Selective EP4 Receptor Antagonist Research Articles

Discovery of Novel, Selective Prostaglandin EP4 Receptor Antagonists with Efficacy in Cancer Models.

Prostaglandin E2 (PGE2) receptor 4 (EP4) is one of four EP receptors commonly upregulated in the tumor microenvironment and plays vital roles in stimulating cell proliferation, invasion, and metastasis. Biochemical blockade of the PGE2-EP4 signaling pathway is a promising strategy for controlling inflammatory and immune related disorders. Recently combination therapies of EP4 antagonists with anti-PD-1 or chemotherapy agents have emerged in clinical studies for lung, breast, colon, and pancreatic cancers. Herein, a novel series of indole-2-carboxamide derivatives were identified as selective EP4 antagonists, and SAR studies led to the discovery of the potent compound 36. Due to favorable pharmacokinetics properties and good oral bioavailability (F = 76%), compound 36 was chosen for in vivo efficacy studies. Compound 36 inhibited tumor growth in a CT-26 colon cancer xenograft better than E7046 and a combination of 36 with capecitabine significantly suppressed tumor growth (TGI up to 94.26%) in mouse models.

Abstract 2330: Discovery of EP4 receptor antagonist KF-0210 for the treatment of cancers

Abstract Background: It is known that tumors with more infiltrated immune cells have better responses to PD-1/PD-L1 antibody therapy. PGE2 is involved in the regulation of the cancer microenvironment and tumor growth, mainly through the EP4 receptor. Inhibition of the EP4 receptor may increase tumor-killing immune cell infiltration and thus increase the response to PD-1/PD-L1 antibody therapy. Here we reported a potent and selective EP4 receptor antagonist, KF-0210, for the treatment of cancers. Methods: Multiple experiments were conducted to characterize KF-0210 and to evaluate its anti-tumor activities in vitro and in vivo, including receptor binding assay, cAMP release in cultured cells, and in vivo pharmacokinetic and toxicity studies. Syngeneic mouse tumor models were used to evaluate the effect of KF-0210 on immune cell infiltration and tumor growth. Results: In the EP4 receptor binding assay, KF-0210 showed a potent binding to EP4 receptor (IC50=18.6nM). Its binding potency to EP4 receptor was >500 fold higher than to other prostaglandin receptors, including EP1, EP2, EP3, IP, DP1, and FP. PGE2-induced cAMP production was inhibited by KF-0210 in MCF-7 cells (IC50 = 1.06 nM) and in Flpin-293-mEP4 cells (IC50 = 0.915 nM). In human PBMCs, KF-0210 increased LPS-induced TNFα release (IC50 = 4.5 nM) in the presence of PGE2. Oral administration of KF-0210 showed superior plasma exposure in rats and mice compared to E7046, another EP4 antagonist under clinical development. In the CT26 mouse model of colorectal cancer, KF-0210 increased the tumor-killing immune cells in the tumor. Tumor growth was attenuated by 16%, 36%, and 42% respectively when treated with 10mg/kg, 30 mg/kg/, and 150 mg/kg KF-0210. In the combination therapy studies, when animals were treated with KF-0210 or anti-PD-1 antibody alone, tumor growth was attenuated by 68% and 83% respectively. While combination treatment of KF-0210 and PD-1 antibody achieved 95% inhibition of tumor growth (P<0.001), superior to the monotherapy of either KF-0210 or PD-1 antibody. Similar results were also observed when KF-0210 was combined with PD-L1 antibody in the same model. In the mouse EMT6 breast cancer model, the combination treatment of KF-0210 and PD-1 antibody also showed a superior effect in inhibiting tumor growth. No significant safety issues were identified in toxicity and safety studies in vivo. Conclusion: KF-0210 is a potent and selective EP4 antagonist with a good PK and safety profile. It shows the inhibitory effect on tumor growth in mouse models of colorectal and breast cancers. The combination treatment of KF-0210 and PD-1/PD-L1 antibodies shows a superior therapeutic effect compared to the monotherapy of each treatment. These results support the clinical development of KF-0210 for cancer therapy, especially the combination therapy with PD-1/PD-L1 antibodies. KF-0210 is currently in phase 1 clinical study and the preliminary data showed good safety and PK profile. Citation Format: Yongqi Deng, Yuan Tian, Tao Rong, Xiangdong Gan, Shuai Qin, Yuanqi Zhou, Xiaomei Wang, Yanlin Jia. Discovery of EP4 receptor antagonist KF-0210 for the treatment of cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2330.

Scaffold Hopping Strategy to Identify Prostanoid EP4 Receptor Antagonists for Cancer Immunotherapy.

