Defining the COX inhibitor selectivity of NSAIDs: implications for understanding toxicity

The hypothesis that the anti-inflammatory activity of NSAIDs derives from COX inhibition is well established. It also underpins the accepted mechanism of the gastrointestinal and renal toxicity of NSAIDs. However, in terms of NSAID-induced cardiovascular toxicity, is COX inhibition then guilty by association? Multiple experimental models of COX-1/COX-2 inhibition have enabled ranking of the relative inhibitory activity of NSAIDs. Inhibition is expressed as an IC(50) value and the index of COX selectivity as the ratio of the IC(50) value for COX-2 and COX-1. These data informed the 'imbalance hypothesis' that the cardiovascular risk of NSAIDs results from an imbalance in the detrimental actions of COX-1-derived thromboxane A(2) and the beneficial actions of COX-2-derived prostacyclin (PGI(2)). Data derived from in vitro models used to generate NSAID IC(50) values are discussed in the context of the difficulties in defining COX selectivity and hence understanding the toxicity of NSAIDs in current clinical use.     

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. 1. Thiazolone and oxazolone series

Selective cyclooxygenase-2 (COX-2) inhibitors have been shown to be potent antiinflammatory agents with fewer side effects than currently marketed nonsteroidal antiinflammatory drugs (NSAIDs). Initial mass screening and subsequent structure-activity relationship (SAR) studies have identified 4b (PD138387) as the most potent and selective COX-2 inhibitor within the thiazolone and oxazolone series of di-tert-butylphenols. Compound 4b has an IC50 of 1.7 microM against recombinant human COX-2 and inhibited COX-2 activity in the J774A.1 cell line with an IC50 of 0.17 microM. It was inactive against purified ovine COX-1 at 100 microM and did not inhibit COX-1 activity in platelets at 20 microM. Compound 4b was also orally active in vivo with an ED40 of 16 mg/kg in the carrageenan footpad edema (CFE) assay and caused no gastrointestinal (GI) damage in rats at the dose of 100 mg/kg but inhibited gastric prostaglandin E2 (PGE2) production in rats' gastric mucosa by 33% following a dose of 100 mg/kg. The SAR studies of this chemical series revealed that the potency and selectivity are very sensitive to minor structural changes. A simple isosteric replacement led to the reversal of selectivity.     

COX-1 and COX-2 inhibition in horse blood by phenylbutazone, flunixin, carprofen and meloxicam: an in vitro analysis.

We report on the inhibitory activity of the NSAIDs meloxicam, carprofen, phenylbutazone and flunixin, on blood cyclooxygenases in the horse using in vitro enzyme-linked assays. As expected, comparison of IC50 indicated that meloxicam and carprofen are more selective inhibitors of COX-2 than phenylbutazone and flunixin; meloxicam was the most advantageous for horses of four NSAIDs examined. However at IC80, phenylbutazone (+134.4%) and flunixin (+29.7%) had greater COX-2 selectivity than at IC50, and meloxicam (-41.2%) and carprofen (-12.9%) had lower COX-2 selectivity than at IC50. We therefore propose that the selectivity of NSAIDs should be assessed at the 80% as well as 50% inhibition level.     

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.     

Evaluation of 2 celecoxib derivatives： analgesic effect and selectivity to cyclooxygenase-2/1

