PGE2 inhibits TIL expansion by disrupting IL-2 signalling and mitochondrial function.

Expansion of antigen-experienced CD8+ T cells is critical for the success of tumour-infiltrating lymphocyte (TIL)-adoptive cell therapy (ACT) in patients with cancer1. Interleukin-2 (IL-2) acts as a key regulator of CD8+ cytotoxic T lymphocyte functions by promoting expansion and cytotoxic capability2,3. Therefore, it is essential to comprehend mechanistic barriers to IL-2 sensing in the tumour microenvironment to implement strategies to reinvigorate IL-2 responsiveness and T cell antitumour responses. Here we report that prostaglandin E2 (PGE2), a known negative regulator of immune response in the tumour microenvironment4,5, is present at high concentrations in tumour tissue from patients and leads to impaired IL-2 sensing in human CD8+ TILs via the PGE2 receptors EP2 and EP4. Mechanistically, PGE2 inhibits IL-2 sensing in TILs by downregulating the IL-2Rγc chain, resulting in defective assembly of IL-2Rß-IL2Rγc membrane dimers. This results in impaired IL-2-mTOR adaptation and PGC1α transcriptional repression, causing oxidative stress and ferroptotic cell death in tumour-reactive TILs. Inhibition of PGE2 signalling to EP2 and EP4 during TIL expansion for ACT resulted in increased IL-2 sensing, leading to enhanced proliferation of tumour-reactive TILs and enhanced tumour control once the cells were transferred in vivo. Our study reveals fundamental features that underlie impairment of human TILs mediated by PGE2 in the tumour microenvironment. These findings have therapeutic implications for cancer immunotherapy and cell therapy, and enable the development of targeted strategies to enhance IL-2 sensing and amplify the IL-2 response in TILs, thereby promoting the expansion of effector T cells with enhanced therapeutic potential.

Restoring metabolism of myeloid cells reverses cognitive decline in ageing.

Ageing is characterized by the development of persistent pro-inflammatory responses that contribute to atherosclerosis, metabolic syndrome, cancer and frailty1-3. The ageing brain is also vulnerable to inflammation, as demonstrated by the high prevalence of age-associated cognitive decline and Alzheimer's disease4-6. Systemically, circulating pro-inflammatory factors can promote cognitive decline7,8, and in the brain, microglia lose the ability to clear misfolded proteins that are associated with neurodegeneration9,10. However, the underlying mechanisms that initiate and sustain maladaptive inflammation with ageing are not well defined. Here we show that in ageing mice myeloid cell bioenergetics are suppressed in response to increased signalling by the lipid messenger prostaglandin E2 (PGE2), a major modulator of inflammation11. In ageing macrophages and microglia, PGE2 signalling through its EP2 receptor promotes the sequestration of glucose into glycogen, reducing glucose flux and mitochondrial respiration. This energy-deficient state, which drives maladaptive pro-inflammatory responses, is further augmented by a dependence of aged myeloid cells on glucose as a principal fuel source. In aged mice, inhibition of myeloid EP2 signalling rejuvenates cellular bioenergetics, systemic and brain inflammatory states, hippocampal synaptic plasticity and spatial memory. Moreover, blockade of peripheral myeloid EP2 signalling is sufficient to restore cognition in aged mice. Our study suggests that cognitive ageing is not a static or irrevocable condition but can be reversed by reprogramming myeloid glucose metabolism to restore youthful immune functions.

Targeted prostaglandin E2 inhibition enhances antiviral immunity through induction of type I interferon and apoptosis in macrophages.

Aspirin gained tremendous popularity during the 1918 Spanish Influenza virus pandemic, 50 years prior to the demonstration of their inhibitory action on prostaglandins. Here, we show that during influenza A virus (IAV) infection, prostaglandin E2 (PGE2) was upregulated, which led to the inhibition of type I interferon (IFN) production and apoptosis in macrophages, thereby causing an increase in virus replication. This inhibitory role of PGE2 was not limited to innate immunity, because both antigen presentation and T cell mediated immunity were also suppressed. Targeted PGE2 suppression via genetic ablation of microsomal prostaglandin E-synthase 1 (mPGES-1) or by the pharmacological inhibition of PGE2 receptors EP2 and EP4 substantially improved survival against lethal IAV infection whereas PGE2 administration reversed this phenotype. These data demonstrate that the mPGES-1-PGE2 pathway is targeted by IAV to evade host type I IFN-dependent antiviral immunity. We propose that specific inhibition of PGE2 signaling might serve as a treatment for IAV.

