Discovery of a novel COX-2 inhibitor as an orally potent anti-pyretic and anti-inflammatory drug: design, synthesis, and structure-activity relationship

Cyclooxygenase (COX) has been considered as a significant pharmacological target because of its pivotal roles in the prostaglandin biosynthesis and following cascades that lead to various (patho)physiological effects. Non-steroidal anti-inflammatory drugs (NSAIDs) that suppress COX activities have been used clinically for the treatment of fever, inflammation, and pain; however, nonselective COX inhibitors exhibit serious side-effects such as gastrointestinal damage because of their inhibitory activities against COX-1. Thus, COX-1 is constitutive and expressed ubiquitously and serves a housekeeping role, while COX-2 is inducible or upregulated by inflammatory/injury stimuli such as interleukin-1β, tumor necrosis factor-α, and lipopolysaccharide in macrophage, monocyte, synovial, liver, and lung, and is associated with prostaglandin E₂ and prostacyclin production that evokes or sustains systemic/peripheral inflammatory symptoms. Also, hypersensitivity of aspirin is a significant concern clinically. Hence, design, synthesis, and structure–activity relationship of [2-{[(4-substituted)-pyridin-2-yl]carbonyl}-(6- or 5-substituted)-1H-indol-3-yl]acetic acid analogues were investigated to discover novel acid-type COX-2 inhibitor as an orally potent new-class anti-pyretic and anti-inflammatory drug. As significant findings, compounds 1–3 demonstrated potent COX-2 inhibitory activities with high selectivities for COX-2 over COX-1 in human cells or whole-blood in vitro, and demonstrated orally potent anti-pyretic activity against lipopolysaccharide-induced systemic-inflammatory fever model in F344 rats. Also compound 1 demonstrated orally potent anti-inflammatory activity against edema formation and a suppressive effect against PGE₂ production in carrageenan-induced peripheral-inflammation model on the paw of SD rats. These results suggest that compounds 1–3 are potential agents for the treatment of inflammatory disease and are useful for further pharmacological COX-2 inhibitor investigations.     

Prostaglandin F2alpha produced by inducible cyclooxygenase may contribute to the resolution of inflammation

Cyclooxygenase-2 may play a role in resolution of carrageenan-induced pleurisy in rats by generating anti-inflammatory prostanoids. Here, we show exudate prostaglandin F2alpha concentrations rise during resolution of this model. These were reduced by the selective cyclooxygenase-2 inhibitor NS-398, which exacerbated inflammation. Concomitant treatment with NS-398 and the synthetic FP receptor agonist fluprostenol reversed this exacerbation. This suggests prostaglandin F2alpha produced by cyclooxygenase-2 contributes to resolution of this inflammatory reaction.     

Possible participation of cyclooxygenase-2 in the recurrence of allergic inflammation in rats

In the recurrence of allergic inflammation in a rat air pouch model, pouch fluid volume, prostaglandin E₂ concentration in the pouch fluid, leukocyte infiltration into the pouch fluid, and granulation tissue weight were markedly increased by the antigen challenge. To clarify the role of cyclooxygenase-2 in the recurrence of allergic inflammation, the time-course of changes in protein levels of cyclooxygenase-1 and cyclooxygenase-2 in the granulation tissue and in the infiltrated leukocytes was examined by Western blot analysis. It was shown that cyclooxygenase-1 levels in the granulation tissue and in the infiltrated leukocytes were not changed by the antigen challenge, but cycoloxygenase-2 levels were increased. Furthermore, treatment with the selective cyclooxygenase-2 inhibitor, NS-398 ([N-2(cyclohexyloxy-4-nitrophenyl]-methanesulfonamide), suppressed the recurrence of allergic inflammation as did the non-selective cyclooxygenase-1/cyclooxygenase-2 inhibitor, indomethacin. The steroidal anti-inflammatory drug, dexamethasone, inhibited the induction of cyclooxygenase-2, and suppressed the allergic inflammation. These findings strongly suggested that cyclooxygenase-2 induced by the antigen challenge plays a role in the recurrence of inflammation induced by the allergic mechanism.     