AIM: To evaluate the analgesic effects of 2 celecoxib derivatives and their inhibitory effects on cyclooxygenase (COX). METHODS: Four antinociceptive assays were used: the acetic acid-induced writhing test, hot plate test, hot tail-flick test and formalin test. Three doses were used in the analgesic assays and ED50 values were calculated. For the selectivity assay, macrophages were incubated with test compounds at various concentrations and then stimulated with calcimycin or lipopolysaccharide (LPS). The amounts of 6-keto-prostaglandin F1alpha(6-keto-PGF1alpha) and prostaglandin E2 (PGE2) in the supernatant were examined by radioimmunoassay (RIA). The selectivity of the test compounds was expressed as the IC(50,COX-1)/IC(50,COX-2) value. RESULTS: Celecoxib and its 2 derivatives had a significant analgesic effect. The ED50 values of celecoxib, PC-406 and PC-407 were 94.2, 67.9, and 63.3 mg/kg, respectively, for the acetic acid-induced writhing test; 104.7, 89.1, and 30.0 mg/kg, respectively, for the hot tail-flick response test; 60.7, 56.7, and 86.2 mg/kg, respectively, for the hot plate response test; 67.1, 55.8, and 68.8 mg/kg, respectively, for the formalin-induced response. That is, the ED50 of PC-406 was the lowest for the formalin and hot plate tests, which focus on changes above the spinal cord level; however, the ED50 of PC-407 was lowest for the tail-flick and writhing tests, which focus on changes at the spinal cord level. Celecoxib and PC-407 inhibited COX-1 with IC50 values of 39.8 and 27.5 nmol/L, respectively. PC-406 inhibited COX-1 with an IC50 value of more than 1000 nmol/L. The IC50 values for the effect of celecoxib, PC-406 and PC-407 on COX-2 were 4.8, 8.9, and 1.9 nmol/L respectively. The IC(50, COX-1)/IC(50,COX-2) ratios for celecoxib and PC-407 were 8.3 and 14.4, respec-tively. For PC-406, the ratio was greater than 112.2. CONCLUSION: Derivatives of celecoxib via substitution with an isopropyl or naphthyl group at the 5 position in the pyrazole ring still have analgesic effects and the ability to selectively inhibit COX-2. Substitution with a naphthyl group may have more effect on the peripheral pain pathway, whereas substitution with an isopropyl group may have more effect on the central pain pathway. This phenomenon occurs partly because substitution with an isopropyl group is more beneficial for COX-2 selectivity than is substitution with a naphthyl group.     

Human whole blood assay for rapid and routine testing of non-steroidal anti-inflammatory drugs (NSAIDs) on cyclo-oxygenase-2 activity

Aim of this study was to present a simple, fast and reliable method to examine the capacity of NSAIDs to inhibiting COX-2 activity that uses rapid (stimulation takes only 5 h compared to other existing protocols) and routine testing. The assay includes elimination of COX-1-activity using ASS (a selective COX-1 inhibitor) and the thromboxane synthetase inhibitor (TXBSI), COX-2 induction via LPS and measurement of PGE(2). Using TXBSI reduces the amount of LPS and results in higher prostaglandin production.Cremophor EL((R))-EtOH was used as vehicle instead of DMSO because within a defined concentration range, Cremophor EL((R))-EtOH allows even very hydrophobic drugs to be solubilized and applied in vitro without cell damage. Cremophor EL((R))-EtOH at 0.2% was optimal as at this relatively low concentration excellent drug dissolution was obtained whereas many hydrophobic substances precipitate in 0.2% DMSO.Our results demonstrate that the IC(50) values for the tested NSAIDs are in the range of published data.     

Comparison of cyclooxygenase inhibitory activity and ocular anti-inflammatory effects of ketorolac tromethamine and bromfenac sodium