PGE2 signaling via the neuronal EP2 receptor increases injury in a model of cerebral ischemia.

The inflammatory prostaglandin E2 (PGE2) EP2 receptor is a master suppressor of beneficial microglial function, and myeloid EP2 signaling ablation reduces pathology in models of inflammatory neurodegeneration. Here, we investigated the role of PGE2 EP2 signaling in a model of stroke in which the initial cerebral ischemic event is followed by an extended poststroke inflammatory response. Myeloid lineage cell-specific EP2 knockdown in Cd11bCre;EP2lox/lox mice attenuated brain infiltration of Cd11b+CD45hi macrophages and CD45+Ly6Ghi neutrophils, indicating that inflammatory EP2 signaling participates in the poststroke immune response. Inducible global deletion of the EP2 receptor in adult ROSA26-CreERT2 (ROSACreER);EP2lox/lox mice also reduced brain myeloid cell trafficking but additionally reduced stroke severity, suggesting that nonimmune EP2 receptor-expressing cell types contribute to cerebral injury. EP2 receptor expression was highly induced in neurons in the ischemic hemisphere, and postnatal deletion of the neuronal EP2 receptor in Thy1Cre;EP2lox/lox mice reduced cerebral ischemic injury. These findings diverge from previous studies of congenitally null EP2 receptor mice where a global deletion increases cerebral ischemic injury. Moreover, ROSACreER;EP2lox/lox mice, unlike EP2-/- mice, exhibited normal learning and memory, suggesting a confounding effect from congenital EP2 receptor deletion. Taken together with a precedent that inhibition of EP2 signaling is protective in inflammatory neurodegeneration, these data lend support to translational approaches targeting the EP2 receptor to reduce inflammation and neuronal injury that occur after stroke.

Prostaglandin EP2 receptor antagonist ameliorates neuroinflammation in a two-hit mouse model of Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) causes substantial medical and societal burden with no therapies ameliorating cognitive deficits. Centralized pathologies involving amyloids, neurofibrillary tangles, and neuroinflammatory pathways are being investigated to identify disease-modifying targets for AD. Cyclooxygenase-2 (COX-2) is one of the potential neuroinflammatory agents involved in AD progression. However, chronic use of COX-2 inhibitors in patients produced adverse cardiovascular effects. We asked whether inhibition of EP2 receptors, downstream of the COX-2 signaling pathway, can ameliorate neuroinflammation in AD brains in presence or absence of a secondary inflammatory stimuli. METHODS: We treated 5xFAD mice and their non-transgenic (nTg) littermates in presence or absence of lipopolysaccharide (LPS) with an EP2 antagonist (TG11-77.HCl). In cohort 1, nTg (no-hit) or 5xFAD (single-hit-genetic) mice were treated with vehicle or TG11-77.HCl for 12 weeks. In cohort 2, nTg (single-hit-environmental) and 5xFAD mice (two-hit) were administered LPS (0.5 mg/kg/week) and treated with vehicle or TG11-77.HCl for 8 weeks. RESULTS: Complete blood count analysis showed that LPS induced anemia of inflammation in both groups in cohort 2. There was no adverse effect of LPS or EP2 antagonist on body weight throughout the treatment. In the neocortex isolated from the two-hit cohort of females, but not males, the elevated mRNA levels of proinflammatory mediators (IL-1ß, TNF, IL-6, CCL2, EP2), glial markers (IBA1, GFAP, CD11b, S110B), and glial proteins were significantly reduced by EP2 antagonist treatment. Intriguingly, the EP2 antagonist had no effect on either of the single-hit cohorts. There was a modest increase in amyloid-plaque deposition upon EP2 antagonist treatment in the two-hit female brains, but not in the single-hit genetic female cohort. CONCLUSION: These results reveal a potential neuroinflammatory role for EP2 in the two-hit 5xFAD mouse model. A selective EP2 antagonist reduces inflammation only in female AD mice subjected to a second inflammatory insult.

