A novel series of EP(4) ligands, based on a benzyl indoline scaffold, has been discovered. It was found that agonism and antagonism in this series can be easily modulated by minor modifications on the benzyl group. The pharmacokinetic, metabolic and pharmacological profiles of these compounds was explored. It was found that these compounds show good pharmacokinetics in rat and are efficacious in pre-clinical models of pain and inflammation.

The discovery of highly potent and selective second generation EP4 antagonist MK-2894 (34d) is discussed. This compound exhibits favorable pharmacokinetic profile in a number of preclinical species and potent anti-inflammatory activity in several animal models of pain/inflammation. It also shows favorable GI tolerability profile in rats when compared to traditional NSAID indomethacin.

Two new series of EP4 antagonists based on naphthalene/quinoline scaffolds have been identified as part of our on-going efforts to develop treatments for inflammatory pain. One series contains an acidic sulfonylurea pharmacophore, whereas the other is a neutral amide. Both series show subnanomolar intrinsic binding potency towards the EP4 receptor, and excellent selectivity towards other prostanoid receptors. While the amide series generally displays poor pharmacokinetic parameters, the sulfonylureas exhibit greatly improved profile. MF-592, the optimal compound from the sulfonylurea series, has a desirable overall preclinical profile that suggests it is suitable for further development.

OBJECTIVE:Prostaglandins (PGs) are found in high levels in the synovial fluid of patients with rheumatoid arthritis, and nonsteroidal blockade of these bioactive lipids plays a role in patient care. The aim of this study was to explore the relative contribution of cyclooxygenase (COX) isoforms and PG species in the autoantibody-driven K/BxN serum-transfer arthritis.METHODS:The prostanoid content of arthritic ankles was assessed in ankle homogenates, and the importance of this pathway was confirmed with pharmacologic blockade. The presence of COX isoforms was assessed by Western blotting and their functional contribution was compared using COX-1-/- and COX-2-/- mice as well as isoform-specific inhibitors. The relative importance of PGE2 and PGI2 (prostacyclin) was determined using mice deficient in microsomal PGE synthase 1 (mPGES-1) and in the receptors for PGI2.RESULTS:High levels of PGE2 and 6-keto-PGF1alpha (a stable metabolite of PGI2) were detected in arthritic joint tissues, correlating strongly with the intensity of synovitis. Pharmacologic inhibition of PG synthesis prevented arthritis and ameliorated active disease. While both COX isoforms were found in inflamed joint tissues, only COX-1 contributed substantially to clinical disease; COX-1-/- mice were fully resistant to disease, whereas COX-2-/- mice remained susceptible. These findings were confirmed by isoform-specific pharmacologic inhibition. Mice lacking mPGES-1 (and therefore PGE2) developed arthritis normally, whereas mice incapable of responding to PGI2 exhibited a significantly attenuated arthritis course, confirming a role of PGI2 in this arthritis model.CONCLUSION:These findings challenge previous paradigms of distinct "housekeeping" versus inflammatory functions of the COX isoforms and highlight the potential pathogenic contribution of prostanoids synthesized via COX-1, in particular PGI2, to inflammatory arthritis.

Prostaglandin E(2) (PGE(2)) plays an important role in the normal physiology of many organ systems. Increased levels of this lipid mediator are associated with many disease states, and it potently regulates inflammatory responses. Three enzymes capable of in vitro synthesis of PGE(2) from the cyclooxygenase metabolite PGH(2) have been described. Here, we examine the contribution of one of these enzymes to PGE(2) production, mPges-2, which encodes microsomal prostaglandin synthase-2 (mPGES-2), by generating mice homozygous for the null allele of this gene. Loss of mPges-2 expression did not result in a measurable decrease in PGE(2) levels in any tissue or cell type examined from healthy mice. Taken together, analysis of the mPGES-2 deficient mouse lines does not substantiate the contention that mPGES-2 is a PGE(2) synthase.

