The Roles Played by FP/EP3 Receptors During Pressure-lowering in Mouse Eyes Mediated by a Dual FP/EP3 Receptor Agonist.

Purpose: We investigated the intraocular pressure (IOP)-lowering effect of topical sepetaprost (SPT), a dual agonist of the FP and EP3 receptors. We explored whether certain receptors mediated the hypotensive effect of SPT and outflow facility changes in C57BL/6 mice (wild-type [WT]) and FP and EP3 receptor-deficient mice (FPKO and EP3KO mice, respectively). Methods: IOP was measured using a microneedle. Outflow facility was measured using a two-level, constant-pressure perfusion method. Results: SPT significantly reduced IOP for 8 hours after administration to WT mice. The 2-hour IOP reductions afforded by latanoprost were 15.3 ± 2.5, 1.8 ± 2.0, and 12.3 ± 2.4% in WT, FPKO, and EP3KO mice, respectively; the SPT figures were 13.6 ± 2.1, 5.9 ± 2.7, and 6.6 ± 2.6%, respectively. Latanoprost-mediated IOP reduction was significantly decreased in FPKO mice, and SPT-mediated IOP reduction was reduced in both FPKO and EP3KO mice. At 6 hours after administration, latanoprost did not significantly reduce the IOP in any tested mouse strain. SPT-mediated IOP reduction was reduced in both FPKO and EP3KO mice. IOP reduction at 6 hours was significantly higher after simultaneous administration of selective FP and EP3 receptor agonists, but IOP did not fall on administration of (only) a selective EP3 receptor agonist. SPT significantly increased outflow facility in WT mice, but less so in FPKO and EP3KO mice. Conclusions: The IOP-lowering effect of SPT lasted longer than that of latanoprost. Our data imply that this may be attributable to augmented outflow facility mediated by the FP and EP3 receptors.

Prostaglandin E2 receptor EP3 subtype in the paraventricular hypothalamic nucleus mediates corticotropin-releasing factor-induced elevation of plasma noradrenaline levels in rats.

Corticotropin-releasing factor (CRF) plays an important role in sympathetic regulation. Centrally administered CRF elevates plasma catecholamine levels, resulting in CRF-dependent hypertension and tachycardia. We previously reported that brain thromboxane A2 mediates CRF-induced elevation of plasma adrenaline levels, whereas prostanoids other than thromboxane A2 mediate elevations in plasma noradrenaline levels. However, the mechanism by which CRF induces elevations in plasma noradrenaline levels remains unknown. Previous studies have revealed that brain prostaglandin (PG) E2, but not other PGs, causes sympathetic activation. In this study, we examined the roles of brain PGE2 and its receptors in CRF-induced elevation of plasma noradrenaline levels in rats. Our results showed that intracerebroventricular pretreatment with an antagonist of the PGE2 receptor EP3 subtype, but not other subtypes, suppressed CRF-induced elevations in plasma noradrenaline levels. We also examined the role of PGE2 and EP3 receptors in the paraventricular hypothalamic nucleus (PVN), the major integrative center for sympathetic regulation, in CRF-induced elevation of plasma noradrenaline levels. Centrally administered CRF increased PGE2 levels in PVN microdialysates, and microinjection of an EP3 receptor agonist into the PVN elevated plasma noradrenaline levels. Bilateral blockade of EP3 receptors in the PVN suppressed the elevation of plasma noradrenaline levels evoked by intracerebroventricular administration and PVN-microinjection of CRF. Our results suggest that CRF stimulates PGE2 release into the PVN that activates EP3 receptors in the PVN, resulting in the elevation of plasma noradrenaline levels.

Crystal structure of the endogenous agonist-bound prostanoid receptor EP3.

Prostanoids are a series of bioactive lipid metabolites that function in an autacoid manner via activation of cognate G-protein-coupled receptors (GPCRs). Here, we report the crystal structure of human prostaglandin (PG) E receptor subtype EP3 bound to endogenous ligand PGE2 at 2.90 Å resolution. The structure reveals important insights into the activation mechanism of prostanoid receptors and provides a molecular basis for the binding modes of endogenous ligands.