Cancer cells can effectively suppress the natural immune response in humans, and prostaglandin E2 (PGE2) is a key mediator in the development of tumor cell resistance to immunotherapy. As a major contributor to PGE2-elicited immunosuppressive activity, the EP4 receptor promotes tumor development and progression in the tumor microenvironment, and the development of selective and potent EP4 receptor antagonists should have promising potential for tumor immunotherapy. Aiming at improving the drug-like properties, a series of 4,7-dihydro-5H-thieno[2,3-c]pyran derivatives were designed and synthesized through a scaffold hopping strategy. The most promising compound 47 exhibited good EP4 antagonistic activity and excellent subtype selectivity, as well as favorable drug-like properties. It effectively suppressed the expression of multiple immunosuppression-related genes in macrophages. Meanwhile, oral administration of compound 47, alone or in combination with anti-PD-1 antibody, significantly enhanced the antitumor immune response and inhibited tumor growth in the mouse CT26 colon carcinoma model.

EP1 receptor antagonism mitigates early and late stage renal fibrosis.

AimRenal fibrosis is a major driver of chronic kidney disease, yet current treatment strategies are ineffective in attenuating fibrogenesis. The cyclooxygenase/prostaglandin system plays a key role in renal injury and holds great promise as a therapeutic target. Here, we used a translational approach to evaluate the role of the PGE2‐EP1 receptor in the pathogenesis of renal fibrosis in several models of kidney injury, including human (fibrotic) kidney slices.MethodsThe anti‐fibrotic efficacy of a selective EP1 receptor antagonist (SC‐19220) was studied in mice subjected to unilateral ureteral obstruction (UUO), healthy and fibrotic human precision‐cut kidney slices (PCKS), Madin‐Darby Canine Kidney (MDCK) cells and primary human renal fibroblasts (HRFs). Fibrosis was evaluated on gene and protein level using qPCR, western blot and immunostaining.ResultsEP1 receptor inhibition diminished fibrosis in UUO mice, illustrated by a decreased protein expression of fibronectin (FN) and α‐smooth muscle actin (αSMA) and a reduction in collagen deposition. Moreover, treatment of healthy human PCKS with SC‐19220 reduced TGF‐β‐induced fibrosis as shown by decreased expression of collagen 1A1, FN and αSMA as well as reduced collagen deposition. Similar observations were made using fibrotic human PCKS. In addition, SC‐19220 reduced TGF‐β‐induced FN expression in MDCK cells and HRFs.ConclusionThis study highlights the EP1 receptor as a promising target for preventing both the onset and late stage of renal fibrosis. Moreover, we provide strong evidence that the effect of SC‐19220 may translate to clinical care since its effects were observed in UUO mice, cells and human kidney slices.

Abstract 6697: DT095895, a selective EP4 receptor antagonist with monotherapy efficacy in syngeneic mouse model(s) and best-in-class properties

Abstract Elevated levels of Prostaglandin E2 (PGE2), an eicosanoid notably synthesized by the cyclooxygenase-2 (COX-2), exert strong immunosuppressive effects in the tumor microenvironment. COX-2-positive solid tumors have the ability to use this pathway as a resistance mechanism, especially to escape from the host immune system, thus limiting the anti-tumor effects of immune checkpoint inhibitors (ICI). These immunosuppressive effects are largely mediated by the EP4 receptor, expressed on multiple immune cells. A novel series of EP4 receptor antagonists has been developed, with improved pharmacokinetic properties when compared to the EP4 receptor antagonists currently being evaluated in clinical trials. An intensive lead optimization program led to the identification of DT095895, a small molecule development candidate with a “best-in class” potential. Its preclinical package will be presented in the poster. DT095895 was assessed in multiple syngeneic mouse tumor models selected for their COX-2 expression profile. Efficacy was seen both in a monotherapy setting, as well as in combination with ICI. Additionally, a specific biomarker program was implemented and validated in order to show target engagement. A phospho-flow murine whole blood assay was set up to assess the ability of DT095895 to inhibit CREB phosphorylation induced by a selective EP4 receptor agonist in CD3+ cells. This biomarker was further developed for human whole blood to support Phase 1 and clinical trials studies. In conclusion, DT095895 is a selective EP4 receptor antagonist and demonstrates strong anti-tumor effects in multiple syngeneic mouse tumor models, both as a monotherapy and in combination with ICI, through the inhibition of the PGE2-induced immunosuppression. DT095895 progresses in regulatory development. Citation Format: Anne-Laure Blayo, Laurène Deshons, Alexia Dumont, Christel Franchet, Célia Halter, Ludovic Herbin, Gaël Hommet, Stanislas Mayer, Alban Bessede, Imane Nafia, Marjorie Sidhoum, Xavier Leroy, Stephan Schann. DT095895, a selective EP4 receptor antagonist with monotherapy efficacy in syngeneic mouse model(s) and best-in-class properties [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6697.