Elevated levels of prostaglandin E-2 (PGE(2)) are often found in colorectal cancers. Thus, nonsteroidal anti-inflammatory drugs, including selective cyclooxygenase-2 (COX-2) inhibitors, are among the most promising chemopreventive agents for colorectal cancer. However, their long-term use is restricted by the occurrence of adverse events believed to be associated with a global reduction in prostaglandin production. In the present study, we evaluated the chemopreventive efficacy of targeting the terminal synthase microsomal PGE(2) synthase 1 (mPGES-1), which is responsible for generating PGE(2), in two murine models of intestinal cancer. We report for the first time that genetic deletion of mPGES-1 in Apc-mutant mice results in marked and persistent suppression of intestinal cancer growth by 66%, whereas suppression of large adenomas (>3 mm) was almost 95%. This effect occurred despite loss of Apc heterozygosity and beta-catenin activation. However, we found that mPGES-1 deficiency was associated with a disorganized vascular pattern within primary adenomas as determined by CD31 immunostaining. We also examined the effect of mPGES-1 deletion on carcinogen-induced colon cancer. The absence of mPGES-1 reduced the size and number of preneoplastic aberrant crypt foci (ACF). Importantly, mPGES-1 deletion also blocked the nuclear accumulation of beta-catenin in ACF, confirming that beta-catenin is a critical target of PGE(2) procarcinogenic signaling in the colon. Our data show the feasibility of targeting mPGES-1 for cancer chemoprevention with the potential for improved tolerability over traditional nonsteroidal anti-inflammatory drugs and selective COX-2 inhibitors.

Microsomal prostaglandin E synthase-1 (mPGES-1) is a terminal prostaglandin E-2 (PGE(2)) synthase in the cyclooxygenase pathway. Inhibitors of mPGES-1 may block PGE(2) production and relieve inflammatory symptoms. To test the hypothesis, we evaluated the antipyretic and analgesic properties of a novel and selective mPGES-1 inhibitor, MF63 [2-(6-chloro-1H-phenanthro[ 9,10-d]imidazol-2-yl) isophthalonitrile], in animal models of inflammation. MF63 potently inhibited the human mPGES-1 enzyme (IC50 = 1.3 nM), with a high degree (>1000-fold) of selectivity over other prostanoid synthases. In rodent species, MF63 strongly inhibited guinea pig mPGES-1 (IC50 = 0.9 nM) but not the mouse or rat enzyme. When tested in the guinea pig and a knock-in (KI) mouse expressing human mPGES-1, the compound selectively suppressed the synthesis of PGE2, but not other prostaglandins inhibitable by nonsteroidal anti-inflammatory drugs (NSAIDs), yet retained NSAID-like efficacy at inhibiting lipopolysaccharide-induced pyresis, hyperalgesia, and iodoacetate-induced osteoarthritic pain. In addition, MF63 did not cause NSAID-like gastrointestinal toxic effects, such as mucosal erosions or leakage in the KI mice or nonhuman primates, although it markedly inhibited PGE(2) synthesis in the KI mouse stomach. Our data demonstrate that mPGES-1 inhibition leads to effective relief of both pyresis and inflammatory pain in preclinical models of inflammation and may be a useful approach for treating inflammatory diseases.

A new series of EP4 antagonists based on a quinoline acylsulfonamide scaffold have been identified as part of our on-going efforts to develop treatments for chronic inflammation. These compounds show subnanomolar intrinsic binding potency towards the EP4 receptor, and excellent selectivity towards other prostanoid receptors. Acceptable pharmacokinetic profiles have also been demonstrated across a series of preclinical species.

Prostaglandin E(2) (PGE(2)) is a major mediator of inflammation and is present at high concentrations in the synovial fluid of rheumatoid arthritis (RA) patients. PGE(2), acting through the EP4 receptor, has both pro- and anti-inflammatory roles in vivo. To shed light on this dual role of PGE(2), we investigated its effects in whole blood and in primary human fibroblast-like synoviocytes (FLS). Gene expression analysis in human leukocytes, confirmed at the protein level, revealed an EP4-dependent inhibition of the expression of genes involved in the IFN-gamma-activation pathway, including IFN-gamma itself. This effect of the PGE(2)/EP4 axis on IFN-gamma is a reciprocal phenomenon since IFN-gamma blocks PGE(2) release and blocks EP receptor expression. The mutually antagonistic relationship between IFN-gamma and PGE(2) extends to downstream cytokine and chemokine release; PGE(2) counters the effects of IFN-gamma, on the release of IP-10, IL-8, TNF-alpha and IL-1beta. To gain further insight into IFN-gamma-mediated cellular events in RA, we assessed the effects of IFN-gamma on gene expression in FLS. We observed an IFN-gamma-dependent up-regulation of macrophage-attracting chemokines, and down-regulation of metalloprotease expression. These results suggest the existence of a mutually antagonistic relationship between PGE(2) and IFN-gamma, which may represent a fundamental mechanism of immune control in diseases such as RA.

