Screening method for nonsteroidal antiinflammatory drugs based on the cyclooxygenase 2 pathway activated by serum-free stimulation in A549 cells

Cyclooxygenase 2 (COX-2) pathway inhibitors were regarded as promising nonsteroidal antiinflammatory drugs (NSAIDs). We discovered that the COX-2 pathway in A549 cells, a human lung cancer cell line, was activated by serum-free stimulation, and a drug screening model for NSAIDs was established based on this principle with simple performance and sufficient reliability. The COX-2 pathway was activated by treating with serum-free medium for 12 h. The activated cells were incubated with NS398 (selective COX-2 inhibitor), SC560 (selective COX-1 inhibitor), acetyl salicylic acid (ASA) (nonselective COX inhibitor) at 37 degrees C for 15 min. Then the cells were incubated with 10 microM of arachidonic acid (AA) for another 30 min prostaglandin E2 and 6-keto-prostaglandin F(1alpha) were assayed in an enzyme immunoassay (EIA). The results showed that the COX-2 pathway was dominant in A549 cells whether activated by serum-free medium or not, and the COX-1 pathway could be ignored. The model accepted the positive inhibition threshold as NS398 2 microM; if a compound (10 microM) inhibited COX-2 pathway more than NS398 (2 microM), it was regarded as a hit. The COX-2 pathway inhibition experiment showed that the Z;-factor of the screening model was 0.62, which suggests that the model is suitable for COX-2 pathway inhibitor screening.     

Nicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone induce cyclooxygenase-2 activity in human gastric cancer cells: Involvement of nicotinic acetylcholine receptor (nAChR) and beta-adrenergic receptor signaling pathways

Induction of cyclooxygenase-2 (COX-2) associates with cigarette smoke exposure in many malignancies. Nicotine and its derivative, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), are the two important components in cigarette smoke that contributes to cancer development. However, the molecular mechanism(s) by which nicotine or NNK promotes gastric carcinogenesis remains largely unknown. We found that nicotine and NNK significantly enhanced cell proliferation in AGS cells that expressed both alpha7 nicotinic acetylcholine receptor (α7 nAChR) and β-adrenergic receptors. Treatment of cells with α-bungarotoxin (α-BTX, α7nAChR antagonist) or propranolol (β-adrenergic receptor antagonist) blocked NNK-induced COX-2/PGE₂ and cell proliferation, while nicotine-mediated cell growth and COX-2/PGE₂ induction can only be suppressed by propranolol, but not α-BTX. Moreover, in contrast to the dependence of growth promoting effect of nicotine on Erk activation, inhibitor of p38 mitogen-activated protein kinase (MAPK) repressed NNK-induced COX-2 upregulation and resulted in suppression of cell growth. In addition, nicotine and NNK mediated COX-2 induction via different receptors to modulate several G1/S transition regulatory proteins and promote gastric cancer cell growth. Selective COX-2 inhibitor (SC-236) caused G1 arrest and abrogated nicotine/NNK-induced cell proliferation. Aberrant expression of cyclin D1 and other G1 regulatory proteins are reversed by blockade of COX-2. These results pointed to the importance of adrenergic and nicotinic receptors in gastric tumor growth through MAPK/COX-2 activation, which may perhaps provide a chemoprevention strategy for cigarette smoke-related gastric carcinogenesis.     

(-)-Nyasol (cis-hinokiresinol), a norneolignan from the rhizomes of <Emphasis Type="Italic">Anemarrhena asphodeloides</Emphasis>, is a broad spectrum inhibitor of eicosanoid and nitric oxide production