Effect of selective inhibition or activation of PGE2 EP1 receptor on glomerulosclerosis.

Prostaglandin E2 (PGE2) is involved in numerous physiological and pathological processes of the kidney via its four receptors. A previous study has suggested that a defect in the PGE2 receptor 1 (EP1) gene markedly suppressed the transforming growth factor-β1 (TGF-β1)-induced mesangial cell (MC) proliferation and extracellular matrix aggregation. Therefore, the present study aimed to adopt a pharmacological method of specifically suppressing or activating the EP1 receptor to further verify and demonstrate these results. The EP1 receptor antagonist SC-19220 and EP1 receptor agonist 17-phenyl-trinor-PGE2 ethyl amide (17-pt-PGE2) were selectively used to treat five-sixths nephrectomy renal fibrosis model mice and TGF-β1-stimulated MCs. An Alpha screen PGE2 assay kit, flow cytometry, western blotting and immunohistochemical techniques were adopted to perform in vivo and in vitro experiments. The present results suggested that compared with the control group, the selective EP1 receptor antagonist SC-19220 improved renal function, markedly reduced the plasma blood urea nitrogen and creatinine levels (P<0.05) and alleviated glomerulosclerosis (P<0.05). By contrast, the EP1 receptor agonist 17-pt-PGE2 aggravated renal dysfunction and glomerulosclerosis (P<0.05). To verify the renal protection mechanisms mediated by suppression of the EP1 receptor, the expression levels of endoplasmic reticulum stress (ERS)-related proteins, including chaperone glucose-regulated protein 78 (GRP78), transient receptor potential channel 1 (TRPC1) and protein kinase R-like endoplasmic reticulum kinase (PERK), were further evaluated histologically. The expression of GRP78, TRPC1 and PERK in the antagonist treatment group were markedly downregulated (P<0.05), whereas those in the agonist treatment group were upregulated (P<0.05). The present in vitro experiments demonstrated that, compared with the control group, the EP1 receptor antagonist suppressed the expression of GRP78, TRPC1 and PERK (P<0.05), reduced the production of PGE2 (P<0.05) and decreased the MC apoptosis rate (P<0.05), thus alleviating TGF-β1-stimulated MC injury. Consequently, consistent with previous results, selectively antagonizing the EP1 receptor improved renal function and mitigated glomerulosclerosis, and its potential mechanism might be associated with the suppression of ERS.

AB0360 EFFICACY AND SAFETY OF THE PROSTAGLANDIN EP4 RECEPTOR ANTAGONIST CR6086 ADDED TO METHOTREXATE IN DMARD-NAÏVE EARLY RA PATIENTS: A PHASE 2 RANDOMIZED CONTROLLED TRIAL

