Patsy Clark

MF63 [2-(6-Chloro-1H-phenanthro[9,10-d]imidazol-2-yl)-isophthalonitrile], a Selective Microsomal Prostaglandin E Synthase-1 Inhibitor, Relieves Pyresis and Pain in Preclinical Models of Inflammation

Microsomal prostaglandin E synthase-1 (mPGES-1) is a terminal prostaglandin E2 (PGE2) synthase in the cyclooxygenase pathway. Inhibitors of mPGES-1 may block PGE2 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 PGE2 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.

Prostacyclin Antagonism Reduces Pain and Inflammation in Rodent Models of Hyperalgesia and Chronic Arthritis

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 PGE2 and PGI2 act as mediators of pain and inflammation. Most of the data indicating a role for PGI2 in this context have been generated in animal models of acute pain. Herein, we describe the role of PGI2 in models of osteoarthritis (OA) and rheumatoid arthritis using a highly selective PGI2 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, PGI2 is involved in the development of chronic inflammation. The results also suggest that the inhibition of PGI2 synthesis by NSAIDs and COX-2 inhibitors, in addition to that of PGE2, contributes to their efficacy in treating the signs of arthritis.

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], a Selective E Prostanoid Receptor 4 Antagonist, Relieves Joint Inflammation and Pain in Rodent Models of Rheumatoid and Osteoarthritis

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 ∼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.

MF498, a selective EP4 antagonist, relieves joint inflammation and pain in rodent models of rheumatoid and osteoarthritis

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 ∼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.

Microsomal Prostaglandin E Synthase-2 Is Not Essential For In Vivo Prostaglandin E2 Biosynthesis

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.

Mutual antagonistic relationship between prostaglandin E2 and IFN-γ: Implications for rheumatoid arthritis

Prostaglandin E2 (PGE2) is a major mediator of inflammation and is present at high concentrations in the synovial fluid of rheumatoid arthritis (RA) patients. PGE2, acting through the EP4 receptor, has both pro‐ and anti‐inflammatory roles in vivo. To shed light on this dual role of PGE2, 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‐γ‐activation pathway, including IFN‐γ itself. This effect of the PGE2/EP4 axis on IFN‐γ is a reciprocal phenomenon since IFN‐γ blocks PGE2 release and blocks EP receptor expression. The mutually antagonistic relationship between IFN‐γ and PGE2 extends to downstream cytokine and chemokine release; PGE2 counters the effects of IFN‐γ, on the release of IP‐10, IL‐8, TNF‐α and IL‐1β. To gain further insight into IFN‐γ‐mediated cellular events in RA, we assessed the effects of IFN‐γ on gene expression in FLS. We observed an IFN‐γ‐dependent up‐regulation of macrophage‐attracting chemokines, and down‐regulation of metalloprotease expression. These results suggest the existence of a mutually antagonistic relationship between PGE2 and IFN‐γ, which may represent a fundamental mechanism of immune control in diseases such as RA.

The discovery of 4-{1-[({2,5-dimethyl-4-[4-(trifluoromethyl)benzyl]-3-thienyl}carbonyl)amino]cyclopropyl}benzoic acid (MK-2894), a potent and selective prostaglandin E2 subtype 4 receptor antagonist.

The discovery of highly potent and selective second generation EP(4) 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.

Structure–activity relationships and pharmacokinetic parameters of quinoline acylsulfonamides as potent and selective antagonists of the EP4 receptor

A new series of EP(4) 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 EP(4) receptor, and excellent selectivity towards other prostanoid receptors. Acceptable pharmacokinetic profiles have also been demonstrated across a series of preclinical species.

A novel series of potent and selective EP4 receptor ligands: Facile modulation of agonism and antagonism

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.

Naphthalene/quinoline amides and sulfonylureas as potent and selective antagonists of the EP4 receptor.

Two new series of EP(4) 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 EP(4) 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.