Prostaglandin E2 (PGE2) is a principal mediator of inflammation in diseases such as rheumatoid arthritis and osteoarthritis. Nonsteroidal anti-inflammatory medications (NSAIDs) and selective cyclooxygenase-2 (COX-2) inhibitors reduce PGE2 production to diminish the inflammation seen in these diseases, but have toxicities that may include both gastrointestinal bleeding and prothrombotic tendencies. In cells, arachidonic acid is transformed into PGE2 via cyclooxygenase (COX) enzymes and terminal prostaglandin E synthases (PGES). Accumulating data suggest that the interaction of various enzymes in the PGE2 synthetic pathway is complex and tightly regulated. In this review, we summarize the synthesis and secretion of PGE2. In particular, we focus on the three isoforms of the terminal PGES, and discuss the potential of targeting PGES as a more precise strategy for inhibiting PGE2 production.

Previous evidence has implicated E prostanoid receptor 4 (EP4) in mechanical hyperalgesia induced by subplantar inflammation. However, its role in chronic arthritis remains to be further defined because previous attempts have generated two conflicting lines of evidence, with one showing a marked reduction of arthritis induced by a collagen antibody in mice lacking EP4, but not EP1-EP3, and the other showing no impact of EP4 antagonism on arthritis induced by collagen. Here, we assessed the effect of a novel and selective EP4 antagonist MF498 [N-{[4-(5,9-diethoxy-6-oxo-6,8-dihydro-7H-pyrrolo[3,4-g]quinolin-7-yl)-3-methylbenzyl]sulfonyl}-2-(2-methoxyphenyl)acetamide] on inflammation in adjuvant-induced arthritis (AIA), a rat model for rheumatoid arthritis (RA), and joint pain in a guinea pig model of iodoacetate-induced osteoarthritis (OA). In the AIA model, MF498, but not the antagonist for EP1, MF266-1 [1-(5-{3-[2-(benzyloxy)-5-chlorophenyl]-2-thienyl}pyridin-3-yl)-2,2,2-trifluoroethane-1,1-diol] or EP3 MF266-3 [(2E)-N-[(5-bromo-2-methoxyphenyl)sulfonyl]-3-[5-chloro-2-(2-naphthylmethyl)phenyl]acrylamide], inhibited inflammation, with a similar efficacy as a selective cyclooxygenase 2 (COX-2) inhibitor MF-tricyclic. In addition, MF498 was as effective as an nonsteroidal anti-inflammatory drug, diclofenac, or a selective microsomal prostaglandin E synthase-1 inhibitor, MF63 [2-(6-chloro-1H-phenanthro[9,10-d]imidazol-2-yl)isophthalonitrile], in relieving OA-like pain in guinea pigs. When tested in rat models of gastrointestinal toxicity, the EP4 antagonist was well tolerated, causing no mucosal leakage or erosions. Lastly, we evaluated the renal effect of MF498 in a furosemide-induced diuresis model and demonstrated that the compound displayed a similar renal effect as MF-tricyclic [3-(3,4-difluorophenyl)-4-(4-(methylsulfonyl)phenyl)-2-(5H)-furanone], reducing furosemide-induced natriuresis by approximately 50%. These results not only suggest that EP4 is the major EP receptor in both RA and OA but also provide a proof of principle to the concept that antagonism of EP4 may be useful for treatment of arthritis.