Anti-inflammatory potency of FR167653, a p38 mitogen-activated protein kinase inhibitor,in mouse models of acute inflammation

The anti-inflammatory effect of FR167653 (1-[7-(4-fluorophenyl)-1,2,3,4-tetrahydro-8-(4-pyridyl)pyrazolo[5,1-c][1,2,4]triazin-2-yl]-2-phenylethanedione sulfate monohydrate), a p38 mitogen-activated protein (MAP) kinase inhibitor, was examined in two mouse models of acute inflammation. Carrageenan-induced paw edema was inhibited by pretreatment with FR167653, anti-tumor necrosis factor (TNF)-alpha antibody, and NS-398 (N-(2-cyclohexyloxy-4-nitrophenyl) methanesulfonamide), a selective cyclooxygenase-2 inhibitor. Carrageenan increased TNF-alpha and prostaglandin E(2) levels in the paw, both of which were suppressed by FR167653. Subcutaneous injection of lipopolysaccharide at the back of mouse caused local increase in vascular permeability determined by leakage of Pontamine sky blue. FR167653 dose-dependently inhibited the lipopolysaccharide-induced plasma leakage. FR167653 also inhibited lipopolysaccharide-induced increases in serum TNF-alpha level, and skin TNF-alpha and prostaglandin E(2) levels at the injection site. On the other hand, FR167653 did not reduce arachidonic acid-induced plasma leakage which is not mediated by cyclooxygenase-2. FR167653 exhibits anti-inflammatory effects against both carrageenan-induced paw edema and lipopolysaccharide-induced plasma leakage through inhibiting the synthesis of inflammatory mediators that are regulated by p38 MAP kinase.     

Inhibition of inducible prostaglandin E<Subscript>2</Subscript> production and cyclooxy-genase-2 expression by curdione from<Emphasis Type="Italic">Curcuma zedoaria</Emphasis>

Overproduction of prostaglandins has been considered in mediation of inflammation and carcinogenic process. On this line, the inhibitors of prostaglandin biosynthetic enzyme cyclooxygenase (COX) have played a role of anti-inflammatory and cancer chemopreventive agents. In our continuous efforts to search anti-inflammatory and chemopreventive agents from natural products, bioassay-guided fractionation led to the isolation of curdione from the rhizome of Curcuma zedoaria with the inhibitory effect on the production of prostaglandin E2 in lipopolysaccharide (LPS)-stimulated mouse macrophage RAW 264.7 cells in a concentration-dependent manner (IC50 = 1.1 microM). Mechanistic studies suggest that the suppression of cyclooxygenase-2 (COX-2) mRNA expression is, at least in part, involved in this inhibitory activity of curdione.     

Effect of a potent cyclooxygenase inhibitor, 5-ethyl-4-methoxy-2-phenylquinoline (KTC-5), on human platelets

Because the metabolites of arachidonic acid participate in many physiopathological responses, including inflammation and platelet aggregation, cyclooxygenase inhibitors are important in the treatment of associated diseases. A biologically active compound, 5-ethyl-4-methoxy-2-phenylquinoline (KTC-5), selectively and concentration dependently inhibited aggregation of platelets from man and ATP release caused by arachidonic acid (200 microM) and collagen (10 microg mL(-1)) without affecting the aggregation caused by thrombin (0.1 U mL(-1)) and U46619 (2 microM). The IC50 value (drug concentration inhibiting maximum response by 50%) of KTC-5 for aggregation induced by arachidonic acid and collagen was 0.11+/-0.04 microM and 0.20+/-0.03 microM, respectively. This inhibitory effect of KTC-5 was reversible and time dependent. KTC-5 specifically inhibited intracellular calcium mobilization initiated by arachidonic acid or collagen without affecting that caused by thrombin or U46619 in human platelets. Furthermore, KTC-5 inhibited thromboxane B2 and prostaglandin D2 formation provoked by arachidonic acid. The IC50 value of KTC-5 for arachidonic-acid-induced thromboxane B2 formation was 0.07+/-0.02 microM. Based on these observations, the data indicated that KTC-5 potently inhibited human platelet aggregation and ATP release mainly via the inhibition of the cyclooxygenase-1 activity. Moreover, KTC-5 inhibited lipopolysaccharide-induced prostaglandin E2 formation in RAW264.7 cells in the presence of external arachidonic acid with an IC50 value of 0.17+/-0.06 microM. Immunoblot analysis showed that KTC-5 did not affect the cyclooxygenase-2 expression in the presence of lipopolysaccharide on RAW264.7 cells. This result indicated that KTC-5 affects the activity of cyclooxygenase-2. According to these data, we concluded that KTC-5 is a cyclooxygenase inhibitor for both subtypes.     