Use of selective PGE2 receptor antagonists on human endometriotic stromal cells and peritoneal macrophages.

Non-hormonal therapeutic strategies for endometriosis are needed. The aim of this study was to characterize the effects of prostaglandin (PG)E2 receptor inhibitors to explore their potential as novel therapeutic strategies for endometriosis. The expression of PGE2 receptors (EP2 and EP4) in donated tissues from human ovarian endometriosis, adenomyosis and peritoneal endometriosis was examined using immunohistochemistry. Human endometriotic stromal cells (ESC) isolated from ovarian endometriotic tissue and peritoneal macrophages were treated with EP2 and EP4 antagonists. cAMP accumulation and the effect of EP antagonists were measured using cAMP assays. DNA synthesis in ESC was detected using bromodeoxyuridine incorporation analysis. Interleukin (IL)-6 and IL-8 protein levels in ESC supernatants were measured using ELISAs. mRNA expression level for aromatase by ESC, and selected cytokines by peritoneal macrophages was measured using RT-PCR. EP2 and EP4 receptors were expressed in cells derived from control and diseased tissue, ovarian endometriotic, adenomyotic and peritoneal lesions. A selective EP2 antagonist reduced DNA synthesis, cAMP accumulation and IL-1ß-induced proinflammatory cytokine secretion and aromatase expression. A selective EP4 antagonist negated IL-1ß-induced IL-6 secretion and aromatase expression. In peritoneal macrophages, EP expression was elevated in endometriosis samples but the EP4 antagonist reduced cAMP levels and expression of vascular endothelial growth factor, chemokine ligand 2 and chemokine ligand 3 mRNA. EP2 and EP4 are functioning in endometriosis lesions and peritoneal macrophages, and their selective antagonists can reduce EP-mediated actions, therefore, the EP antagonists are potential therapeutic agents for controlling endometriosis.

A rat model of organophosphate-induced status epilepticus and the beneficial effects of EP2 receptor inhibition.

This review describes an adult rat model of status epilepticus (SE) induced by diisopropyl fluorophosphate (DFP), and the beneficial outcomes of transient inhibition of the prostaglandin-E2 receptor EP2 with a small molecule antagonist, delayed by 2-4 h after SE onset. Administration of six doses of the selective EP2 antagonist TG6-10-1 over a 2-3 day period accelerates functional recovery, attenuates hippocampal neurodegeneration, neuroinflammation, gliosis and blood-brain barrier leakage, and prevents long-term cognitive deficits without blocking SE itself or altering acute seizure characteristics. This work has provided important information regarding organophosphate-induced seizure related pathologies in adults and revealed the effectiveness of delayed EP2 inhibition to combat these pathologies.

Manganese exposure caused reproductive toxicity of male mice involving activation of GnRH secretion in the hypothalamus by prostaglandin E2 receptors EP1 and EP2.

Exposure to manganese (Mn) can cause male reproductive damage and lead to abnormal secretion of sex hormones. Gonadotropin-releasing hormone (GnRH) plays an important role in the neuromodulation of vertebrate reproduction. Astrocytes can indirectly regulate the secretion of GnRH by binding paracrine prostaglandin E2 (PGE2) specifically to the EP1 and EP2 receptors on GnRH neurons. Prior studies assessed the abnormal secretion of GnRH caused by Mn exposure, but the specific mechanism has not been reported in detail. This study investigated the effects of Mn exposure on the reproductive system of male mice to clarify the role of PGE2 in the abnormal secretion of GnRH in the hypothalamus caused by exposure to Mn. Our data demonstrate that antagonizing the EP1 and EP2 receptors of PGE2 can restore abnormal levels of GnRH caused by Mn exposure. Mn exposure causes reduced sperm count and sperm shape deformities. These findings suggest that EP1 and EP2, the receptors of PGE2, may be the key to abnormal GnRH secretion caused by Mn exposure. Antagonizing the PGE2 receptors may reduce reproductive damage caused by Mn exposure.

