Synthesis and evaluation of γ-lactam analogs of PGE2 as EP4 and EP2/EP4 agonists.

To identify topically effective EP4 agonists and EP2/EP4 dual agonists with excellent subtype selectivity, further optimization of the 16-phenyl ω-chain moiety of the γ-lactam 5-thia prostaglandin E analog and the 2-mercaptothiazole-4-carboxylic acid analog were undertaken. Rat in vivo evaluation of these newly identified compounds as their poly (lactide-co-glycolide) microsphere formulation, from which sustained release of the test compound is possible, led us to discover compounds that showed efficacy in a rat bone fracture healing model after its topical administration without serious influence on blood pressure and heart rate. A structure-activity relationship study is also presented.

Augmentation of receptor-mediated adenylyl cyclase activity by Gi-coupled prostaglandin receptor subtype EP3 in a Gbetagamma subunit-independent manner.

We previously demonstrated that the mouse EP3beta receptor and its C-terminal tail-truncated receptor (abbreviated T-335) expressed in Chinese hamster ovary cells showed agonist-dependent and fully constitutive Gi activity in forskolin-stimulated cAMP accumulation, respectively. Here we examined the effect of the EP3beta receptor or T-335 receptor on adenylyl cyclase activity stimulated by the Gs-coupled EP2 subtype receptor in COS-7 cells. As a result, sulprostone, a selective EP3 agonist, dose dependently augmented butaprost-stimulated adenylyl cyclase activity in EP3beta receptor- or T-335 receptor-expressing COS-7 cells. However, such adenylyl cyclase augmentation was not attenuated by either pertussis toxin treatment or expression of the PH domain of rat betaARK1, which serves as a scavenger of Gbetagamma subunits, but was partially attenuated by treatment with either 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)ester, an intracellular Ca(2+) chelator, or W-7, a calmodulin inhibitor. These findings suggest that the C-terminal tail of the EP3beta receptor is not essentially involved in activation of EP2 receptor-stimulated adenylyl cyclase in a Ca(2+)/calmodulin-dependent but Gbetagamma subunit-independent manner.

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 E2 may induce hyperthermia through EP1 receptor in the anterior wall of the third ventricle and neighboring preoptic regions.

We have previously reported that intracerebroventricular injection of prostaglandin E2 (PGE2) induces hyperthermia possibly through EP1 receptors in the rat. In the present study, to determine the sites in the brain where PGE2 induces hyperthermia through EP1 receptors, we microinjected an EP1 receptor agonist, 17-phenyl-omega-trinor PGE2 (17-Ph-PGE2, 100 ng) into different sites in the rat brain and observed the colonic temperature (Tco) for 2 h in a 23 +/- 1 degrees C environment. Responsive sites where 17-Ph-PGE2 (100 ng) produced a rise in the Tco of more than 1.1 degrees C within 60 min after injection were found in the medial preoptic area, the subchiasmatic portion of the median preoptic nucleus, the anterior wall of the third ventricle (A3V) and the ventral portion of the diagonal band of Broca. Among these sites, the A3V was the most responsive. In contrast, microinjection of neither butaprost (an EP2 agonist, 100 ng) nor M&B28767 (an EP3 agonist, 100 ng) into these four sites had any effect on the Tco. Intracerebroventricular pretreatment with SC-19220 (an EP1 antagonist, 100 microg) inhibited the rise in the Tco which was induced by microinjection of PGE2 (50 ng) into the A3V. These results thus suggest that PGE2 induces hyperthermia by stimulating EP1 receptors in the A3V and the neighboring preoptic region.

The effect of the E2 prostaglandin enprostil, and the somatostatin analogue SMS 201 995, on the growth of a human gastric cell line, MKN45G.

The effect of enprostil and the somatostatin analogue SMS 201 995 on the growth of a clonal variant of the human gastric adenocarcinoma cell line, MKN45, was studied. The derived cell line grew twice as fast as MKN45 when grown as a xenograft line in nude mice. However, it did not respond trophically to gastrin either in vitro or in vivo (unlike MKN45) although it possessed the same number of gastrin receptors as the parental line. Gastrin production by the cell line during in vitro culture was twice that of MKN45; thus, the cell line was denoted MKN45G. When MKN45G was grown as xenografts in nude mice (n = 10/group), enprostil (20 micrograms/kg/day) significantly inhibited tumour growth when administered continuously by an osmotic mini-pump from day 1 to day 7 of a 20-day experiment, and induced tumour regression when administered from day 7 to day 14. Enprostil reduced postprandial serum gastrin levels when administered from day 7 to day 14 and prevented gastrin release by MKN45 in vitro. SMS 201 995 at doses of 25 and 240 micrograms/kg/day induced tumour regression when administered from day 1 to day 7 and the former dose reduced post-prandial serum gastrin levels at day 5. Gastrin release by MKN45G was not affected by SMS 201 995 in vitro, thus its effect may not be mediated directly via gastrin, requiring interaction between other hormones or growth factors in the in vivo situation.