To assess the anti-inflammatory activity of constituents from the rhizomes of Anemarrhena asphodeloides, (-)-nyasol {cis-hinokiresinol, 4,4-[1Z,3R]-3-ethenyl-1-propene-1,3-diyl]bisphenol} was isolated and its anti-inflammatory activity was examined in lipopolysaccharide (LPS)-treated RAW 264.7 cells and A23187-treated RBL-1 cells. In vivo activity was measured using carrageenan-induced paw edema assay. At > 1 μM, (-)-nyasol significantly inhibited cyclooxygenase-2 (COX-2)-mediated PGE₂ production and inducible nitric oxide synthase (iNOS)-mediated NO production in LPS-treated RAW 264.7 cells, a mouse macrophage-like cell line, but did not affect the expression levels of COX-2 and iNOS. (-)-Nyasol also inhibited 5-lipoxygenase (5-LOX)-mediated leukotriene production in A23187-treated RBL-1 cells. Furthermore, (-)-nyasol potently inhibited carrageenan-induced paw edema in mice (28.6–77.1% inhibition at 24–120 mg/kg). Therefore, (-)-nyasol is a potential new lead compound and may contribute to the anti-inflammatory action of A. asphodeloides, possibly by inhibiting COX-2, iNOS and 5-LOX.     

Amide derivatives of benzene-sulfonanilide,pharmaceutical composition thereof and method for cancer treatment using the same (US20120095092)

A series of sulfonamide derivatives incorporating an aromatic scaffold based on 1,4-phenylene diamine was prepared by a succession of five reactions. The lead molecule of these compounds was nimesulide (4-nitro-2-phenoxymethane-sulfoanilide), a cyclooxygenase (COX) inhibitor acting on both COX-1 and 2 isoforms, with some selectivity for COX-2. The new compounds from the patent are claimed to act as efficient antitumor agents, against a multitude of tumor types, such as leukemia, non-small cell lung cancer, colon cancer, brain cancer, melanoma, ovarian cancer, etc., although no COX inhibition data or other rationale to explain their biological effects are provided. As these compounds incorporate a methanesulfonamide moiety it is not unexpected that they may also appreciably inhibit tumor-associated carbonic anhydrases (CAs), such as isoforms CA IX and XII.     

Amide derivatives of benzene-sulfonanilide, pharmaceutical composition thereof and method for cancer treatment using the same (US20120095092)

A series of sulfonamide derivatives incorporating an aromatic scaffold based on 1,4-phenylene diamine was prepared by a succession of five reactions. The lead molecule of these compounds was nimesulide (4-nitro-2-phenoxymethane-sulfoanilide), a cyclooxygenase (COX) inhibitor acting on both COX-1 and 2 isoforms, with some selectivity for COX-2. The new compounds from the patent are claimed to act as efficient antitumor agents, against a multitude of tumor types, such as leukemia, non-small cell lung cancer, colon cancer, brain cancer, melanoma, ovarian cancer, etc., although no COX inhibition data or other rationale to explain their biological effects are provided. As these compounds incorporate a methanesulfonamide moiety it is not unexpected that they may also appreciably inhibit tumor-associated carbonic anhydrases (CAs), such as isoforms CA IX and XII.     

Synthesis, structure-activity relationships, and in vivo evaluations of substituted di-tert-butylphenols as a novel class of potent, selective, and orally active cyclooxygenase-2 inhibitors. 2. 1,3,4- and 1,2,4-thiadiazole series

Two isoforms of the cyclooxygenase (COX) enzyme have been identified: COX-1, which is expressed constitutively, and COX-2, which is induced in inflammation. Recently, it has been shown that selective COX-2 inhibitors have antiinflammatory activity and lack the GI side effects typically associated with NSAIDs. Initial mass screening and subsequent SAR studies have identified 6b (PD164387) as a potent, selective, and orally active COX-2 inhibitor. It had IC50 values of 0.14 and 100 microM against recombinant human COX-2 and purified ovine COX-1, respectively. It inhibited COX-2 activity in the J774A.1 cell line with an IC50 of 0.18 microM and inhibited COX-1 activity in platelets with an IC50 of 3.1 microM. The choline salt of compound 6b was also orally active in vivo with an ED40 of 7. 1 mg/kg in the carrageenan footpad edema (CFE) assay. In vivo studies in rats at a dose of 100 mg/kg showed that this compound inhibited gastric prostaglandin E2 (PGE2) production in gastric mucosa by 77% but caused minimal GI damage. SAR studies of this chemical series revealed that the potency and selectivity are very sensitive to minor structural changes.     