A new pyrazolo pyrimidine derivative inhibitor of cyclooxygenase-2 with anti-angiogenic activity

In a previous study, we reported a new pyrazolo pyrimidine derivative, N(4)-benzyl-N(6),N(6)-dimethyl-1-1(tert-butyl)-1H-pyrazolo[3,4-d]pyrimidine-6,4-diamine (DPP), which inhibited potently cyclooxygenase-2 activity in intact cell assays with minor activity against cyclooxygenase-1 (IC(50)=0.9 nM for cyclooxygenase-2 versus IC(50)=59.6 nM for cyclooxygenase-1). In the present work, this behaviour was confirmed in vivo by using the 24-h zymosan-injected mouse air pouch model (ID(50)=1.36 nM/pouch for prostaglandin E(2) level). We also studied the possible beneficial effect of DPP in the angiogenesis-dependent murine air pouch granuloma and rat paw carrageenan-induced hyperalgesia models. DPP exerted analgesic and anti-angiogenic (52% reduction in angiogenesis at 10 mg/kg, i.p.) effects that may be associated with inhibition of cyclooxygenase-2 activity.     

The analgesic effect profile of FR122047, a selective cyclooxygenase-1 inhibitor, in chemical nociceptive models

The pharmacological profile of the analgesic agent, 1-[(4, 5-bis(4-methoxyphenyl)-2-thiazoyl)carbonyl]-4-methylpiperazine hydrochloride (FR122047), was investigated. In recombinant human cyclooxygenase enzyme assays, the inhibition of prostaglandin E(2) formation by FR122047 was 2300 times more selective for cyclooxygenase-1 than cyclooxygenase-2. Oral administration of FR122047 (3.2-100 mg/kg) dose dependently reduced the phase 2 response (10-60 min) of the formalin test in rats. This effect was 3 times less potent than that of indomethacin. FR122047 (1-32 mg/kg; p. o.) showed a dose-dependent analgesic effect against the acetic acid-induced writhing response in mice. Furthermore, FR122047 (0. 01-10 mg/kg, p.o.) inhibited the increase in 6-keto prostaglandin F(1alpha) level in acetic acid-injected mouse peritoneal cavity. However, a selective cyclooxygenase-2 inhibitor, NS-398, had no effect in these cyclooxygenase-1 sensitive pain models. These results suggest that FR122047, a selective cyclooxygenase-1 inhibitor, shows an analgesic effect in chemical nociceptive models and may be a useful analgesic agent.     

Evidence for release of prostaglandin F2 alpha in contractile response of guinea pig trachea to norepinephrine

The concentration (10(-8)-10(-5) M)-dependent contractile response of norepinephrine was completely inhibited by cyclooxygenase inhibitors but not by a thromboxane synthetase inhibitor. The amount of prostaglandin F2 alpha was significantly increased in the presence of norepinephrine (10(-5) M). Norepinephrine increased the amount of prostaglandin D2, although not significantly. The norepinephrine-evoked prostaglandin release was inhibited by tolazoline (3 x 10(-5) M). These results suggest that norepinephrine evoked the release of prostaglandins, mainly prostaglandin F2 alpha to induce the contraction of the guinea pig trachea.     

Relationship between prostaglandin E2 and vascular endothelial growth factor (VEGF) in angiogenesis in human vascular endothelial cells

To address the role of prostaglandin E2 (PGE2) in tube formation of endothelial cells and the relationships between the action of PGE2 and vascular endothelial growth factor (VEGF), cultured human umbilical vein endothelial cells (HUVECs) were used to evaluate tube formation on Matrigel and the expression of angiogenesis-related genes. PGE2 treatment stimulated the tube-like formation of HUVECs. Whereas VEGF-induced tube formation was significantly suppressed by ETYA, an inhibitor of arachidonic acid metabolism, or SU5614, an inhibitor of VEGF-receptor tyrosine kinase, the stimulatory effect of PGE2 was observed in the presence of ETYA or SU5614. Thus, PGE2 counteracted both ETYA- and SU5614-induced blockage of angiogenesis in the presence of VEGF. VEGF induced cyclooxygenase (COX) -2 mRNA expression in HUVECs and increased the PGE2 concentration in the medium. PGE2 treatment enhanced the expression of VEGF mRNA. These findings suggest that PGE2 directly stimulates angiogenesis, apart from VEGF signaling, and further induces VEGF expression in HUVECs. In addition, the effect of VEGF on angiogenesis may be mediated, in part, by PGE2 secretion.     