[Effect of 11-deoxyprostaglandins E on the reproductive system]. / Vliianie 11-dezoksi-prostaglandinov E na reproduktivnuiu sistemu.

The pharmacological properties of 4 derivatives of 11-desoxy-prostaglandins E (uterotonic, luteolytic, hypotensive, anti-inflammatory, diarrhogenic ones and also acute toxicity) were studied. Compound I possessed the uterotonic activity which decreased with an alpha-chain elongation and a radical introduction into a benzene ring. Compounds I and II exhibited the luteotropic properties at low doses and the luteolytic ones at high doses. The compounds under study had the hypotensive activity due to the presence of a benzene ring in a W-chain.

[Biological properties of 11-deoxyprostaglandin E1]. / Issledovanie nekotorykh biologicheskikh svoistv 11-dezoksiprostaglandina E1.

A study was made of 11-desoxyprostaglandin E1 (11-desoxy-PGE1) effect on bronchial resistance, gastric secretion, indomethacin-induced ulcer in rats and adrenalin-produced lipolysis in rat epididymal adipose tissue in vitro. The drug exerted dose-dependent bronchodilator effect in doses of 1 to 100 micrograms/kg. ED50 was 18.66 micrograms/kg. The bronchodilator effect of 11-desoxy-PGE1 was 46.5 to 50.5% of alupent activity within 60 minutes and 97.8% for a period of 60 to 90 minutes. The drug administration in the presence of bronchial spasm considerably reduces the bronchodilator effect duration (down to 5 to 15 minutes). 11-desoxy-PGE1 produces dose-dependent decrease in gastric secretion (ED50 = 4000 micrograms/kg) and hydrogen ion concentration in rat gastric juice (ED50 = 2000 micrograms/kg). The use of 1-desoxy-PGE1 in rats prior to indomethacin injection protects the gastric mucosa, diminishing erosion number. ED50 was equal to 50 micrograms/kg. The drug exerts inhibitory effect on adrenalin-induced lipolysis of the epididymal adipose tissue in vitro. The most pronounced action of 11-desoxy-PGE1 is its bronchodilator effect manifesting after the minimum dose use (1 microgram/kg).

[Steroid and nonsteroid postcoital contraceptives]. / Steroidnye i nesteroidnye kontratseptivy postkoital'nogo deistviia.

It has been shown experimentally that a combined use of estrogen-norsteroids prostaglandins and neurotropic substances during preimplantation period produces the most potent contraceptive effect. Analysis of the action mode of combined small doses of steroids, prostaglandins and neurotropic drugs suggests that the contraceptive action is underlain by the inhibitory effect on incretion produced by the luteinizing hormone, this effect being in its turn a reason for variation in the amount and condition of the developing fetuses.

Ovarian activity, and total gonadotrophin and gonadotrophin releasing hormone levels in response to some synthetic non-steroidal ovulating agents in the catfish, Heteropneustes fossilis (Bloch).

Effects of clomiphene citrate, cyclofenil and prostaglandins (PGE1 and PGF2 alpha) on ovarian 32P uptake, on gonadotrophin levels in the pituitary gland and blood serum and on a gonadotrophin releasing hormone-like (GnRH-like) substance in the hypothalamus were investigated in Heteropneustes fossilis. These drugs were very effective in increasing the serum level of gonadotrophin with a subsequent increase in ovarian 32P uptake in sham-hypophysectomized recipients. All the drugs except cyclofenil failed to stimulate 32P incorporation by the ovary in hypophysectomized fish. Clomiphene citrate and cyclofenil also induced a significant increase in the GnRH-like factor in the hypothalamus of H. fossilis. Such a response was not obtained in fish treated with PGE1 and PGF2 alpha. It seems likely that the action of clomiphene is routed through the hypothalamo-pituitary-ovarian axis and that of prostaglandins directly through the pituitary-ovarian axis. The action of cyclofenil is bimodal; one effect like that of clomiphene and the other direct upon the ovary probably by increasing its sensitivity to the available gonadotrophin.

Combined effect of prostaglandins and an aortic proteoglycan on platelet aggregation and plasma clotting.

The individual and combined effects of PGD2, PGI2 and an aortic proteoglycan on human platelet aggregation and plasma clotting were studied. PGI2 was at least 10 times more potent than PGD2 in inhibiting platelet aggregation. Small doses of prostaglandins inhibited ADP- and thrombin-induced aggregation, but only prolonged aggregation time without affecting the extent of arachidonic acid (AA)-induced aggregation. Small doses of prostaglandins did not affect thrombin-induced clotting of PRP. Large doses of prostaglandins abolished platelet aggregation and prolonged the onset of thrombin-induced clotting. The aortic proteoglycan (APG) had no appreciable effect on ADP- or AA-induced aggregation. Small doses of APG abolished thrombin-induced clotting, while large doses of APG suppressed both clotting and aggregation induced by thrombin. PGI2 and PGD2 showed additive inhibition of platelet aggregation regardless how the aggregation was induced. APG and prostaglandins showed additive inhibition of only thrombin-induced aggregation. APG, but not any of the prostaglandins, prolonged clotting time of PPP. This prolongation was not potentiated by PGI2 or PGD2.