Pharmacological characterization of a selective COX-2 inhibitor MF-tricyclic, [3-(3,4-difluorophenyl)-4-(4-(methylsulfonyl)phenyl)-2-(5H)-furanone], in multiple preclinical species

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 PGE₂ production with an IC₅₀ (COX-2) of 0.6 and 2.8 μM, respectively. By comparison, the compound displayed a much weaker activity on clot-induced formation of thromboxane with an IC₅₀ (COX-1) of > 10 μM (guinea pigs) and 23 μM (rabbits). In keeping with the in vitro potency data, the compound significantly inhibited interleukin-1 beta (IL-1β) -induced PGE₂ formation in the rabbit synovium at plasma concentrations near the whole blood assay IC₅₀ 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 PGE₂ accumulation in the air pouch dose-dependently (ED₅₀ = 0.5 mg/kg) without affecting stomach PGE₂ 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 PGE₂ 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.     

(2E,6Z,10E)-7-hydroxymethyl-3,11,15-trimethyl-2,6,10,14-hexadecatetraen-1-ol (Plaunotol) increases cyclooxygenase-2 expression via nuclear factor kappaB and cyclic AMP response element in rat gastric epithelial cells

Plaunotol, [(2E,6Z,10E)-7-hydroxymethyl-3,11,15-trimethyl-2,6,10,14-hexadecatetraen-1-ol], a gastroprotective agent, increases the prostaglandin production in the gastric mucosa and accelerates ulcer healing. The precise mechanisms underlying the gastroprotective actions by plaunotol are not known. On the other hand, cyclooxygenase (COX)-2 is a key enzyme in PGE(2) production and its induction is thought to have an important role in ulcer healing. We investigated the mechanism of plaunotol-mediated COX-2 induction in rat gastric epithelial (RGM1) cells. We used a PGE(2) enzyme-linked immunoassay kit and Western blot analysis to measure PGE(2) production and COX-2 induction with plaunotol treatment in serum-starved RGM1 cells. In addition, gel-shift assay, Western blot analysis and a reporter assay were performed to observe which Cox-2 promoter was involved in plaunotol-induced Cox-2 expression. The findings indicated that plaunotol treatment dose-dependently increased COX-2 expression and PGE(2) production. The nuclear factor kappaB (NF-kappaB) and cyclic AMP response element (CRE) sites of the COX-2 gene promoter were critical to plaunotol-mediated COX-2 expression. In conclusion, plaunotol induced COX-2 expression and increased PGE(2) production in serum-starved RGM1 cells via activation of the NF-kappaB and CRE sites of Cox-2 gene promoters.     

Inhibition of mouse osteoblast proliferation and prostaglandin E2 synthesis by Ulmus davidiana Planch (Ulmaceae)

Ulmus davidiana Planch (Ulmaceae) (UD) is a widely used Korean herbal medicine that has been used historically in anti-inflammatory and anticancer therapy. Since UD has been known to have anti-inflammatory and protective effects on damaged tissue, inflammation and bone among other functions, this study was undertaken to address whether the water extract of the bark of UD could modulate proliferation of mouse osteoblasts in vitro and to investigate its effect on cyclooxygenase-2 (COX-2), which converts arachidonic acid to prostaglandin E2 (PGE2). Mouse osteoblasts were tested in vitro for growth inhibition, proliferating cell nuclear antigen (PCNA) expression, and COX-2 activity and expression after treatment with UD extract. Its effects were compared with those of indomethacin (a nonselective COX inhibitor) and celecoxib (a selective COX-2 inhibitor). UD demonstrated a strong growth inhibition in tested mouse osteoblasts. The IC50s were 10 μg/ml for UD, 6 μM for celecoxib and 42 μM for indomethacin. UD, as well as celecoxib and indomethacin, suppressed PCNA expression and PGE2 synthesis in osteoblasts. UD inhibited COX-2 expression, whereas celecoxib inhibited COX-2 activity directly. UD selectively and effectively inhibits osteoblasts cell growth in vitro. Inhibition of PGE2 synthesis via suppression of COX-2 expression may be responsible for its anti-inflammatory activity.     

