% and natural killer (NK) activity by 53%. Addition of the phosphodiesterase inhibitors, theophylline, RO20-1724, or dipyridamole, decreased both the Con A response and NK activity by at least an additional 30%. We also demonstrate that treatment with dm-PGE2 and theophylline in vivo is more effective than either compound alone in inhibiting NK activity of both untreated mice and mice treated with polyinosinic-polycytidylic acid. These studies support the hypothesis that the immunosuppressive effect of dm-PGE2 is mediated by cAMP and suggest that treatment with a combination of dm-PGE2 and phosphodiesterase inhibitors can augment this immunosuppressive effect.

Investigation of the relationship between melting-related parameters and in vitro drug release from vaginal suppositories.

The effect of temperature on drug release from meteneprost potassium vaginal suppositories was investigated using a dissolution test based on the USP I apparatus. Comparison of the dissolution results with the DSC melting behaviour revealed that drug release was extremely slow until melting of the suppository was essentially complete. The melting behaviour of the meteneprost potassium suppositories was also varied by preparing suppositories from bases with higher and broader melting ranges. The observed dissolution behaviour (at 37 degrees C) confirmed that drug release increased as the melting temperature of a particular suppository decreased. Differential scanning calorimetry, viscosity and dilatometry methods were used to characterize the suppository melting process. The effects of suppository melting range, melt temperature and composition were investigated with respect to in vitro drug release. Methodology for the HPLC determination of meteneprost in suppositories and in dissolution media are also reported.

Clinical trials with RU 486 (mifepristone): an update.

This paper reviews much of the clinical data obtained with the antiprogestin RU 486 (mifepristone) in the fields of obstetrics and gynecology. To interrupt early pregnancy (less than 50 days of amenorrhea) RU 486 as a single dose of 600 mg followed 36-48 hours later by a prostaglandin derivative, constitutes an alternative to vacuum aspiration or dilatation and curettage (D and C). All three methods have comparable efficacy, provided that they are performed in centers with adequate medical facilities. RU 486 is the first antiprogestational steroid available for clinical purposes. Its pharmacological properties have been reviewed in detail elsewhere (1,2). The present paper reviews the main clinical data obtained during trials monitored by Roussel Uclaf.

Termination of early pregnancy using mifepristone in combination with prostaglandin analogs.

Mifepristone, (RU 486), is an antiprogesterone which, when administered alone will terminate early pregnancy in 80% of cases. Prostaglandins at high doses have also been used to terminate pregnancy but there are often unacceptable side effects. The combined treatment of mifepristone with low doses of prostaglandin analogs appears to be an effective method for termination of pregnancy, with a high success rate and a reduction in the side effects observed with prostaglandins.

Development of a vaginal gel containing 9-deoxo-16,16-dimethyl-9-methylene PGE2 for cervical dilatation and pregnancy termination.

A stable hydrophilic gel for vaginal administration containing 9-deoxo-16,16-dimethyl-9-methylene PGE2 (9-methylene PGE2) was developed and its clinical usefulness for preoperative cervical dilatation and for termination of first and second trimester pregnancy evaluated in 521 pregnant patients admitted to the hospital for therapeutic abortion. Following vaginal administration of 3 mg of 9-methylene PGE2 gel a peak plasma level of between 3.5 and 10 ng/ml was found 3 to 6 hours following treatment. The "bioavailability" of the drug was in the order of 25-30%. 9-methylene PGE2 was found to be equally effective as 1 mg Cervagem for preoperative cervical dilatation. With a pretreatment period of 3 hours side effects were rare with both compounds. If the pretreatment period was extended to 12 hours the degree of cervical dilatation, but also the frequency of side effects increased significantly. Repeated administration of 9-methylene PGE2 was found to be effective (96% complete abortion) in terminating very early pregnancy provided the total dose was 10 mg or more. During second trimester the minimum effective dose was 4.5 mg of the compound repeated every fourth hour. The results of the present study have shown that with the new gel formulation the amount of 9-methylene PGE2 needed to terminate first and second trimester pregnancy was approximately ten times less in comparison with the previously used lipid base suppositories. The treatment was also associated with a low frequency of side effects.

16,16-Dimethyl prostaglandin E2 delays collagen formation in nutritional injury in rat liver.

Chronic nutritional injury was induced in rats by a high-fat, lipotrope-deficient diet. The hepatoprotective effect of 16,16-dimethyl prostaglandin E2 on the deposition of collagen and fat was assessed by histological evaluation and measurement of hydroxyproline. Dose-response studies established that optimal protection was achieved by the twice daily administration of 16,16-dimethyl prostaglandin E2 at 100 micrograms per kg (subcutaneous) or 250 micrograms per kg (oral). 16,16-Dimethyl prostaglandin E2 and a crystalline analog [(p-acetamidobenzamido)phenyl ester of 16,16-dimethyl prostaglandin E2 significantly delayed both the deposition of collagen and the increase in hepatic hydroxyproline content. There was an excellent correlation between the histological assessment of collagen and the biochemical measurement of hydroxyproline. These data provide a rationale for the evaluation of prostaglandins in the treatment of human liver disease.

Prostaglandin vaginal suppositories in non pregnant women requiring cervical dilatation prior to hysteroscopy.

A randomized doubleblind investigation of prostaglandin vaginal suppositories prior to hysteroscopy was undertaken in 30 non-pregnant women. PGE2 in 14 of 15 treated patients we found a softening and a dilatation of the cervical canal, but with a relatively high frequency of side effects-nausea, vomiting and diarrhea. No serious bleeding or side effects were observed.

Cervical dilation before first-trimester elective abortion: a controlled comparison of meteneprost, laminaria, and hypan.

Preoperative cervical dilation makes first-trimester abortion safer, but increases costs and inconvenience to patients if extra visits are required. Eighty-four pregnant primigravid volunteers who requested first-trimester abortion were assigned randomly to one of four study groups: placebo, 16 dimethyl prostaglandin E2 (PGE2) vaginal suppository, 4-mm hypan plastic dilator, or small Laminaria japonica tent. This grouping was done to determine which of three modalities is most effective for cervical dilation within three hours before abortion. After three to four hours of treatment, hypan achieved greater cervical dilation among primigravid women and only modestly increased side effects, as compared with 16 dimethyl PGE2 suppositories or laminaria tents.

[Cervical dilatation by use of a vaginal suppository of meteneprost before voluntary interruption of the pregnancy in the first trimester]. / Dilatation cervicale par ovule vaginal de Méténéprost avant les IVG du premier trimestre.

Instrumental dilatation of the cervix prior to first trimester absorption may be difficult and source of complications. Pharmacological dilatation is proposed in a prospective random study using vaginal suppository containing 10 mg of 9-deoxo, 16-16 dimethyl, 9 methylene PGE2 (Meteneprost). Mean cervical dilatation, 3 hours after treatment, was significantly higher in the treated group (8.1 vs 6 mm) and additional dilatation was facilitated by cervical softening. Side effects occurred in most of the treated patients (uterine pain) but on a minor scale. This procedure may be considered as effective, safe and easy.