Background:MTX is the first line treatment in early RA. There is robust evidence from cohort studies, but less from RCTs, that a “window of opportunity” exists over 12-16 weeks symptom duration. CR6086 is a selective prostaglandin EP4 receptor antagonist, with an immunomodulatory profile.Objectives:To test efficacy and safety of CR6086 added to MTX in early RA, DMARD-naïve patients.Methods:Patients with RA (ACR/EULAR 2010 criteria), &lt; 1 year from symptom onset and naïve to DMARDs were randomized to oral CR6086 30, 90, 180mg, or placebo bid and oral MTX (20mg weekly) for 13 weeks (NCT03163966). Primary endpoint was the ACR20 response rate: 240 patients were needed to detect a difference among groups, with 50% responders on placebo and 70% on the 90mg CR6086 target dose. Pairwise comparisons of proportions were performed, with nonresponder imputation for withdrawals. A subgroup of patients underwent dynamic contrast-enhanced (DCE) MRI for quantification of synovitis at MCP and wrist joints, evaluated as DEMRIQ-ME and DEMRIQ-vol.Results:The ITT population included all 244 randomized patients receiving at least one dose of study drugs (59 CR6086 30mg/MTX, 60 CR6086 90mg/MTX, 63 CR6086 180mg/MTX, 62 placebo/MTX). Safety was good with no increased rate of infections or other disorders; however, there were more minor upper GI adverse events (AEs) with CR6086, and increased dropouts due to AEs with the 180mg dose (9/63, 14.3% vs 1.7-3.4% in other groups). There were more ACR20 responders with MTX monotherapy than predicted (59.7%) and thus the 10.3% difference with the 90mg target dose (70.0%) was not significant. The low 30mg dose was no better than placebo (55.9%), while the high 180mg dose did not provide additional benefit compared with 90mg (74.0% net of dropouts). CR6086 90mg and 180mg induced a significant improvement in MRI, compared with placebo (Fig. 1). In a post-hoc analysis in patients &lt; 6 months from symptom onset (ACR definition of early RA: 98/244, 40.2%), MTX monotherapy exerted a large 76% ACR20 response rate that precluded potentiation. Conversely, in patients of 6-12 months disease duration (146/244, 59.8%) ACR20 responders were 48.6% with MTX monotherapy vs 68.4% with 90mg, i.e. a 19.8% difference as postulated, with proportional differences in secondary endpoints (Tab. 1).Conclusion:There was no benefit demonstrated for CR6086 added to MTX in the study cohort as a whole. However, in a post-hoc analysis, enhanced responses were observed with CR6086 90mg bid added to MTX in patients &gt;6 months disease duration. This generated the hypothesis that addition of CR6086 90mg bid may benefit in RA patients initiating MTX after the window of opportunity, to be tested in further studies.Table 1.Patient characteristics &amp; pregnancy outcomesSymptom onset &lt;12 months(principal analysis)Symptom onset 6-12 months(post-hoc analysis)*Placebo+MTX(N=62)CR6086 90mg+MTX(N=60)Placebo+MTX(N=37)CR6086 90mg+MTX(N=38)ACR20, %59.7%70.0%48.6%68.4%ACR50, %33.9%38.3%29.7%39.5%ACR70, %17.7%23.3%10.8%28.9%DAS28 (CRP) &lt;2.6, %12.9%20.0%8.1%18.4%CDAI ≤2.8, %8.1%11.7%5.4%15.8%SDAI ≤3.3, %6.5%10.0%2.7%15.8%Boolean-based remission, %6.5%6.7%2.7%10.5%*In patients with symptom onset &lt;6 months, MTX monotherapy exerted a large 76% ACR20 response, and correspondingly high secondary efficacy parameters, precluding potentiation in this subsetFigure 1.Change in MRI (DEMRIQ-ME#) after 13 weeksDisclosure of Interests:Karel Pavelka Consultant of: Abbvie, MSD, BMS, Egis, Roche, UCB, Medac, Pfizer, Biogen, Speakers bureau: Abbvie, MSD, BMS, Egis, Roche, UCB, Medac, Pfizer, Biogen, Ivanova Delina2 Delina: None declared, Minodora Mazur: None declared, Massimo D’Amato Employee of: Rottapharm Biotech, GIAMPAOLO GIACOVELLI Employee of: Rottapharm Biotech, Federica Girolami Employee of: Rottapharm Biotech, Marek Krogulec: None declared, René Østgård: None declared, Asger Reinstrup Bihlet Shareholder of: Nordic Bioscience A/S., Olga Kubassova Shareholder of: IAG, Image Analysis Group, Consultant of: Novartis, Takeda, Lilly, Employee of: IAG, Image Analysis Group, Lucio Rovati Shareholder of: Rottapharm Biotech, Employee of: Rottapharm Biotech, Peter C. Taylor Grant/research support from: Celgene, Eli Lilly and Company, Galapagos, and Gilead, Consultant of: AbbVie, Biogen, Eli Lilly and Company, Fresenius, Galapagos, Gilead, GlaxoSmithKline, Janssen, Nordic Pharma, Pfizer Roche, and UCB

Pharmacological properties of ASP7657, a novel, potent, and selective prostaglandin EP4 receptor antagonist.

We determined the pharmacologic profile of ASP7657, trans-4-[({[1-(quinolin-2-ylmethyl)-5-(trifluoromethyl)-1H-indol-7 yl] carbonyl} amino) methyl] cyclohexanecarboxylic acid methanesulfonate (1:1), a newly synthesized selective E-type prostaglandin (EP)4 receptor antagonist using several in vitro and in vivo experiments. ASP7657 exhibited high affinity for rat and human EP4 receptors, with Ki values of 6.02nM and 2.21nM, respectively. In addition, ASP7657 potently inhibited the PGE2-induced cyclic adenosine monophosphate (cAMP) increase in Chinese hamster ovary (CHO) cells expressing rat EP4 receptors and human lymphoblastoid T (Jurkat) cells, with IC50 values of 0.86nM and 0.29nM, respectively. In contrast, ASP7657 did not inhibit the PGE2-induced intracellular calcium increase in HEK293 cells expressing rat EP1 and EP3 receptors, or cAMP increase in CHO cells expressing rat EP2 receptors. ASP7657 showed good pharmacokinetic properties following oral dosing and dose-dependently antagonized the prostaglandin (PG)E2-mediated inhibition of lipopolysaccharide-induced tumor necrosis factor-α release from rat whole blood culture. In addition, 4weeks repeated oral administration of ASP7657 dose-dependently attenuated albuminuria in type 2 diabetic mice; these effects were significant at doses of 0.01mg/kg or higher. These results demonstrate that ASP7657 is a potent and selective EP4 receptor antagonist that may be useful in future studies to help clarify the physiological and pathophysiological roles of PG.