11-Oxoeicosatetraenoic acid is a cyclooxygenase-2/15-hydroxyprostaglandin dehydrogenase-derived antiproliferative eicosanoid

Previously, we established that 11(R)-hydroxy-5,8,12,14-(Z,Z,E,Z)-eicosatetraenoic acid (HETE) was a significant cyclooxygenase (COX)-2-derived arachidonic acid (AA) metabolite in epithelial cells. Stable isotope dilution chiral liquid chromatography (LC)-electron capture atmospheric pressure chemical ionization (ECAPCI)/mass spectrometry (MS) was used to quantify COX-2-derived eicosanoids in the human colorectal adenocarcinoma (LoVo) epithelial cell line, which expresses both COX-2 and 15-hydroxyprostaglandin dehydrogenase (15-PGDH). 11(R)-HETE secretion reached peak concentrations within minutes after AA addition before rapidly diminishing, suggesting further metabolism had occurred. Surprisingly, recombinant 15-PGDH, which is normally specific for oxidation of eicosanoid 15(S)-hydroxyl groups, was found to convert 11(R)-HETE to 11-oxo-5,8,12,14-(Z,Z,E,Z)-eicosatetraenoic acid (ETE). Furthermore, LoVo cell lysates converted 11(R)-HETE to 11-oxo-ETE and inhibition of 15-PGDH with 5-[[4-(ethoxycarbonyl)phenyl]azo]-2-hydroxy-benzeneacetic acid (CAY10397) (50 μM) significantly suppressed endogenous 11-oxo-ETE production with a corresponding increase in 11(R)-HETE. These data confirmed COX-2 and 15-PGDH as enzymes responsible for 11-oxo-ETE biosynthesis. Finally, addition of AA to the LoVo cells resulted in rapid secretion of 11-oxo-ETE into the media, reaching peak levels within 20 min of starting the incubation. This was followed by a sharp decrease in 11-oxo-ETE levels. Glutathione (GSH) S-transferase (GST) was found to metabolize 11-oxo-ETE to the 11-oxo-ETE-GSH (OEG)-adduct in LoVo cells, as confirmed by LC-MS/MS analysis. Bromodeoxyuridine (BrdU)-based cell proliferation assays in human umbilical vein endothelial cells (HUVECs) revealed that the half-maximal inhibitory concentration (IC(50)) of 11-oxo-ETE for inhibition of HUVEC proliferation was 2.1 μM. These results show that 11-oxo-ETE is a novel COX-2/15-PGDH-derived eicosanoid, which inhibits endothelial cell proliferation with a potency that is similar to that observed for 15d-PGJ(2).     

Cyclooxygenase-2 mediates interleukin-6 upregulation by vomitoxin (deoxynivalenol) in vitro and in vivo

Interleukin-6 (IL-6) is a central mediator of immunotoxicity that is associated with exposure to the trichothecene vomitoxin (VT). The purpose of this investigation was to test the hypothesis that the inducible cyclooxygenase-2 (COX-2) and its metabolites contribute to VT-induced IL-6 upregulation. VT at 100 to 250 ng/ml readily induced COX-2 protein expression in the RAW 264.7 murine macrophage cell line. Superinduction of lipopolysaccharide (LPS)-mediated IL-6 production by VT in these cells was significantly reduced by the COX inhibitors indomethacin and NS-398, whereas the inhibitors did not affect direct induction of IL-6 by LPS alone. Mice that had been gavaged orally with 5 and 25 mg/kg VT exhibited elevated COX-2 mRNA expression in Peyer's patches and spleen with peak induction occurring 2 h after VT exposure. IL-6 mRNA was also induced by VT in vivo, however, peak expression occurred from 2 to 4 h after toxin exposure, suggesting that maximal COX-2 gene upregulation preceded or was concurrent with that for IL-6. Also consistent with a putative contributory role for COX-2 was the finding that both induction of splenic IL-6 mRNA and serum IL-6 by VT were significantly reduced by pretreating mice with the COX inhibitors indomethacin or NS-398. Finally, COX-2 knockout mice showed significantly reduced splenic IL-6 mRNA and serum IL-6 responses to oral VT exposure compared to their parental wild type. Taken together, these in vitro and in vivo data suggest that VT-induced COX-2 gene expression and resultant COX-2 metabolites contributed, in part, to subsequent upregulation of IL-6 gene expression, which has been previously shown to be a hallmark of VT-mediated immunotoxicity.     