Crystal structure of misoprostol bound to the labor inducer prostaglandin E2 receptor.

Misoprostol is a life-saving drug in many developing countries for women at risk of post-partum hemorrhaging owing to its affordability, stability, ease of administration and clinical efficacy. However, misoprostol lacks receptor and tissue selectivities, and thus its use is accompanied by a number of serious side effects. The development of pharmacological agents combining the advantages of misoprostol with improved selectivity is hindered by the absence of atomic details of misoprostol action in labor induction. Here, we present the 2.5 Å resolution crystal structure of misoprostol free-acid form bound to the myometrium labor-inducing prostaglandin E2 receptor 3 (EP3). The active state structure reveals a completely enclosed binding pocket containing a structured water molecule that coordinates misoprostol's ring structure. Modeling of selective agonists in the EP3 structure reveals rationales for selectivity. These findings will provide the basis for the next generation of uterotonic drugs that will be suitable for administration in low resource settings.

Prostaglandin E2 increases migration and proliferation of human glioblastoma cells by activating transient receptor potential melastatin 7 channels.

Recent studies showed that both prostaglandin E2 (PGE2) and transient receptor potential melastatin 7 (TRPM7) play important roles in migration and proliferation of human glioblastoma cells. In this study, we tested the association between PGE2 and TRPM7. We found that PGE2 increased TRPM7 currents in HEK293 and human glioblastoma A172 cells. The PGE2 EP3 receptor antagonist L-798106 abrogated the PGE2 stimulatory effect, while EP3 agonist 17-phenyl trinor prostaglandin E2 (17-pt-PGE2) mimicked the effect of PEG2 on TRPM7. The TRPM7 phosphotransferase activity-deficient mutation, K1646R had no effect on PGE2 induced increase of TRPM7 currents. Inhibition of protein kinase A (PKA) activity by Rp-cAMP increased TRPM7 currents. TRPM7 PKA phosphorylation site mutation S1269A abolished the PGE2 effect on TRPM7 currents. PGE2 increased both mRNA and membrane protein expression of TRPM7 in A172 cells. Knockdown of TRPM7 by shRNA abrogated the PGE2 stimulated migration and proliferation of A172 cells. Blockage of TRPM7 with 2-aminoethoxydiphenyl borate (2-APB) or NS8593 had a similar effect as TRPM7-shRNA. In conclusion, our results demonstrate that PGE2 activates TRPM7 via EP3/PKA signalling pathway, and that PGE2 enhances migration and proliferation of human glioblastoma cells by up-regulation of the TRPM7 channel.

Bioanalysis of sulprostone, a prostaglandin E2 analogue and selective EP3 agonist, in monkey plasma by liquid chromatography-tandem mass spectrometry.

Sulprostone is a potent prostaglandin E2 (PGE2) analogue and one of the first identified selective G-protein-coupled receptor 3 (EP3) agonists. It has been investigated as a potential antiulcer agent and frequently used in the research of EP3 antagonist. To assist pharmacokinetic and pharmacodynamic studies, a rapid and sensitive LC-MS/MS method was developed and qualified for the quantitation of sulprostone in monkey plasma. Using electrospray ionization mass spectrometry, an ammonium adduct in positive mode was chosen for analysis which had seven times of the sensitivity of the depronated ion in negative mode. Latanoprost, a prostaglandin F2α analogue, was used as the internal standard while good sensitivity and chromatography were obtained on a 2.6 µm core-shell column with pentafluorophenyl stationary phase. An assay dynamic range of 2 to 4000 ng/mL was achieved with a sample volume of 25 µL plasma on a Sciex API4000 instrument with simple protein precipitation. Several esterase inhibitors including sodium fluoride (NaF), phenylmethanesulfonyl fluoride (PMSF), diisopropylfluorophosphate (DFP), paraoxon and dichlorvos as well as wet ice conditions were explored for the stabilization of sulprostone in monkey plasma. The developed method was successfully applied for the evaluation of pharmacokinetics of sulprostone after intravenous administration of 0.5 mg/kg to cynomolgus monkey.