EP2 and EP4 receptor antagonists: Impact on cytokine production and ß2 -adrenergic receptor desensitization in human airway smooth muscle.

Prostaglandin E2 (PGE2 ) is a key prostanoid known to have both proinflammatory and anti-inflammatory impact in the context of chronic respiratory diseases. We hypothesize that these opposing effects may be the result of different prostanoid E (EP) receptor-mediated signaling pathways. In this study, we focus on two of the four EP receptors, EP2 and EP4 , as they are known to induce cyclic adenosine monophosphate (cAMP)-dependent signaling pathways. Using primary human airway smooth muscle (ASM) cells, we first focussed on the PGE2 -induced production of two cAMP-dependent proinflammatory mediators: interleukin 6 (IL-6) and cyclo-oxygenase 2 production. We show that PGE2 -induced IL-6 protein secretion occurs via an EP2 -mediated pathway, in a manner independent of receptor-mediated effects on messenger RNA (mRNA) expression and temporal activation kinetics of the transcription factor cAMP response element binding. Moreover, stimulation of ASM with PGE2 did not establish a positive, receptor-mediated, feedback loop, as mRNA expression for EP2 and EP4 receptors were not upregulated and receptor antagonists were without effect. Our studies revealed that the EP2 , but not the EP4 , receptor is responsible for ß2 -adrenergic desensitization induced by PGE2 . We demonstrate that PGE2 -induced heterologous receptor desensitization responsible for tachyphylaxis to short- (salbutamol) or long- (formoterol) ß2 -agonists (measured by cAMP release) can be reversed by the EP2 receptor antagonist PF-04418948. Importantly, this study highlights that inhibiting the EP2 receptor restores ß2 -adrenergic receptor function in vitro and offers an attractive novel therapeutic target for treating infectious exacerbations in people suffering from chronic respiratory diseases in the future.

Prostaglandin E2 Receptor Antagonist with Antimicrobial Activity against Methicillin-Resistant Staphylococcus aureus.

Polymicrobial intra-abdominal infections (IAI) involving Candida albicans and Staphylococcus aureus are associated with severe morbidity and mortality (∼80%). Our laboratory discovered that the immunomodulatory eicosanoid prostaglandin E2 (PGE2) plays a key role in the lethal inflammatory response during polymicrobial IAI using a mouse model of infection. In studies designed to uncover key PGE2 biosynthesis/signaling components involved in the response, selective eicosanoid enzyme inhibitors and receptor antagonists were selected and prescreened for antimicrobial activity against C. albicans or S. aureus Unexpectedly, we found that the EP4 receptor antagonist L-161,982 had direct growth-inhibitory effects on S. aureusin vitro at the physiological concentration required to block the PGE2 interaction with EP4 This antimicrobial activity was observed with methicillin-sensitive S. aureus and methicillin-resistant S. aureus (MRSA) strains, with the MIC and minimum bactericidal concentration values for planktonic cells being 50 µg/ml and 100 µg/ml, respectively. In addition, L-161,982 inhibited S. aureus biofilm formation and had activity against preformed mature biofilms. More importantly, treatment of mice with L-161,982 following intraperitoneal inoculation with a lethal dose of MRSA significantly reduced the bioburden and enhanced survival. Furthermore, L-161,982 protected mice against the synergistic lethality induced by coinfection with C. albicans and S. aureus The antimicrobial activity of L-161,982 is independent of EP4 receptor inhibitory activity; an alternative EP4 receptor antagonist exerted no antimicrobial or protective effects. Taken together, these findings demonstrate that L-161,982 has potent antimicrobial activity against MRSA and may represent a significant therapeutic alternative in improving the prognosis of mono- or polymicrobial infections involving MRSA.

Peripherally Restricted, Highly Potent, Selective, Aqueous-Soluble EP2 Antagonist with Anti-Inflammatory Properties.