Comparison of developmental toxicity of selective and non-selective cyclooxygenase-2 inhibitors in CRL:(WI)WUBR Wistar rats--DFU and piroxicam study

BACKGROUND: Cyclooxygenase (COX) inhibitors are one of the most often ingested drugs during pregnancy. Unlike general toxicity data, their prenatal toxic effects were not extensively studied before. The aim of the experiment was to evaluate the developmental toxicity of the non-selective (piroxicam) and selective (DFU; 5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl) phenyl-2(5H)-furanon) COX-2 inhibitors. METHODS: Drugs were separately, orally once daily dosed to pregnant rats from day 8 to 21 (GD1=plug day). Doses were set at 0.3, 3.0 and 30.0mg/kg for piroxicam and 0.2, 2.0 and 20.0mg/kg for DFU. Fetuses were delivered on GD 21 and routinely examined. Comprehensive clinical and developmental measurements were done. The pooled statistical analysis for ventricular septal (VSD) and midline (MD) defects was performed for rat fetuses exposed to piroxicam, selective and non-selective COX-2 inhibitor based on present and historic data. RESULTS: Maternal toxicity, intrauterine growth retardation, and increase of external and skeletal variations were found in rats treated with the highest dose of piroxicam. Decrease of fetal length was the only signs of the DFU developmental toxicity observed in pups exposed to the highest compound dose. Lack of teratogenicity was found in piroxicam and DFU-exposed groups. Prenatal exposure to non-selective COX inhibitors increases the risk of VSD and MD when compared to historic control but not with selective COX-2 inhibitors. CONCLUSION: Both selective and non-selective COX-2 inhibitors were toxic for rats fetuses when administered in the highest dose. Unlike DFU, piroxicam was also highly toxic to the dams. Prenatal exposure to selective COX-2 inhibitors does not increase the risk of ventricular septal and midline defects in rat when compared to non-selective drugs and historic control.     

Synthesis,anti-inflammatory activity,and molecular docking studies of some novel Mannich bases of the 1,3,4-oxadiazole-2(3H)-thione scaffold

A series of novel ibuprofen and salicylic acid-based 3,5-disubstituted-1,3,4-oxadiazole-2(3H)-thione derivatives was synthesized, and they were evaluated as potential anti-inflammatory agents. Following the structure identification studies employing IR, ¹H nuclear magnetic resonance (NMR), ¹³C NMR, and elemental analysis, the title compounds were tested by cyclooxygenase (COX)-1 and COX-2 inhibition assays concomitant to lipopolysaccharide (LPS)-induced nitric oxide and prostaglandin production prevention experiments. The results indicated that the majority of the compounds displayed either a superior or comparable activity in preventing both LPS-induced NO production and COX-1 activity in comparison to the activities of the reference molecules. Furthermore, docking studies were also performed to reveal possible interactions with the COX enzymes.     

Inhibition of angiogenic tubule formation and induction of apoptosis in human endothelial cells by the selective cyclooxygenase-2 inhibitor 5-bromo-2-(4-fluorophenyl)-3-(methylsulfonyl) thiophene (DuP-697)