Synthesis and evaluation of a potent, well-balanced EP2/EP3 dual agonist.

A highly potent and well-balanced dual agonist for the EP2 and EP3 receptors is described. Optimization of the lead compound was accomplished in consideration of the relative agonist activity against each EP subtype receptor and the pharmacokinetic profile. As the result, 2-[(2-{(1R,2R)-2-[(1E,4S)-5-cyclopentyl-4-hydroxy-4-methyl-1-penten-1-yl]-5-oxocyclopentyl}eth-yl)thio]-1,3-thiazole-4-carboxylic acid (10) showed excellent potency (human EC50 EP2 = 1.1 nM, EP3 = 1.0 nM) with acceptable selectivity over the EP1 and EP4 subtypes (>2000-fold). Further fine-tuning of compound 10 led to identification of ONO-8055 as a clinical candidate. ONO-8055 was effective at an extremely low dose (0.01 mg/kg, po, bid) in rats, and dose-dependently improved voiding dysfunction in a monkey model of underactive bladder (UAB). ONO-8055 is expected to be a novel and highly promising drug for UAB.

Prostaglandin E2 facilitates neurite outgrowth in a motor neuron-like cell line, NSC-34.

Prostaglandin E2 (PGE2) exerts various biological effects by binding to E-prostanoid receptors (EP1-4). Although recent studies have shown that PGE2 induces cell differentiation in some neuronal cells such as mouse DRG neurons and sensory neuron-like ND7/23 cells, it is unclear whether PGE2 plays a role in differentiation of motor neurons. In the present study, we investigated the mechanism of PGE2-induced differentiation of motor neurons using NSC-34, a mouse motor neuron-like cell line. Exposure of undifferentiated NSC-34 cells to PGE2 and butaprost, an EP2-selective agonist, resulted in a reduction of MTT reduction activity without increase the number of propidium iodide-positive cells and in an increase in the number of neurite-bearing cells. Sulprostone, an EP1/3 agonist, also significantly lowered MTT reduction activity by 20%; however, no increase in the number of neurite-bearing cells was observed within the concentration range tested. PGE2-induced neurite outgrowth was attenuated significantly in the presence of PF-0441848, an EP2-selective antagonist. Treatment of these cells with dibutyryl-cAMP increased the number of neurite-bearing cells with no effect on cell proliferation. These results suggest that PGE2 promotes neurite outgrowth and suppresses cell proliferation by activating the EP2 subtype, and that the cAMP-signaling pathway is involved in PGE2-induced differentiation of NSC-34 cells.

Prostanoid EP3 receptor agonist sulprostone enhances pacemaker activity of colonic interstitial cells of Cajal.

EP receptor activation by PGE2 regulates gastrointestinal motility by modulating smooth muscle contractility. Interstitial cells of Cajal (ICCs) are pacemaker cells that regulate smooth muscle activity. We aimed to determine effects of the EP3 receptor agonist sulprostone on pacemaker potentials in colonic ICCs. We performed a whole cell patch clamp, RT-PCR, and Ca2+ imaging in cultured ICCs from mouse colon. Sulprostone depolarized the membrane and increased pacemaker frequency. EP3 receptor antagonist blocked these sulprostone-induced effects. EP3 receptors were expressed in ANO1-positive ICCs. Phospholipase C inhibitor or Ca2+-ATPase inhibitor from the endoplasmic reticulum blocked the sulprostone-induced effects and sulprostone increased intracellular Ca2+ ([Ca2+]i) oscillations. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blockers also suppressed the sulprostone-induced effects. Sulprostone enhanced pacemaker activity through EP3 receptors by activating HCN channels via the [Ca2+]i release pathway. Therefore, EP3 receptor activation in ICCs may modulate colonic motility and could be a therapeutic target for enhancing colonic GI motility.