Analgesic and anti-inflammatory properties of novel, selective, and potent EP4 receptor antagonists.

Prostaglandin (PG) E2 is the key driver of inflammation associated with arthritic conditions. Inhibitors of PGE 2 production (NSAIDs and Coxibs) are used to treat these conditions, but carry significant side effect risks due to the inhibition of all prostanoids that play important physiological function. The activities of PGE 2 are transduced through various receptor sub‐types. Prostaglandin E2 type 4 receptor (EP4) is associated with the development of inflammation and autoimmunity. We therefore are interested in identifying novel EP4 antagonists to treat the signs and symptoms of arthritis without the potential side effects of PGE 2 modulators such as NSAIDs and Coxibs. Novel EP4 antagonists representing distinct chemical scaffolds were identified using a variety of in vitro functional assays and were shown to be selective and potent. The compounds were shown to be efficacious in animal models of analgesia, inflammation, and arthritis.

Synthesis and evaluation of 18F-labeled CJ-042794 for imaging prostanoid EP4 receptor expression in cancer with positron emission tomography

The potent and selective prostanoid EP4 receptor antagonist CJ-042794 was radiolabeled with 18F, and evaluated for imaging EP4 receptor expression in cancer with positron emission tomography (PET). The fluorination precursor, arylboronic acid pinacol ester 4, was prepared in 4 steps with 42% overall yield. 18F-CJ-042794 was synthesized via a copper-mediated 18F-fluorination reaction followed by base hydrolysis, and was obtained in 1.5±1.1% (n=2) decay-corrected radiochemical yield. PET/CT imaging and biodistribution studies in mice showed that 18F-CJ-042794 was excreted through both renal and hepatobiliary pathways with significant retention in blood. The EP4-receptor-expressing LNCaP prostate cancer xenografts were clearly visualized in PET images with 1.12±0.08%ID/g (n=5) uptake value and moderate tumour-to-muscle contrast ratio (2.73±0.22) at 1h post-injection. However, the tumour uptake was nonspecific as it could not be blocked by co-injection of cold standard, precluding the application of 18F-CJ-042794 for PET imaging of EP4 receptor expression in cancer.

Discovery of AAT-008, a novel, potent, and selective prostaglandin EP4 receptor antagonist

Starting from acylsufonamide HTS hit 2, a novel series of para-N-acylaminomethylbenzoic acids was identified and developed as selective prostaglandin EP4 receptor antagonists. Structural modifications on lead compound 4a were explored with the aim of improving potency, physicochemical properties, and animal PK predictive of QD (once a day) dosing regimen in human. These efforts led to the discovery of the clinical candidate AAT-008 (4j), which exhibited significantly improved pharmacological profiles over grapiprant (1).

Computational study of diarylcyclopentene derivatives as selective prostaglandin EP1 receptor antagonist: QSAR approach

2D quantitative structure–activity relationships (2D QSAR) studies were performed on a series of diarylcyclopentene derivatives as prostaglandin EP1 receptor antagonist. To establish the relationship between Prostaglandin EP1 receptor and diarylcyclopentene derivatives, a 2D-QSAR model based on individual, estate numbers, structural, electro topological and baumann alignment descriptors parameters was developed. The best model-1 correlation coefficient with r2 = 0.8101, significant cross validated correlation coefficient (q2 = 0.7491) and for external test set pred_r2 = 0.7812 developed by Partial Least Squares method. The QSAR model reveals that hydroxyl and methoxy group at R position on the diarylcyclopentene moiety is responsible for improving the prostaglandin EP1 receptor activity. The results of 2D QSAR studies were used to design new molecules and to predict their prostaglandin EP1 receptor activity using the developed models.