There are indications that inhibitors of the cyclooxygenase-2 (COX-2) enzyme may cause inhibition of angiogenesis, proliferation of endothelial cells and induce apoptosis in cell systems. The concentrations of inhibitors required for such effects are however much higher than those needed to inhibit COX-2, suggesting that the latter may not be involved in these actions of the drugs. We have however generated data that strongly indicates a critical role for COX-2 suppression in the inhibition of angiogenesis and induction of apoptosis in human cultured umbilical vein endothelial cells (HUVECs) by the selective cyclooxygenase-2 (COX-2) inhibitor 5-bromo-2-(4-fluorophenyl)-3-(methylsulfonyl) thiophene (DuP-697). DuP-697 concentration-dependently inhibited prostaglandin E(2) (PGE(2)) production by HUVECs and at its known IC(50) for COX-2 inhibition of 10 nM inhibited basal and vascular endothelial cell growth factor (VEGF)-induced PGE(2) production by 80% and 85% respectively. DuP-697 also induced apoptosis as shown by FACs analysis, an increase in chromatin condensation and DNA laddering in HUVECS treated with the drug. Moreover, these effects were reversed by PGE(2) and by VEGF. In parallel studies, DuP-697 induced caspases 3, 8 and 9, with the caspase-3 specific inhibitor N-Acetyl-Asp-Glu-Val-Asp-al (DEVD-CHO) blocking the induction of apoptosis. Capillary-like tubule formation by HUVECs cultured on Matrigel was inhibited by DuP-697 and this inhibition was prevented by PGE(2) but not by DEVD-CHO. These results indicate that the induction of apoptosis and inhibition of tubule formation by DuP-697 involves the inhibition of COX-2 and that whereas the induction of apoptosis is caspase-dependent, the inhibition of tubule formation occurs through a caspase-independent mechanism.     

4-(4-cycloalkyl/aryl-oxazol-5-yl)benzenesulfonamides as selective cyclooxygenase-2 inhibitors: enhancement of the selectivity by introduction of a fluorine atom and identification of a potent, highly selective, and orally active COX-2 inhibitor JTE-522(1)

A series of 4-(4-cycloalkyl/aryl-oxazol-5-yl)benzenesulfonamide derivatives were synthesized and evaluated for their abilities to inhibit cyclooxygenase-2 (COX-2) and cyclooxygenase-1 (COX-1) enzymes. In this series, substituent effects at the ortho position to the sulfonamide group on the phenyl ring were examined. Most substituents reduced or lost both COX-2 and COX-1 activities. In contrast, introduction of a fluorine atom preserved COX-2 potency and notably increased COX1/COX-2 selectivity. This work led to the identification of a potent, highly selective, and orally active COX-2 inhibitor JTE-522 [9d, 4-(4-cyclohexyl-2-methyloxazol-5-yl)-2-fluorobenzenesulfonamide], which is currently in phase II clinical trials for the treatment of rheumatoid arthritis, osteoarthritis, and acute pain.     

Design, synthesis and pharmacological evaluation of 4-[2-alkylthio-5（4）-（4- substitutedphenyl）imidazole-4（5）yl]benzenesulfonamides as selective COX-2 inhibitors

Aim： To design and synthesize a series of benzenesulfonamide derivatives, 4-[2- alkylthio-5 （4）-（4-substitutedphenyl）imidazole-4（5）-yl]benzenesul fonamides （4a-4j）, which are intended to act as cyclooxygenase-2 （COX-2） inhibitors with good COX-2 inhibitor activity, and which will exert anti-inflammatory activities in vivo. Methods： Benzenesulfonamide derivatives were designed and synthesized through multi-step chemical reactions. All the synthesized compounds were evaluated in an in vitro assay. The active compound 4a-4f was selected for further evaluation in a carrageenan-induced rat paw edema model. Results： Docking studies showed that compound 4 bind into the primary binding site of COX-2 with the sulfonamide SO2NH2 moiety interacting with the secondary pocket amino acid residues. In the in vitro assay, compound 4 inhibited COX-2 with an inhibition concentration IC50 value of 1.23-8 nmol/L, compared to celecoxib with IC50 value of 1.5 nmol/L. Compound 4b and 4c had good potency and selectivity in comparison to the celecoxib. In the in vivo model, compound 4a-4f exhibited a moderate potency to inhibit 50% carrageenan-induced paw edema with value of 1.58-4.3 mg/kg. In the latter experiment, compound 4c was the most active compound. Conclusion： The antiinflammatory effects obtained for compound 4a-4j could be due to the presence of fluorine or hydrogen substituents in the para position of the phenyl ring of these compounds.