Opposing effects of prostaglandin E2 receptors EP3 and EP4 on mouse and human ß-cell survival and proliferation.

OBJECTIVE: Hyperglycemia and systemic inflammation, hallmarks of Type 2 Diabetes (T2D), can induce the production of the inflammatory signaling molecule Prostaglandin E2 (PGE2) in islets. The effects of PGE2 are mediated by its four receptors, E-Prostanoid Receptors 1-4 (EP1-4). EP3 and EP4 play opposing roles in many cell types due to signaling through different G proteins, Gi and GS, respectively. We previously found that EP3 and EP4 expression are reciprocally regulated by activation of the FoxM1 transcription factor, which promotes ß-cell proliferation and survival. Our goal was to determine if EP3 and EP4 regulate ß-cell proliferation and survival and, if so, to elucidate the downstream signaling mechanisms. METHODS: ß-cell proliferation was assessed in mouse and human islets ex vivo treated with selective agonists and antagonists for EP3 (sulprostone and DG-041, respectively) and EP4 (CAY10598 and L-161,982, respectively). ß-cell survival was measured in mouse and human islets treated with the EP3- and EP4-selective ligands in conjunction with a cytokine cocktail to induce cell death. Changes in gene expression and protein phosphorylation were analyzed in response to modulation of EP3 and EP4 activity in mouse islets. RESULTS: Blockade of EP3 enhanced ß-cell proliferation in young, but not old, mouse islets in part through phospholipase C (PLC)-γ1 activity. Blocking EP3 also increased human ß-cell proliferation. EP4 modulation had no effect on ex vivo proliferation alone. However, blockade of EP3 in combination with activation of EP4 enhanced human, but not mouse, ß-cell proliferation. In both mouse and human islets, EP3 blockade or EP4 activation enhanced ß-cell survival in the presence of cytokines. EP4 acts in a protein kinase A (PKA)-dependent manner to increase mouse ß-cell survival. In addition, the positive effects of FoxM1 activation on ß-cell survival are inhibited by EP3 and dependent on EP4 signaling. CONCLUSIONS: Our results identify EP3 and EP4 as novel regulators of ß-cell proliferation and survival in mouse and human islets ex vivo.

Role of prostanoid EP3/1 receptors in mechanisms of emesis and defaecation in ferrets.

Prostanoid EP receptor agonists are used for a number of clinical indications but may be associated with gastric disturbance. In the present studies we used the ferret and sulprostone (30µg/kg, i.p.) to investigate the role of EP3/1 receptors in mechanisms of emesis and defaecation. The emetic response was antagonized significantly by (+)-(2S,3S)-3-(2-methoxybenzylamino)-2-phenlypiperidine hydrochloride (CP-99,994; 10mg/kg, i.p.; P<0.05), but not by metoclopramide (0.3 and 3mg/kg), ondansetron (0.1 and 1mg/kg), or scopolamine (3mg/kg); promethazine (3mg/kg) potentiated emesis by approximately 82% (P<0.05). Out of the drugs tested, only scopolamine (3mg/kg) reduced significantly the defaecatory and/or tenesmus response (P<0.05). Bilateral abdominal vagotomy was ineffective to reduce sulprostone (30µg/kg, i.p.)-induced emesis and defaection and/or tenesmus. However, sulprostone (10µg, i.c.v.) administered into the fourth ventricle was emetic but did not induce defaection or tenesmus. These data suggests that the action of sulprostone to induce emesis and defaecation and/or tenesmus is largely independent of the abdominal vagal system, with emesis involving central mechanisms. Emetic mechanisms appear dissociated from those mediating defaecation and/or tenesmus.

EP3 activation facilitates bladder excitability via HCN channels on ICCs.