The prostaglandin E2 receptor, EP2, plays an important role in physiology and in a variety of pathological conditions. Studies indicate that EP2 is pro-inflammatory in chronic peripheral and central nervous system disease and cancer models. Thus, targeting the EP2 receptor with small molecules could be a therapeutic strategy for treating inflammatory diseases and cancer. We recently reported a novel class of competitive antagonists of the EP2 receptor. However, earlier leads displayed low selectivity against the DP1 prostanoid receptor, moderate plasma half-life, and low aqueous solubility, which renders them suboptimal for testing in animal models of disease. We now report a novel compound TG8-69, which has suitable drug-like properties. We present synthesis, lead-optimization studies, pharmacological characterization, and anti-inflammatory properties of this compound that support its use in chronic peripheral inflammatory diseases, including rheumatoid arthritis, endometriosis, and cancer, in which EP2 appears to play a pathogenic role.

EP2 receptor plays pivotal roles in generating mechanical hyperalgesia after lengthening contractions.

We previously demonstrated that nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF) were upregulated after lengthening contractions (LC) in exercised muscle through B2 bradykinin receptor activation and cyclooxygenase (COX)-2 upregulation, respectively, and that these trophic factors sensitized nociceptors resulting in mechanical hyperalgesia (delayed-onset muscle soreness, DOMS). Here, we examined the prostaglandin receptor subtype involved in DOMS. The mechanical withdrawal threshold of the exercised muscle was measured before and after LC in rats administered prostaglandin E2 receptor (EP) antagonists before LC, or in wild-type (WT), EP2 knockout (EP2-/- ), and IP knockout (IP-/- ) mice. The change in expression of NGF, GDNF, or COX-2 mRNA was examined using real-time RT-PCR in the muscle in EP2-/- and WT mice. None of the antagonists to EP1, EP3, and EP4 receptors (ONO-8713, ONO-AE5-599, and ONO-AE3-208, respectively) induced a significant difference in DOMS compared with controls in rats. WT and IP-/- mice developed mechanical hyperalgesia after LC, but EP2-/- mice did not. Upregulation of NGF, GDNF, and COX-2 mRNA was observed after LC in WT mice but not in EP2-/- mice. Injecting an EP2 agonist (ONO-AE1-259-01) into the mouse muscle increased expression of COX-2 mRNA. These results suggest that EP2 contributes to generating mechanical hyperalgesia through positive feedback upregulation of COX-2 expression in muscle after LC.

Molecular and preclinical basis to inhibit PGE2 receptors EP2 and EP4 as a novel nonsteroidal therapy for endometriosis.

Endometriosis is a debilitating, estrogen-dependent, progesterone-resistant, inflammatory gynecological disease of reproductive age women. Two major clinical symptoms of endometriosis are chronic intolerable pelvic pain and subfertility or infertility, which profoundly affect the quality of life in women. Current hormonal therapies to induce a hypoestrogenic state are unsuccessful because of undesirable side effects, reproductive health concerns, and failure to prevent recurrence of disease. There is a fundamental need to identify nonestrogen or nonsteroidal targets for the treatment of endometriosis. Peritoneal fluid concentrations of prostaglandin E2 (PGE2) are higher in women with endometriosis, and this increased PGE2 plays important role in survival and growth of endometriosis lesions. The objective of the present study was to determine the effects of pharmacological inhibition of PGE2 receptors, EP2 and EP4, on molecular and cellular aspects of the pathogenesis of endometriosis and associated clinical symptoms. Using human fluorescent endometriotic cell lines and chimeric mouse model as preclinical testing platform, our results, to our knowledge for the first time, indicate that selective inhibition of EP2/EP4: (i) decreases growth and survival of endometriosis lesions; (ii) decreases angiogenesis and innervation of endometriosis lesions; (iii) suppresses proinflammatory state of dorsal root ganglia neurons to decrease pelvic pain; (iv) decreases proinflammatory, estrogen-dominant, and progesterone-resistant molecular environment of the endometrium and endometriosis lesions; and (v) restores endometrial functional receptivity through multiple mechanisms. Our novel findings provide a molecular and preclinical basis to formulate long-term nonestrogen or nonsteroidal therapy for endometriosis.