Selective type 2 cyclooxygenase (COX-2) inhibitors are often used in preclinical studies without potency and selectivity data in the experimental species. To address this issue, we assessed a selective COX-2 inhibitor MF-tricyclic in four commonly used species, namely mice, rats, guinea pigs and rabbits, in the present study. In both the guinea pig and rabbit whole blood assay, the compound inhibited lipopolysaccharide (LPS)-induced PGE(2) production with an IC(50) (COX-2) of 0.6 and 2.8 microM, respectively. By comparison, the compound displayed a much weaker activity on clot-induced formation of thromboxane with an IC(50) (COX-1) of >10 microM (guinea pigs) and 23 microM (rabbits). In keeping with the in vitro potency data, the compound significantly inhibited interleukin-1 beta (IL-1beta) -induced PGE(2) formation in the rabbit synovium at plasma concentrations near the whole blood assay IC(50) for COX-2 but much lower than that for COX-1. MF-tricyclic was also potent and selective toward COX-2 in mice, inhibiting carrageenan-induced PGE(2) accumulation in the air pouch dose-dependently (ED(50)=0.5 mg/kg) without affecting stomach PGE(2) levels. In rats, MF-tricyclic was found to be effective in three standard in vivo assays utilized for assessing COX-2 inhibitors, namely, LPS-induced pyresis, carrageenan-induced paw edema and adjuvant-induced arthritis at the doses that did not inhibit stomach PGE(2) levels. Similar to that in rats, the compound displayed pharmacological efficacy in mice, guinea pigs and rabbits when tested in the LPS pyresis model. Our data reveal that MF-tricyclic has the desired biochemical and pharmacological properties for selective COX-2 inhibition in all four test species.

A liquid chromatography/high-field asymmetric waveform ion mobility spectrometry/tandem mass spectrometry (LC-FAIMS-MS/MS) semi-quantitative method was developed for the simultaneous determination of prostaglandin (PG) E2, PGD2, PGF(2alpha), 6-keto-PGF(1alpha), and thromboxane (TX) B2. Diluted samples containing these prostanoids and their tetra-deuterium-substituted internal standards were analyzed by LC followed by either selected reaction monitoring (LC-SRM) or FAIMS and selected reaction monitoring (LC-FRM). FAIMS reduced background noise, separated the isobaric ions PGE2 and PGD2, and separated dynamically interchanging TXB2 anomers. This is the first report of the separation of interconverting anomers by FAIMS. Generally, the LC-FRM chromatograms were more selective than the LC-SRM chromatograms. Its potential was demonstrated by analysis of prostanoids in guinea pig lumbar spinal cord homogenate.

The inhibition of prostaglandin (PG) synthesis is at the center of current anti-inflammatory therapies. Because cyclooxygenase-2 (COX-2) inhibitors and nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit the formation of multiple PGs, there is currently a strong focus on characterizing the role of the different PGs in the inflammation process and development of arthritis. Evidence to date suggests that both PGE(2) and PGI(2) act as mediators of pain and inflammation. Most of the data indicating a role for PGI(2) in this context have been generated in animal models of acute pain. Herein, we describe the role of PGI(2) in models of osteoarthritis (OA) and rheumatoid arthritis using a highly selective PGI(2) receptor (IP, Ptgir) antagonist and IP receptor-deficient mice. In the rat OA model using monoiodoacetate injection into the knee joint, the IP antagonist reduced pain with an efficacy approaching that of the NSAID diclofenac. In a chronic model of inflammatory arthritis, collagen-antibody induced arthritis model in mice, IP receptor-deficient mice displayed a 91% reduction in arthritis score. Interestingly, pretreatment with the IP [N-[4-(imidazolidin-2-ylideneamino)-benzyl]-4-methoxy-benzamide] antagonist in this model also caused a significant reduction of the symptoms, whereas administration of the compound after the initiation of arthritis had no detectable effect. Our data indicate that, in addition to its role in acute inflammation, PGI(2) is involved in the development of chronic inflammation. The results also suggest that the inhibition of PGI(2) synthesis by NSAIDs and COX-2 inhibitors, in addition to that of PGE(2), contributes to their efficacy in treating the signs of arthritis.