EP3 is a receptor for prostaglandin E2 (PGE2), and although its effect on bladder excitability has attracted considerable attention, the underlying mechanism remains unclear. To investigate whether the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in the interstitial cells of Cajal (ICCs) of the bladder are involved in the effect of EP3 activation on bladder excitability, wild-type mice, HCN1 knockout (HCN1-/-) mice and rats were used in our study. Double immunofluorescence staining and immunoprecipitation assays demonstrated the interaction between EP3 and the HCN channels. Sulprostone is a selective agonist of EP3. The current density of HCN channels was enhanced by sulprostone or PGE2 using whole-cell patch clamping. Western blot analyses showed that the expression levels of HCN1 and HCN4 were higher in bladders that had undergone intravesical instillation with sulprostone than in bladders treated with normal saline (NS). Both PGE2 and sulprostone increased the calcium concentration of the ICCs, and their effects were inhibited by ZD7288 (antagonist of HCN channels) treatment. In bladder detrusor strip testing, both PGE2 and sulprostone enhanced the amplitude of the bladder detrusor in HCN1-/- mice; however, these effects were less than those in the wild-type mice. Furthermore, the effects of PGE2 and sulprostone were inhibited by ZD7288. Taken together, our results indicate that EP3 is expressed in bladder ICCs and facilitates bladder excitability via HCN channels. This study provides more comprehensive insights into the mechanism between inflammation and bladder excitability and highlights methods that can resolve bladder hyperactivity.

Structural features of subtype-selective EP receptor modulators.

Prostaglandin E2 is a potent endogenous molecule that binds to four different G-protein-coupled receptors: EP1-4. Each of these receptors is a valuable drug target, with distinct tissue localisation and signalling pathways. We review the structural features of EP modulators required for subtype-selective activity, as well as the structural requirements for improved pharmacokinetic parameters. Novel EP receptor subtype selective agonists and antagonists appear to be valuable drug candidates in the therapy of many pathophysiological states, including ulcerative colitis, glaucoma, bone healing, B cell lymphoma, neurological diseases, among others, which have been studied in vitro, in vivo and in early phase clinical trials.

The EP1/EP3 receptor agonist 17-pt-PGE2 acts as an EP4 receptor agonist on endothelial barrier function and in a model of LPS-induced pulmonary inflammation.

Endothelial dysfunction is a hallmark of inflammatory conditions. We recently demonstrated that prostaglandin (PG)E2 enhances the resistance of pulmonary endothelium in vitro and counteracts lipopolysaccharide (LPS)-induced pulmonary inflammation in vivo via EP4 receptors. The aim of this study was to investigate the role of the EP1/EP3 receptor agonist 17-phenyl-trinor-(pt)-PGE2 on acute lung inflammation in a mouse model. In LPS-induced pulmonary inflammation in mice, 17-pt-PGE2 reduced neutrophil infiltration and inhibited vascular leakage. These effects were unaltered by an EP1 antagonist, but reversed by EP4 receptor antagonists. 17-pt-PGE2 increased the resistance of pulmonary microvascular endothelial cells and prevented thrombin-induced disruption of endothelial junctions. Again, these effects were not mediated via EP1 or EP3 but through activation of the EP4 receptor, as demonstrated by the lack of effect of more selective EP1 and EP3 receptor agonists, prevention of these effects by EP4 antagonists and EP4 receptor knock-down by siRNA. In contrast, the aggregation enhancing effect of 17-pt-PGE2 in human platelets was mediated via EP3 receptors. Our results demonstrate that 17-pt-PGE2 enhances the endothelial barrier in vitro on pulmonary microvascular endothelial cells, and accordingly ameliorates the recruitment of neutrophils, via EP4 receptors in vivo. This suggests a beneficial effect of 17-pt-PGE2 on pulmonary inflammatory diseases.

Prostaglandin E2 Reduces Cardiac Contractility via EP3 Receptor.