Modulation by endothelin-1 of lipopolysaccharide-induced cyclooxygenase 2 expression in mouse peritoneal macrophages.

We investigated the modulation by endothelin-1 of lipopolysaccharide-induced cyclooxygenase 2 expression and prostaglandin E2 production by mouse peritoneal macrophages. Our previous report showed that endothelin-1 at concentrations above 10(-11) M induced cyclooxygenase 2 expression through mainly endothelin ET(B) receptors and that an endothelin ET(B) receptor-mediated process was not involved in cyclooxygenase 2 activation in macrophages stimulated by lipopolysaccharide for 4 h. In the present study, when macrophages were stimulated by lipopolysaccharide for 12 h in the presence of endothelin-1 (10(-15) to 10(-8) M), cyclooxygenase 2 expression and prostaglandin E2 production were enhanced by 1.2- to 1.6-fold. The endothelin ET(B) receptor selective antagonist, BQ788 (N-cis-2,6-dimethylpiperidino-carbonyl-L-gamma-methyl-leucyl-D-L-m ethoxycarbonyl-tryptophanyl-norleucine), significantly inhibited this synergistic effect of endothelin-1. In addition, the cyclooxygenase 2-selective inhibitor, NS398 (N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide), also suppressed this effect. Western blot analysis showed that the endothelin ET(B) receptor was up-regulated by lipopolysaccharide in a time- and concentration-dependent manner, and that this up-regulation was inhibited by NS398. From these results, we conclude that endothelin-1 promotes lipopolysaccharide-induced cyclooxygenase 2 activation in the delayed phase through endothelin ET(B) receptors up-regulated by lipopolysaccharide.     

Role of MAP kinases in regulating expression of antioxidants and inflammatory mediators in mouse keratinocytes following exposure to the half mustard, 2-chloroethyl ethyl sulfide

Dermal exposure to sulfur mustard causes inflammation and tissue injury. This is associated with changes in expression of antioxidants and eicosanoids which contribute to oxidative stress and toxicity. In the present studies we analyzed mechanisms regulating expression of these mediators using an in vitro skin construct model in which mouse keratinocytes were grown at an air-liquid interface and exposed directly to 2-chloroethyl ethyl sulfide (CEES), a model sulfur mustard vesicant. CEES (100-1000 µM) was found to cause marked increases in keratinocyte protein carbonyls, a marker of oxidative stress. This was correlated with increases in expression of Cu,Zn superoxide dismutase, catalase, thioredoxin reductase and the glutathione S-transferases, GSTA1-2, GSTP1 and mGST2. CEES also upregulated several enzymes important in the synthesis of prostaglandins and leukotrienes including cyclooxygenase-2 (COX-2), microsomal prostaglandin E synthase-2 (mPGES-2), prostaglandin D synthase (PGDS), 5-lipoxygenase (5-LOX), leukotriene A₄ (LTA₄) hydrolase and leukotriene C₄ (LTC₄) synthase. CEES readily activated keratinocyte JNK and p38 MAP kinases, signaling pathways which are known to regulate expression of antioxidants, as well as prostaglandin and leukotriene synthases. Inhibition of p38 MAP kinase suppressed CEES-induced expression of GSTA1-2, COX-2, mPGES-2, PGDS, 5-LOX, LTA₄ hydrolase and LTC₄ synthase, while JNK inhibition blocked PGDS and GSTP1. These data indicate that CEES modulates expression of antioxidants and enzymes producing inflammatory mediators by distinct mechanisms. Increases in antioxidants may be an adaptive process to limit tissue damage. Inhibiting the capacity of keratinocytes to generate eicosanoids may be important in limiting inflammation and protecting the skin from vesicant-induced oxidative stress and injury.     