Neurophysiological mechanisms of bradykinin-evoked mucosal chloride secretion in guinea pig small intestine

To investigate the mechanism for bradykinin (BK) to stimulate intestinal secretomotor neurons and intestinal chloride secretion. Muscle-stripped guinea pig ileal preparations were mounted in Ussing flux chambers for the recording of short-circuit current (Isc). Basal Isc and Isc stimulated by BK when preincubated with the BK receptors antagonist and other chemicals were recorded using the Ussing chamber system. Prostaglandin E2 (PGE2) production in the intestine was determined by enzyme immunologic assay (EIA). Application of BK or B2 receptor (B2R) agonist significantly increased the baseline Isc compared to the control. B2R antagonist, tetrodotoxin and scopolamine (blockade of muscarinic receptors) significantly suppressed the increase in Isc evoked by BK. The BK-evoked Isc was suppressed by cyclooxygenase (COX)-1 or COX-2 specific inhibitor as well as nonselective COX inhibitors. Preincubation of submucosa/mucosa preparations with BK for 10 min significantly increased PGE2 production and this was abolished by the COX-1 and COX-2 inhibitors. The BK-evoked Isc was suppressed by nonselective EP receptors and EP4 receptor antagonists, but selective EP1 receptor antagonist did not have a significant effect on the BK-evoked Isc. Inhibitors of PLC, PKC, calmodulin or CaMKII failed to suppress BK-induced PGE2 production. The results suggest that BK stimulates neurogenic chloride secretion in the guinea pig ileum by activating B2R, through COX increasing PGE2 production. The post-receptor transduction cascade includes activation of PLC, PKC, CaMK, IP3 and MAPK.

Detection and quantification of the selective EP4 receptor antagonist CJ-023423 (grapiprant) in canine plasma by HPLC with spectrofluorimetric detection

Grapiprant, a novel pharmacologically active ingredient, acts as a selective EP4 receptor antagonist whose physiological ligand is prostaglandin E2 (PGE2). It is currently under development for use in humans and dogs for the control of pain and inflammation associated with osteoarthritis. The aim of the present study was to develop an easy and sensitive method to quantify grapiprant in canine plasma and to apply the method in a canine patient. Several parameters, both in the extraction and detection method were evaluated. The final mobile phase consisted of ACN:AcONH4 (20mM) solution, pH 4 (70:30, v/v) at a flow rate of 1mL/min. The elution of grapiprant and IS (metoclopramide) was carried out in isocratic mode through a Synergi Polar-RP 80A analytical column (150mm×4.6mm). The best excitation and emission wavelengths were 320 and 365nm, respectively. Grapiprant was extracted from the plasma using CHCl3, which gave a recovery of 88.1±10.22% and a lower limit of quantification (LLOQ) of 10ng/mL. The method was validated in terms of linearity, limit of detection (LOD), LLOQ, selectivity, accuracy and precision, extraction recovery, stability, and inter-laboratory cross validation, according to international guidelines. The chromatographic runs were specific with no interfering peaks at the retention times of the analyte and IS, as confirmed by HPLC-MS experiments. In conclusion, this was a simple and effective method using HPLC-FL to detect grapiprant in plasma, which may be useful for future pharmacokinetic studies.

Intracerebral hemorrhage outcomes following selective blockade or stimulation of the PGE2 EP1 receptor.

BackgroundInflammation following intracerebral hemorrhage (ICH) significantly contributes to secondary brain damage and poor outcomes. Prostaglandin E2 (PGE2) is known to modulate neuroinflammatory responses and is upregulated in response to brain injury as a result of changes in inducible cyclooxygenase 2 (COX-2) and the membrane-bound type of PGE synthase. Inhibition of COX-2 activity has been reported to attenuate ICH-induced brain injury; however, the clinical utility of such drugs is limited due to the potential for severe side effects. Therefore, it is now important to search for downstream targets capable of preferentially modulating PGE2 signaling, and the four E prostanoid receptors, EP1-4, which are the main targets of PGE2, remain a viable therapeutic option. We have previously shown that EP1 receptor deletion aggravates ICH-induced brain injury and impairs functional recovery, thus the current study aimed to elaborate on these results by including a pharmacologic approach targeting the EP1 receptor.ResultsChronic post-treatment with the selective EP1 receptor antagonist, SC-51089, increased lesion volume by 30.1 ± 14.5% (p < 0.05) and treatment with the EP1 agonist, 17-pt-PGE2, improved neuromuscular functional recovery on grip strength (p < 0.01) and hanging wire (p < 0.05) behavioral testing. To begin identifying the mechanisms involved in EP1-mediated neuroprotection after ICH, histology was performed to assess ferric iron content, neuroinflammation, leukocyte transendothelial migratory potential, and peripheral neutrophil and immunoglobulin infiltration. Following ICH, mice treated with the antagonist displayed increased ferric iron (p < 0.05) and cortical microgliosis (p < 0.05), whereas treatment with the agonist decreased cortical (p < 0.01) and striatal (p < 0.001) astrogliosis, leukocyte transendothelial migratory potential (p < 0.01), neutrophil infiltration (p < 0.05), and blood brain barrier breakdown (p < 0.05).ConclusionsIn agreement with our previous results, selective antagonism of the EP1 receptor aggravated ICH-induced brain injury. Furthermore, EP1 receptor agonism improved anatomical outcomes and functional recovery. Thus, the present data continues to reinforce a putative role for EP1 as a new and more selective therapeutic target for the treatment of ICH that could reduce the side effects associated with COX-2 inhibition while still exploiting the beneficial effects.