BACKGROUND: Prostaglandin E2 (PGE2) EP receptors EP3 and EP4 signal via decreased and increased cAMP production, respectively. Previously, we reported that cardiomyocyte-specific EP4 knockout mice develop dilated cardiomyopathy with reduced ejection fraction. Thus, we hypothesized that PGE2 increases contractility via EP4 but decreases contractility via EP3. METHODS AND RESULTS: The effects of PGE2 and the EP1/EP3 agonist sulprostone on contractility were examined in the mouse Langendorff preparation and in adult mouse cardiomyocytes. Isolated hearts of adult male C57Bl/6 mice were perfused with PGE2 (10(-6) M) or sulprostone (10(-6) M) and compared with vehicle. Both PGE2 and sulprostone decreased +dp/dt (P<0.01) and left ventricular developed pressure (P<0.001) with reversal by an EP3 antagonist. In contrast, the EP4 agonist had the opposite effect. Adult mouse cardiomyocytes contractility was also reduced after treatment with either PGE2 or sulprostone for 10 minutes. We then examined the acute effects of PGE2, sulprostone, and the EP4 agonist on expression of phosphorylated phospholamban and sarcoendoplasmic reticulum Ca(2+)-ATPase 2a in adult mouse cardiomyocytes using Western blot. Treatment with either PGE2 or sulprostone decreased expression of phosphorylated phospholamban corrected to total phospholamban, whereas treatment with the EP4 agonist had the opposite effect. Sarcoendoplasmic reticulum Ca(2+)-ATPase 2a expression was unaffected. Finally, we examined the effect of these compounds in vivo using pressure-volume loops. Both PGE2 and sulprostone decreased +dp/dt, whereas the EP4 agonist increased +dp/dt. CONCLUSIONS: Contractility is reduced via the EP3 receptor but increased via EP4. These effects may be mediated through changes in phospholamban phosphorylation and has relevance to detrimental effects of inflammation.

Therapeutic effects of human gingiva-derived mesenchymal stromal cells on murine contact hypersensitivity via prostaglandin E2-EP3 signaling.

BACKGROUND: The immunomodulatory and anti-inflammatory functions of human gingiva-derived mesenchymal stromal cells (GMSCs) have been demonstrated in contact hypersensitivity (CHS) models; however, their therapeutic effect during the late phase of CHS has been poor. METHODS: The murine CHS model was induced by applying oxazolone to the ears of mice. Mesenchymal stromal cells were applied via two methods (intravenous or local injection) at three time points: 1 day before sensitization, 1 day before challenge, or 1 h after challenge. Prostaglandin E2 (PGE2) and sulprostone were administered subcutaneously 1 h after challenge. RESULTS: The application of GMSCs, bone marrow mesenchymal stem cells, and adipose-derived stem cells all effectively suppressed CHS; however, GMSC treatment exhibited the greatest efficacy. Local injection of GMSCs led to a more marked attenuation of CHS compared with intravenous injection, especially during the late phase of CHS, and this manifested as decreased infiltration of inflammatory cells, suppression of the levels of various proinflammatory cytokines, reconstruction of the disrupted Th1/Th2 balance, and upregulation of regulatory T cells in the allergen contact areas. Pretreatment with indomethacin significantly abrogated the GMSC-mediated immunosuppressive effects, while PGE2 application reversed the effects of indomethacin pretreatment of GMSCs. Moreover, GMSC administration promoted the expression of EP3, a prostaglandin E receptor, and the application of sulprostone, an agonist of EP3, significantly attenuated CHS to a similar degree as that of GMSC administration. CONCLUSIONS: GMSCs have reproducible and powerful immunomodulatory functions. Local injection of GMSCs is the superior mode for therapeutic application. PGE2-EP3 signaling plays an important role in the immunomodulatory functions of GMSCs in murine CHS.

An EP2 Agonist Facilitates NMDA-Induced Outward Currents and Inhibits Dendritic Beading through Activation of BK Channels in Mouse Cortical Neurons.