The effect of endothelin-1 on lipopolysaccharide-induced cyclooxygenase 2 expression in association with prostaglandin E(2)

We demonstrated previously that endothelin-1 (10(-14) to 10(-8) M) promotes lipopolysaccharide-induced cyclooxygenase 2 expression and prostaglandin E(2) production through endothelin ET(B) receptors effects which are up-regulated by lipopolysaccharide. In the present study, we confirmed these findings and showed that prostaglandin E(2) (10(-6) to 10(-5) M) inhibited the lipopolysaccharide plus endothelin-1-induced cyclooxygenase 2 expression more profoundly as compared to its inhibition of the lipopolysaccharide-induced cyclooxygenase 2 expression. The endothelin ET(B) receptor selective antagonist, N-cis-2, 6-dimethylpiperidino-carbonyl-L-gamma-methyl-leucyl-D-L-methoxy carbon yl-tryptophanyl-D-norleucine (BQ788), partly inhibited this suppression. Interestingly, the expression of endothelin ET(B) receptors in macrophages was increased by lipopolysaccharide plus prostaglandin E(2) (10(-8) to 10(-5) M) about 1.6-fold compared with that evoked by lipopolysaccharide stimulation alone. We also showed that treatment with endothelin-1 at 10(-14) M (15 min) elevated an intracellular cyclic AMP concentration in macrophages stimulated by lipopolysaccharide or lipopolysaccharide plus prostaglandin E(2) (10(-6) M) for 6 h, and the elevation in the latter cells was more pronounced. These results suggested that endothelin-1 shows an opposite modulation of lipopolysaccharide-induced cyclooxygenase 2 expression in macrophages through endothelin ET(B) receptors, depending on the level of extracellular prostaglandin E(2), and the changes of intracellular cyclic AMP by endothelin-1 may be involved in this mechanism.     

COX-2-mediated PGD2 synthesis regulates phosphatidylcholine biosynthesis in rat renal papillary tissue

Phosphatidylcholine (PC) is the major membrane phospholipid in mammalian cells. Previous works from our laboratory demonstrated a close metabolic relationship between the maintenance of PC biosynthesis and the prostaglandins endogenously synthesized by cyclooxygenase (COX) in rat renal papilla. In the present work, we studied the COX isoform involved in papillary PC biosynthesis regulation. The incorporation of [methyl-3H]choline and [32P]orthophosphate to PC was determined in the absence and presence of SC-560 and NS-398, COX-1 and COX-2 specific inhibitors. PC synthesis was highly sensitive to COX-2 inhibition, while COX-1 inhibition only reduced PC synthesis at high SC-560 concentration. The analysis of choline-containing metabolites showed that COX-2 inhibition affected the formation of CDP-choline intermediary. The evaluation of PC biosynthetic enzymes revealed that microsomal, as well as nuclear, CTP:phosphocholine cytidylyltransferase (CCT), and nuclear-CDP-choline:1,2-diacylglycerol cholinephosphotransferase (CTP) activities were affected by COX-2 inhibition. The addition of exogenous prostaglandin D(2) (PGD(2)) restored nuclear-CCT and -CPT activities but not microsomal CCT. Papillary synthesis of PGD(2) was only detected in nuclear fraction where it was blocked by COX-2 inhibitor NS-398, but not by COX-1 inhibitor. All together, the present results demonstrated that COX-2-mediated PGD(2) synthesis is a PC biosynthesis regulator in rat renal papilla. Considering the importance of the maintenance of PC biosynthesis for the preservation of cell membrane homeostasis to ensure cell viability, and the extensive use of COX-2 inhibitors in therapeutics, the present results could have great pharmacological implications, and can constitute a biochemical explanation for the nephrotoxic effect of non-steroidal anti-inflammatory drugs.     

Involvement of secretory phospholipase A2 activity in the zymosan rat air pouch model of inflammation.