Prostanoid EP2 Receptors Are Up-Regulated in Human Pulmonary Arterial Hypertension: A Key Anti-Proliferative Target for Treprostinil in Smooth Muscle Cells.

Prostacyclins are extensively used to treat pulmonary arterial hypertension (PAH), a life-threatening disease involving the progressive thickening of small pulmonary arteries. Although these agents are considered to act therapeutically via the prostanoid IP receptor, treprostinil is the only prostacyclin mimetic that potently binds to the prostanoid EP2 receptor, the role of which is unknown in PAH. We hypothesised that EP2 receptors contribute to the anti-proliferative effects of treprostinil in human pulmonary arterial smooth muscle cells (PASMCs), contrasting with selexipag, a non-prostanoid selective IP agonist. Human PASMCs from PAH patients were used to assess prostanoid receptor expression, cell proliferation, and cyclic adenosine monophosphate (cAMP) levels following the addition of agonists, antagonists or EP2 receptor small interfering RNAs (siRNAs). Immunohistochemical staining was performed in lung sections from control and PAH patients. We demonstrate using selective IP (RO1138452) and EP2 (PF-04418948) antagonists that the anti-proliferative actions of treprostinil depend largely on EP2 receptors rather than IP receptors, unlike MRE-269 (selexipag-active metabolite). Likewise, EP2 receptor knockdown selectively reduced the functional responses to treprostinil but not MRE-269. Furthermore, EP2 receptor levels were enhanced in human PASMCs and in lung sections from PAH patients compared to controls. Thus, EP2 receptors represent a novel therapeutic target for treprostinil, highlighting key pharmacological differences between prostacyclin mimetics used in PAH.

Differential expression of E-type prostanoid receptors 2 and 4 in microglia stimulated with lipopolysaccharide.

BACKGROUND: Cyclooxygenase-2 (COX-2) is induced under inflammatory conditions, and prostaglandin E2 (PGE2) is one of the products of COX activity. PGE2 has pleiotropic actions depending on the activation of specific E-type prostanoid EP1-4 receptors. We investigated the involvement of PGE2 and EP receptors in glial activation in response to an inflammatory challenge induced by LPS. METHODS: Cultures of mouse microglia or astroglia cells were treated with LPS in the presence or absence of COX-2 inhibitors, and the production of PGE2 was measured by ELISA. Cells were treated with PGE2, and the effect on LPS-induced expression of TNF-α messenger RNA (mRNA) and protein was studied in the presence or absence of drug antagonists of the four EP receptors. EP receptor expression and the effects of EP2 and EP4 agonists and antagonists were studied at different time points after LPS. RESULTS: PGE2 production after LPS was COX-2-dependent. PGE2 reduced the glial production of TNF-α after LPS. Microglia expressed higher levels of EP4 and EP2 mRNA than astroglia. Activation of EP4 or EP2 receptors with selective drug agonists attenuated LPS-induced TNF-α in microglia. However, only antagonizing EP4 prevented the PGE2 effect demonstrating that EP4 was the main target of PGE2 in naïve microglia. Moreover, the relative expression of EP receptors changed during the course of classical microglial activation since EP4 expression was strongly depressed while EP2 increased 24 h after LPS and was detected in nuclear/peri-nuclear locations. EP2 regulated the expression of iNOS, NADPH oxidase-2, and vascular endothelial growth factor. NADPH oxidase-2 and iNOS activities require the oxidation of NADPH, and the pentose phosphate pathway is a main source of NADPH. LPS increased the mRNA expression of the rate-limiting enzyme of the pentose pathway glucose-6-phosphate dehydrogenase, and EP2 activity was involved in this effect. CONCLUSIONS: These results show that while selective activation of EP4 or EP2 exerts anti-inflammatory actions, EP4 is the main target of PGE2 in naïve microglia. The level of EP receptor expression changes from naïve to primed microglia where the COX-2/PGE2/EP2 axis modulates important adaptive metabolic changes.

Pharmacokinetics and estimated bioavailability of grapiprant, a novel selective prostaglandin E2 receptor antagonist, after oral administration in fasted and fed dogs.