IL-1β reduces tonic contraction of mesenteric lymphatic muscle cells, with the involvement of cycloxygenase-2 and prostaglandin E2.

The lymphatic system maintains tissue homeostasis by unidirectional lymph flow, maintained by tonic and phasic contractions within subunits, 'lymphangions'. Here we have studied the effects of the inflammatory cytokine IL-1β on tonic contraction of rat mesenteric lymphatic muscle cells (RMLMC). We measured IL-1β in colon-conditioned media (CM) from acute (AC-CM, dextran sodium sulfate) and chronic (CC-CM, T-cell transfer) colitis-induced mice and corresponding controls (Con-AC/CC-CM). We examined tonic contractility of RMLMC in response to CM, the cytokines h-IL-1β or h-TNF-α (5, 10, 20 ng·mL(-1) ), with or without COX inhibitors [TFAP (10(-5) M), diclofenac (0.2 × 10(-5) M)], PGE2 (10(-5) M)], IL-1-receptor antagonist, Anakinra (5 μg·mL(-1) ), or a selective prostanoid EP4 receptor antagonist, GW627368X (10(-6) and 10(-7) M). Tonic contractility of RMLMC was reduced by AC- and CC-CM compared with corresponding control culture media, Con-AC/CC-CM. IL-1β or TNF-α was not found in Con-AC/CC-CM, but detected in AC- and CC-CM. h-IL-1β concentration-dependently decreased RMLMC contractility, whereas h-TNF-α showed no effect. Anakinra blocked h-IL-1β-induced RMLMC relaxation, and with AC-CM, restored contractility to RMLMC. IL-1β increased COX-2 protein and PGE2 production in RMLMC.. PGE2 induced relaxations in RMLMC, comparable to h-IL-1β. Conversely, COX-2 and EP4 receptor inhibition reversed relaxation induced by IL-1β. The IL-1β-induced decrease in RMLMC tonic contraction was COX-2 dependent, and mediated by PGE2 . In experimental colitis, IL-1β and tonic lymphatic contractility were causally related, as this cytokine was critical for the relaxation induced by AC-CM and pharmacological blockade of IL-1β restored tonic contraction.

Discovery and characterization of a potent and selective EP4 receptor antagonist.

EP4 is a prostaglandin E2 receptor that is a target for potential anti-nociceptive therapy. Described herein is a class of amphoteric EP4 antagonists which reverses PGE2-induced suppression of TNFα production in human whole blood. From this class, a potent and highly bioavailable compound (6) has been selected for potential clinical studies. EP4 binding and functional data, selectivity, and pharmacokinetic properties of this compound are included.

Role of the prostaglandin E2 EP1 receptor in traumatic brain injury.

Brain injuries promote upregulation of so-called proinflammatory prostaglandins, notably prostaglandin E2 (PGE2), leading to overactivation of a class of its cognate G-protein-coupled receptors, including EP1, which is considered a promising target for treatment of ischemic stroke. However, the role of the EP1 receptor is complex and depends on the type of brain injury. This study is focused on the investigation of the role of the EP1 receptor in a controlled cortical impact (CCI) model, a preclinical model of traumatic brain injury (TBI). The therapeutic effects of post-treatments with a widely studied EP1 receptor antagonist, SC-51089, were examined in wildtype and EP1 receptor knockout C57BL/6 mice. Neurological deficit scores (NDS) were assessed 24 and 48 h following CCI or sham surgery, and brain immunohistochemical pathology was assessed 48 h after surgery. In wildtype mice, CCI resulted in an obvious cortical lesion and localized hippocampal edema with an associated significant increase in NDS compared to sham-operated animals. Post-treatments with the selective EP1 receptor antagonist SC-51089 or genetic knockout of EP1 receptor had no significant effects on cortical lesions and hippocampal swelling or on the NDS 24 and 48 h after CCI. Immunohistochemistry studies revealed CCI-induced gliosis and microglial activation in selected ipsilateral brain regions that were not affected by SC-51089 or in the EP1 receptor-deleted mice. This study provides further clarification on the respective contribution of the EP1 receptor in TBI and suggests that, under this experimental paradigm, the EP1 receptor would have limited effects in modulating acute neurological and anatomical pathologies following contusive brain trauma. Findings from this protocol, in combination with previous studies demonstrating differential roles of EP1 receptor in ischemic, neurotoxic, and hemorrhagic conditions, provide scientific background and further clarification of potential therapeutic application of prospective prostaglandin G-protein-coupled receptor drugs in the clinic for treatment of TBI and other acute brain injuries.