1. In the zymosan rat air pouch model of inflammation we have assessed the time dependence of phospholipase A2 (PLA2) accumulation in the inflammatory exudates as well as cell migration, myeloperoxidase activity, prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) levels. 2. A significant increase in PLA2 activity was detected in 1,200 g supernatants of exudates 8 h after injection of zymosan into rat air pouch. This event coincided with peaks in cell accumulation (mainly neutrophils) and myeloperoxidase activity in exudates and was preceded by a rise in eicosanoid levels. 3. This enzyme (without further purification) behaved as a secretory type II PLA2 with an optimum pH at 7-8 units, lack of selectivity for arachidonate release and dependence on mM calcium concentrations for maximal activity. 4. The PLA2 inhibitors manoalide and scalaradial inhibited this enzyme activity in vitro in a concentration-dependent manner. Scalaradial also inhibited zymosan stimulated myeloperoxidase release in vitro. 5. Injection of the marine PLA2 inhibitor scalaradial together with zymosan into the pouch at doses of 0.5, 1 and 5 mumol per pouch resulted in a dose-dependent inhibition of PLA2 activity in exudates collected 8 h later. Myeloperoxidase levels and cell migration were also decreased, while eicosanoid levels were not modified. 6. Colchicine administration to rats prevented infiltration and decreased PLA2 levels in the 8 h zymosan-injected air pouch. 7. These results indicate that during inflammatory response to zymosan in the rat air pouch a secretory PLA2 activity is released into the exudates. The source of this activity is mainly the neutrophil which migrates into the pouch. 8. Scalaradial exerts anti-inflammatory effects in the zymosan air pouch.     

Effects of a selective cyclooxygenase-2 inhibitor, celecoxib, on bone resorption and osteoclastogenesis in vitro

The effects of an important new anti-inflammatory agent, the selective cyclooxygenase-2 inhibitor celecoxib, on bone resorption and osteoclastogenesis elicited by the inflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), the endotoxin lipopolysaccharide (LPS), and the systemic hormones 1alpha,25-dihydroxyvitamin D(3) and parathyroid hormone were examined in vitro. Bone resorption was evaluated by measuring calcium released into the culture medium in a neonatal mouse calvarial bone organ culture. Osteoclastogenesis was evaluated by measuring tartrate-resistant acid phosphatase activity in the cells in cocultures of bone marrow cells and osteoblastic cells and in macrophage-colony-stimulating factor-dependent bone marrow cell cultures. Celecoxib (0.1 microM) completely inhibited the calcium release induced by IL-1beta, TNF-alpha, and LPS. The resorptive effect of 1alpha,25-dihydroxyvitamin D(3) was inhibited partially by celecoxib. In contrast, celecoxib did not inhibit the calcium release elicited by parathyroid hormone or prostaglandin E(2). Celecoxib (0.1 microM) also markedly inhibited osteoclastogenesis induced by these stimulators of bone resorption except for PGE(2) in the coculture system, whereas it failed to inhibit osteoclastogenesis in macrophage-colony-stimulating factor-dependent bone marrow cell cultures. These results indicate that, under certain conditions, cyclooxygenase-2-dependent prostaglandin synthesis is critical for the bone resorption induced by IL-1beta, TNF-alpha, and LPS, and for the osteoclastogenesis induced by these pro-inflammatory molecules and calciotropic hormones. The prevention of prostaglandin synthesis by inflammatory cytokines in bone cells could contribute to the efficacy of celecoxib in preventing bone loss in rheumatoid arthritis.     

Sodium salicylate enhances the expression of cyclooxygenase-2 in endotoxin-stimulated human mononuclear cells.

The effects of salicylate on the expression of cyclooxygenases, and on prostaglandin E(2) biosynthesis were examined in human peripheral blood mononuclear cells. Peripheral blood mononuclear cells were incubated in the presence of endotoxin, which induced expression of cyclooxygenase-2 protein, and caused a time-dependent increase of immunoreactive prostaglandin E(2) in the supernatant. The cycooxygenase-2 selective inhibitor N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide (NS-398, 1 microM) suppressed the endotoxin-induced increase of prostaglandin E(2), without significantly affecting the expression of cyclooxygenase-1 or cyclooxygenase-2. In peripheral blood mononuclear cells exposed to endotoxin (18 h), 1.0 and 3.0 mM sodium salicylate reduced the prostaglandin E(2) concentration of the supernatant, and, at the same time, stimulated cyclooxygenase-2 expression. After a subsequent 2 h incubation of peripheral blood mononuclear cells in drug-free medium, prostaglandin E(2) concentrations in samples that had been exposed to endotoxin together with 1.0 or 3.0 mM salicylate were significantly higher than in samples exposed to endotoxin alone. These results show that salicylate can enhance the expression of cyclooxygenase-2 in endotoxin-exposed peripheral blood mononuclear cells and at the same time reduce prostaglandin E(2) formation. After washout and removal of salicylate-induced cyclooxygenase inhibition, increased cyclooxygenase-2 expression resulted in enhanced prostaglandin E(2) formation. It seems possible that under certain conditions salicylate-induced stimulation of cyclooxygenase-2 expression may contribute to its clinical pharmacological profile.     