AIMS: To assess the effect of food intake on the pharmacokinetics of grapiprant administered orally at 2 mg/kg, and to estimate its oral bioavailability in dogs. METHODS: Eight healthy female Labrador Retriever dogs, aged 4-10 years were used. In the initial trial two dogs were administered a 0.5 mg/kg I/V bolus of grapiprant dissolved in ethanol. In the second trial, six dogs were assigned to two treatment groups, using a randomised cross-over design, and received 2 mg/kg of grapiprant orally, as pure powder, after fasting for 12 hours or after being fed. Blood samples were collected at preassigned times up to 36 hours after administration, and concentrations of grapiprant in plasma determined using validated high performance liquid chromatography. RESULTS: After I/V administration in the two dogs the terminal half life was 5.30 and 6.06 hours, clearance was 444 and 476 mL/hours/kg, and volume of distribution was 3,642 and 3,883 mL/kg. Compared with fasted dogs, oral administration in fed dogs resulted in reduced median peak concentrations in plasma (1,598 vs. 614 ng/mL) and delayed median time of peak concentration (1.0 vs. 3.0 hours). The estimated bioavailability in fasted and fed dogs was 111.9 and 59.1%, respectively. Concentrations of grapiprant in plasma following oral administration, in either fed or fasted dogs, remained higher than 164 ng/mL for up to 6 hours. This concentration has been estimated to be the minimal effective concentration required to control pain in dogs. CONCLUSION AND RELEVANCE: Oral administration of 2 mg/kg grapiprant in fed and fasted dogs resulted in different pharmacokinetics of the drug, but did not influence the length of time when concentrations in plasma exceeded theoretical effective concentrations. Further studies are necessary to verify these findings using pharmacokinetic-pharmacodynamic studies and in clinical subjects.

Efficient T-cell priming and activation requires signaling through prostaglandin E2 (EP) receptors.

Understanding the regulation of T-cell responses during inflammation and auto-immunity is fundamental for designing efficient therapeutic strategies against immune diseases. In this regard, prostaglandin E2 (PGE2) is mostly considered a myeloid-derived immunosuppressive molecule. We describe for the first time that T cells secrete PGE2 during T-cell receptor stimulation. In addition, we show that autocrine PGE2 signaling through EP receptors is essential for optimal CD4(+) T-cell activation in vitro and in vivo, and for T helper 1 (Th1) and regulatory T cell differentiation. PGE2 was found to provide additive co-stimulatory signaling through AKT activation. Intravital multiphoton microscopy showed that triggering EP receptors in T cells is also essential for the stability of T cell-dendritic cell (DC) interactions and Th-cell accumulation in draining lymph nodes (LNs) during inflammation. We further demonstrated that blocking EP receptors in T cells during the initial phase of collagen-induced arthritis in mice resulted in a reduction of clinical arthritis. This could be attributable to defective T-cell activation, accompanied by a decline in activated and interferon-γ-producing CD4(+) Th1 cells in draining LNs. In conclusion, we prove that T lymphocytes secret picomolar concentrations of PGE2, which in turn provide additive co-stimulatory signaling, enabling T cells to attain a favorable activation threshold. PGE2 signaling in T cells is also required for maintaining long and stable interactions with DCs within LNs. Blockade of EP receptors in vivo impairs T-cell activation and development of T cell-mediated inflammatory responses. This may have implications in various pathophysiological settings.

Inhibition of the prostaglandin receptor EP2 following status epilepticus reduces delayed mortality and brain inflammation.