Synthesis and biological properties of 9-deoxo-16,16-dimethyl-9-methylene-PGE2.

The in vivo monkey uterine stimulating potency of 9-deoxy-16,16-dimethyl-9-methylene-PGE2 is similar to that of 16,16-dimethyl-PGE2 and approximately 15 times that of PGE2. Low doses of this compound stimulated uterine contractions when administered vaginally. Pregnancy was terminated prematurely following subcutaneous, intramuscular or vaginal suppository treatment. Estimates of potential for gastrointestinal side effects using the rat enteropooling assay and in vivo monkey effects indicate that diarrhea will be substantially reduced with retention of uterine stimulating potency.

Effect of sex hormones on HL-RH-degrading hypothalamic enzyme system during estrus cycle in rats.

Basal activity of L-cysteine arylamidase, an LH-RH-degrading enzyme, was determined in the hypothalamus of rats at 4 h intervals throughout the 4-day estrus cycle. The activity of the enzyme system fluctuated during the four estrus stages in a circadian rhythm with maximal values in the night and lowest values at noon. The injection of increasing doses of estradiol-17 beta at various estrus stages caused a moderate stimulation of enzyme activity with no relationship to endogenous hormone levels or estrus stage. The highest activation of the hypothalamic enzyme in response to the application of progesterone occurred at such periods of the cycle when endogenous plasma progesterone is known to be low, and vice versa. When luteinizing hormone or prostaglandin E2 were injected i. v. during the various estrus stages maximal stimulation could be observed at diestrus. The LH-RH-degrading L-cystine arylamidase in the hypothalamus of the rat seems to play a modulating role in the regulation of the tonic LH release during the estrus cycle, but apparently does not influence the events governing the preovulatory LH-peak.

Effects of prostaglandin E1 on the metabolism in rat parathyroid gland in vitro.

We investigated some effects of prostaglandin E1 on the metabolism of rat parathyroid glands using a culture system containing basal Eagle's medium supplemented with 5--10% heat-inactivated rat serum. Rat parathyroid glands incorporate [3H]fucose and 14C-labeled amino acids into cellular glycoproteins and secrete some of these into the culture medium. Gel filtration chromatography separates these glycoproteins into three classes, the smallest of which (peak 3) is secreted with immunoreactive parathyroid hormone. In cultures of 48 h, prostaglandin E1 (1 microgram/ml) specifically inhibits the secretion of peak 3 and of parathyroid hormone but has no effect on the incorporation of [3H]fucose, 14C-labeled amino acids, or [3H]uridine into parathyroid glands. Cytochalasin B inhibits the secretion of parathyroid hormone and the incorporation of isotopic fucose and amino acids. Cortisol stimulates incorporation of [3H]fucose and the secretion of parathyroid hormone even in the presence of inhibitory doses of prostaglandin E1. It is concluded that, in organ culture, prostaglandin E1 inhibits the secretion of parathyroid hormone and of a specific glycoprotein the function of which may be related to the secretion of the hormone.

Comparison of the lytic effects of four prostaglandin analogues in the chacma baboon (Papio ursinus ursinus).

Six healthy, cycling female chacma baboons (Papio ursinus ursinus) were used to determine the luteolytic effects of four prostaglandin analogues. The compounds were administered according to a study design which made provision for adequate controls. Five to seven days following ovulation, venous blood was collected and the baboon given a single intramuscular injection of a compound at a recommended dose. Blood was then collected serially every 3 h for three samples and again at 24, 48, and 72 h to determine the continued effect of the prostaglandin analogues on corpus luteum production of progesterone. It was found that PGF2alpha-1,15-lactone, 11alpha(15S)-17-phenyl-18,19,20-trinor-ent-PGE2 methyl ester and 17-phenyl-18,19,20-trinor-PGF2alpha exhibited definite luteolytic potential in this species. Equivocal results were obtained with (15S)-15-methyl-PGF2Alpha THAM and its toxic qualities resulted in the demise of three animals.

Synthesis and gastrointestinal pharmacology of some 15- and 16- modified (+/-)-11-deoxyprostaglandins.

The synthesis and gastrointestinal pharmacology of some 11-deoxyprostaglandin E1 analogues are described with results analysed for selectivity from side effects. 11-Deoxygenation reduced potency relative to PGE2 but, as has been reported for natural PGs, 15- or 16-methyl analogues were more potent than the unsubstituted parent compound in the order 16-methyl greater than 15-methyl greater than 16,16-dimethyl. The results suggest that a complex interaction between C-15 and C-16 in methyl analogues affects their profile of activity, but that none of the modifications studied conferred a substantial potency or selectivity advantage over PGE2.