Distinction of microsomal prostaglandin E synthase-1 (mPGES-1) inhibition from cyclooxygenase-2 inhibition in cells using a novel, selective mPGES-1 inhibitor

Inflammation-induced microsomal prostaglandin E synthase-1 (mPGES-1) is the terminal enzyme that synthesizes prostaglandin E₂ (PGE₂) downstream of cyclooxygenase-2 (COX-2). The efficacy of nonsteroidal anti-inflammatory drugs and COX-2 inhibitors in the treatment of the signs and symptoms of osteoarthritis, rheumatoid arthritis and inflammatory pain, largely attributed to the inhibition of PGE₂ synthesis, provides a rationale for exploring mPGES-1 inhibition as a potential novel therapy for these diseases. Toward this aim, we identified PF-9184 as a novel mPGES-1 inhibitor. PF-9184 potently inhibited recombinant human (rh) mPGES-1 (IC₅₀ = 16.5 ± 3.8 nM), and had no effect against rhCOX-1 and rhCOX-2 (>6500-fold selectivity). In inflammation and clinically relevant biological systems, mPGES-1 expression, like COX-2 expression was induced in cell context- and time-dependent manner, consistent with the kinetics of PGE₂ synthesis. In rationally designed cell systems ideal for determining direct effects of the inhibitors on mPGES-1 function, but not its expression, PF-9184 inhibited PGE₂ synthesis (IC₅₀ in the range of 0.5-5 μM in serum-free cell and human whole blood cultures, respectively) while sparing the synthesis of 6-keto-PGF_1α (PGF_1α) and PGF_2α. In contrast, as expected, the selective COX-2 inhibitor, SC-236, inhibited PGE₂, PGF_1α and PGF_2α synthesis. This profile of mPGES-1 inhibition, distinct from COX-2 inhibition in cells, validates mPGES-1 as an attractive target for therapeutic intervention.     

Inhibitory action of ethanol on cyclooxygenase-2 expression through suppression of the extracellular signal-related kinase-mediated pathway in rat alveolar macrophages

The purpose of the present study was to elucidate the effects of ethanol on expression of cyclooxygenase in alveolar macrophages. Rat alveolar macrophages were collected by bronchoalveolar lavage and stimulated by lipopolysaccharide. Lipopolysaccharide-induced cyclooxygenase-2 expression and prostaglandin E2 production were inhibited by ethanol (100-200 mM) in a concentration-dependent manner. Ethanol at 100-200 mM concentration-dependently inhibited cyclooxygenase-2 mRNA expression. Lipopolysaccharide-stimulated phosphorylation of both extracellular signal-related kinase and p38 mitogen-activated protein kinase was also significantly inhibited by ethanol (50-200 mM). Cyclooxygenase-2 expression was significantly inhibited by U0126 but was not affected by SB203580. In the presence of SB203580, lipopolysaccharide-induced cyclooxygenase-2 expression was also inhibited by ethanol (50-200 mM). On the other hand, cyclooxygenase-1 was expressed constitutively in alveolar macrophages and cyclooxygenase-1 expression was affected neither by lipopolysaccharide nor ethanol. Lipopolysaccharide-induced expression of inducible nitric oxide synthase in alveolar macrophages was not affected by ethanol at 50-200 mM. These results suggest that lipopolysaccharide-induced expression of cyclooxygenase-2 is mediated by extracellular signal-related kinase but not by p38 kinase and that ethanol selectively attenuates cyclooxygenase-2 expression mainly by inhibiting activation of extracellular signal-related kinase.