Prostaglandin E2 is now widely recognized to play critical roles in brain inflammation and injury, although the responsible prostaglandin receptors have not been fully identified. We developed a potent and selective antagonist for the prostaglandin E2 receptor subtype EP2, TG6-10-1, with a sufficient pharmacokinetic profile to be used in vivo. We found that in the mouse pilocarpine model of status epilepticus (SE), systemic administration of TG6-10-1 completely recapitulates the effects of conditional ablation of cyclooxygenase-2 from principal forebrain neurons, namely reduced delayed mortality, accelerated recovery from weight loss, reduced brain inflammation, prevention of blood-brain barrier opening, and neuroprotection in the hippocampus, without modifying seizures acutely. Prolonged SE in humans causes high mortality and morbidity that are associated with brain inflammation and injury, but currently the only effective treatment is to stop the seizures quickly enough with anticonvulsants to prevent brain damage. Our results suggest that the prostaglandin receptor EP2 is critically involved in neuroinflammation and neurodegeneration, and point to EP2 receptor antagonism as an adjunctive therapeutic strategy to treat SE.

Prostaglandin E2 inhibits mast cell-dependent bronchoconstriction in human small airways through the E prostanoid subtype 2 receptor.

BACKGROUND: Inhaled prostaglandin (PG) E2 might inhibit asthmatic responses, but the mechanisms involved remain undefined. OBJECTIVE: We sought to characterize the direct and indirect effects of PGE2 on human small airways with particular reference to the receptors mediating the responses. METHODS: Contraction and relaxation were studied in isolated human bronchi with an inner diameter of 1 mm or less. RESULTS: Low concentrations of PGE2 (0.01-1 µmol/L) relaxed the bronchi precontracted by histamine. The bronchodilator response was inhibited by the E prostanoid (EP) subtype 4 receptor antagonist ONO-AE3-208 but unaffected by the EP2 receptor antagonist PF-04418948. Higher concentrations of PGE2 (10-100 µmol/L) contracted the small airways. However, the TP receptor agonists U-46,619, PGF2α, and PGD2 were more potent than PGE2. Moreover, the bronchoconstrictor responses to PGE2 and all other tested prostanoids, including the EP1/EP3 receptor agonist 17-phenyl trinor PGE2 and the partial FP receptor agonist AL-8810, were uniformly abolished by the TP receptor antagonist SQ-29,548. In the presence of TP and EP4 antagonists, PGE2 inhibited the mast cell-mediated bronchoconstriction resulting from anti-IgE challenge. Measurement of the release of histamine and cysteinyl leukotrienes documented that this bronchoprotective action of PGE2 was mediated by the EP2 receptor, unrelated to bronchodilation, and increased with time of exposure. CONCLUSION: The pharmacology of PGE2 in isolated human small airways was different from its profile in animal models. This first demonstration of powerful EP2 receptor-mediated inhibition of IgE-dependent contractions in human airways introduces a new selective target for the treatment of asthma. This EP2 control of mast cell-mediated bronchoconstriction is presumably exaggerated in patients with aspirin-exacerbated respiratory disease.

Pharmacokinetic comparison of oral tablet and suspension formulations of grapiprant, a novel therapeutic for the pain and inflammation of osteoarthritis in dogs.

A new anti-inflammatory drug for pain (grapiprant) was recently shown to have minimal side effects following chronic (9-month) daily oral dose of 6 or 50 mg/kg suspension. The current study compares the pharmacokinetics of the formulation used in the chronic safety study to those of the tablet formulation that will be marketed upon FDA approval. Sixteen Beagle dogs were randomized to receive single doses of either 6 or 50 mg/kg grapiprant as both suspension and table formulations within a cross-over design with a 15-day washout. Clinical observations were vomiting in one high-dose suspension dog and loose stools in two dogs, one in each 6 mg/kg formulation group. For both formulations, grapiprant reached a maximum concentration within two hours. The tablet formulation had better bioavailability, with AUClast values 34% higher at 6 mg/kg and 64% higher at 50 mg/kg compared to the suspension. Results on Day 0 were similar to those reported on Day 15, suggesting little to no accumulation. Using conversion factors of 1.34 and 1.64, these findings suggest that the 6 and 50 mg/kg suspension doses are equivalent to 4.5 and 30 mg/kg tableted doses, respectively. Combining these findings with the 9-month safety study demonstrates that safety was evaluated at doses approximately 15-fold above the demonstrated therapeutic dose of 2 mg/kg and 10-fold over the 'safety dose', defined as the maximum dose a dog of any body weight could receive when dosed at 2 mg/kg with whole or half-tablets.