Prostaglandin E2 (PGE2), a major metabolite of arachidonic acid produced by cyclooxygenase pathways, exerts its bioactive responses by activating four E-prostanoid receptor subtypes, EP1, EP2, EP3, and EP4. PGE2 enables modulating N-methyl-D-aspartate (NMDA) receptor-mediated responses. However, the effect of E-prostanoid receptor agonists on large-conductance Ca(2+)-activated K(+) (BK) channels, which are functionally coupled with NMDA receptors, remains unclear. Here, we showed that EP2 receptor-mediated signaling pathways increased NMDA-induced outward currents (I NMDA-OUT), which are associated with the BK channel activation. Patch-clamp recordings from the acutely dissociated mouse cortical neurons revealed that an EP2 receptor agonist activated I NMDA-OUT, whereas an EP3 receptor agonist reduced it. Agonists of EP1 or EP4 receptors showed no significant effects on I NMDA-OUT. A direct perfusion of 3,5'-cyclic adenosine monophosphate (cAMP) through the patch pipette facilitated I NMDA-OUT, which was abolished by the presence of protein kinase A (PKA) inhibitor. Furthermore, facilitation of I NMDA-OUT caused by an EP2 receptor agonist was significantly suppressed by PKA inhibitor. Finally, the activation of BK channels through EP2 receptors facilitated the recovery phase of NMDA-induced dendritic beading in the primary cultured cortical neurons. These results suggest that a direct activation of BK channels by EP2 receptor-mediated signaling pathways plays neuroprotective roles in cortical neurons.

Promising Effects of a Novel EP2 and EP3 Receptor Dual Agonist, ONO-8055, on Neurogenic Underactive Bladder in a Rat Lumbar Canal Stenosis Model.

PURPOSE: We investigated whether the novel EP (prostaglandin E2) receptor agonist ONO-8055 would improve the lower urinary tract dysfunction of neurogenic underactive bladder in a rat lumbar spinal canal stenosis model. MATERIALS AND METHODS: First, we studied the agonistic effect of ONO-8055 on EP receptors in EP receptor expressing CHO (Chinese hamster ovary) cells using the increase in the intracellular calcium level and intracellular cAMP (cyclic adenosine monophosphate) production as indicators of receptor activation. The effects of ONO-8055 on bladder and urethral strips from normal rats were then investigated. Finally, the effects of ONO-8055 on bladder and urethral function in rats with lumbar spinal canal stenosis were evaluated by awake cystometry and intraurethral perfusion pressure, respectively. The effects of tamsulosin and distigmine on urethral pressure were also evaluated. RESULTS: ONO-8055 is a highly potent and selective agonist for EP2 and EP3 receptors on CHO cells. While this compound contracted bladder strips, it relaxed urethral strips. Awake cystometry showed that ONO-8055 significantly decreased bladder capacity, post-void residual urine and voiding pressure. Compared with vehicle, tamsulosin and ONO-8055 significantly decreased urethral pressure. CONCLUSIONS: ONO-8055 decreased post-void residual urine, probably by decreasing bladder capacity. The decrease in voiding pressure probably resulted from the lowered urethral pressure due to relaxation of the urethra. Thus, the novel EP2 and EP3 receptor dual agonist ONO-8055 has the potential to improve neurogenic underactive bladder.

Discovery of highly potent dual EP(2) and EP(3) agonists with subtype selectivity.

The cyclic carbamate derivatives, 2-{[2-((4S)-4-{(1E,3R)-8-fluoro-3-hydroxy-4,4-dimethyl-1-octenyl}-2-oxo-1,3-oxazolidin-3-yl)ethyl]sulfanyl}-1,3-thiazole-4-carboxylic acid (5) and 2-{[2-((4S)-4-{(1E,3R)-3-[1-(4-fluorobutyl)cyclobutyl]-3-hydroxy-1-propenyl}-2-oxo-1,3-oxazolidin-3-yl)ethyl]sulfanyl}-1,3-thiazole-4-carboxylic acid (7) were identified as the first potent dual EP2 and EP3 agonists with selectivity against the EP1 and EP4 subtypes. Compounds 5 and 7 demonstrated highly potent dual EP2 and EP3 agonist activity with EC50 values of 10nM or less. In addition, these compounds possess structural features distinct from natural prostaglandins, such as a cyclic carbamate moiety, a dimethyl or cyclobutyl group and a terminal fluorine atom.