EP2 and EP4 receptors mediate PGE2 induced relaxation in murine colonic circular muscle: Pharmacological characterization

BackgroundProstaglandin E2 (PGE2) is a regulator of gastrointestinal motility that might be involved in impaired motor function associated to gut inflammation. The aim of the present work is to pharmacologically characterize responses to exogenous and endogenous PGE2 in the mouse colon targeting EP2 and EP4 receptors. MethodsWild type (WT) and EP2 receptor knockout (EP2-KO) mice were used to characterize PGE2 and butaprost (EP2 receptor agonist) effects on smooth muscle resting membrane potential and myogenic contractility in circularly oriented colonic preparations. ResultsIn WT animals, PGE2 and butaprost concentration-dependently inhibited spontaneous contractions and hyperpolarized smooth muscle cells. Combination of both EP2 (PF-04418948 0.1μM) and EP4 receptor antagonists (L-161,982 10μM) was needed to block both electrical and mechanical PGE2 responses. Butaprost inhibitory responses (both electrical and mechanical) were totally abolished by PF-04418948 0.1μM. In EP2-KO mice, PGE2 (but not butaprost) concentration-dependently inhibited spontaneous contractions and hyperpolarized smooth muscle cells. In EP2-KO mice, PGE2 inhibition of spontaneous contractility and hyperpolarization was fully antagonized by L-161,982 10μM. In WT animals, EP2 and EP4 receptor antagonists caused a smooth muscle depolarization and an increase in spontaneous mechanical activity. ConclusionsPGE2 responses in murine circular colonic layer are mediated by post-junctional EP2 and EP4 receptors. PF-04418948 and L-161,982 are selective EP2 and EP4 receptor antagonists that inhibit PGE2 responses. These antagonists might be useful pharmacological tools to limit prostaglandin effects associated to dismotility in gut inflammatory processes.

Peripheral prostaglandin E2 prolongs the sensitization of nociceptive dorsal root ganglion neurons possibly by facilitating the synthesis and anterograde axonal trafficking of EP4 receptors

Prostaglandin E2 (PGE2), a well-known pain mediator enriched in inflamed tissues, plays a pivotal role in the genesis of chronic pain conditions such as inflammatory and neuropathic pain. PGE2-prolonged sensitization of nociceptive dorsal root ganglion (DRG) neurons (nociceptors) may contribute to the transition from acute to chronic pain. However, the underlying cellular mechanisms are poorly understood. In this study, we tested the hypothesis that facilitating synthesis and anterograde axonal trafficking of EP receptors contribute to PGE2-prolonged nociceptor sensitization. Intraplantar (i.pl.) injection of a stabilized PGE2 analog, 16,16 dimethyl PGE2 (dmPGE2), in a dose- and time-dependent manner, not only elicited primary tactile allodynia which lasted for 1d, but also prolonged tactile allodynia evoked by a subsequent i.pl. injection of dmPGE2 from 1d to 4d. Moreover, the duration of tactile allodynia was progressively prolonged following multiple sequential i.pl. injections of dmPGE2. Co-injection of the selective EP1 or EP4 receptor antagonist, the inhibitors of cAMP, PKA, PKC, PKCε or PLC as well as an interleukin-6 (IL-6) neutralizing antiserum differentially blocked primary tactile allodynia elicited by the 1st dmPGE2 and the prolonged tactile allodynia evoked by the 2nd dmPGE2, suggesting the involvement of these signaling events in dmPGE2-induced nociceptor activation and sensitization. Co-injection of a selective COX2 inhibitor or two EP4 antagonists prevented or shortened inflammagen-prolonged nociceptor sensitization. I.pl. injection of dmPGE2 or carrageenan time-dependently increased EP4 levels in L4-6 DRG neurons and peripheral nerves. EP4 was expressed in almost half of IB4-binding nociceptors of L4-6 DRG. Taken together, our data suggest that stimulating the synthesis and anterograde axonal trafficking to increase EP4 availability at the axonal terminals of nociceptors is likely a novel mechanism underlying PGE2-prolonged nociceptor sensitization. Blocking COX2/PGE2/EP4 signaling at an earlier stage of inflammation or injury is crucial for preventing the transition from acute pain to a chronic state.