OBJECTIVE: To compare the cyclooxygenase (COX) activity and anti-inflammatory effects of the nonsteroidal anti-inflammatory drugs (NSAIDs) ketorolac tromethamine (ketorolac) and bromfenac sodium (bromfenac). METHODS: Cyclooxygenase activity and selectivity was determined in vitro by measuring prostaglandin E(2) (PGE(2)) production following incubation of varying concentrations of NSAID with human recombinant COX-1 or COX-2 and arachidonic acid. Anti-inflammatory effects were evaluated in a rabbit model in which an ocular inflammatory response was induced by intravenous injection of 10 microg/kg lipopolysaccharide (LPS). In study animals, one eye was treated with 50 microL (+/-) ketorolac 0.4% (Acular LS) or bromfenac 0.09% (Xibrom) and the other eye with 50 microL buffered saline. In control animals, both eyes were treated with vehicle. All animals were treated twice: 2 hours and 1 hour before LPS. MAIN OUTCOME MEASURES: PGE(2) production in vitro, measured by enzyme immunoassay; fluorescein isothiocyanate (FITC)-dextran leakage into the anterior chamber, measured by fluorophotometry; aqueous PGE(2) levels in vivo, measured by ELISA immunoassay. RESULTS: Ketorolac was six times more active against COX-1 (IC(50) = 0.02 microM) than COX-2 (IC(50) = 0.12 microM) while bromfenac was approximately 32 times more active against COX-2 (IC(50) = 0.0066 microM) than COX-1 (IC(50) = 0.210 microM). In the animal model, both drugs resulted in nearly complete inhibition of FITC-dextran leakage and PGE(2) production in the anterior chamber of treated eyes. There was also a 79% inhibition (p < 0.001) of FITC-dextran leakage in the contralateral eyes of bromfenac-treated rabbits, and a 22.5% inhibition (not statistically significant) in the contralateral eyes of ketorolac-treated rabbits. CONCLUSIONS: Ketorolac is relatively COX-1 selective while bromfenac is potently selective for COX-2 over COX-1. In the animal model, both ketorolac 0.4% and bromfenac 0.09% demonstrated maximal anti-inflammatory activity in treated eyes. Only bromfenac 0.09% had a significant effect on the contralateral eye, suggesting possible systemic absorption of this drug.     

Evaluation of COX-1/COX-2 selectivity and potency of a new class of COX-2 inhibitors

A new class of selective cyclooxygenase-2 (COX-2) inhibitors has been identified by high throughput screening. Structurally distinct from previously described selective COX-2 inhibitors, these benzopyrans contain a carboxylic acid function and CF3 functionality. The compound SC-75,416 is a representative of this class. A range if in vitro and in vivo tests were employed to characterize its potency and selectivity. Using human recombinant enzymes, this compound displays a concentration that provides 50% inhibition (IC50) of 0.25 microM for COX-2 and 49.6 microM for COX-1. A mutation of the side pocket residues in COX-2 to COX-1 had little effect on potency suggesting that these inhibitors bind in a unique manner in COX-2 distinct from COX-2 inhibiting diaryl heterocycles. Using rheumatoid arthritic synovial cells stimulated with interleukin-1beta (IL-1beta) and washed platelets the compound displayed IC50 of 3 nM and 400 nM respectively. Potency and selectivity was maintained but predictably right shifted in whole blood with IC50 of 1.4 microM for lipopolysaccharide (LPS) stimulated induction of COX-2 and >200 microM for inhibition of platelet thromboxane production. SC-75,416 is 89% bioavailable and its in vivo half life is sufficient for once a day dosing. In the rat air pouch model of inflammation, the compound inhibited PGE2 production with an effective dose that provides 50% inhibition (ED50) of 0.4 mg/kg, while sparing gastric prostaglandin E2 (PGE2) production with an ED50 of 26.5 mg/kg. In a model of acute inflammation and pain caused by carrageenan injection into the rat paw, the compound reduced edema and hyperalgesia with ED50s of 2.7 and 4 mg/kg respectively. In a chronic model of arthritis the compound demonstrated an ED50 of 0.081 mg/kg and an ED(80) of 0.38 mg/kg. In a model of neuropathic pain, SC-75,416 had good efficacy. This compound's unique chemical structure and effect on COX enzyme binding and activity as well as its potency and selectivity may prove useful in treating pain and inflammation.     

A model with original conditions and materials to test potential inhibitors of cyclooxygenase-2 versus cyclooxygenase-1 in isolated human blood cells

The inflammatory process can be influenced by an enzyme known as cyclooxygenase, which exists in two isoforms: COX-1, the constitutive form, and COX-2, which is only synthesized in the case of inflammation and has negative side effects. To decrease these undesirable effects, current research seeks to discover compounds that selectively inhibit COX-2 versus COX-1. When added to the samples, arachidonic acid activates the synthesis of COX-2. This explains why the two isoforms proceed in different ways. As for COX-1, human lymphocytes and monocytes were activated with 50 microM of calcium ionophore for 1 h at 37 degrees C with 50 microM of arachidonic acid, and the quantity of thromboxane B(2) (TxB(2)) was measured by radioimmuno-assay (RIA). As for COX-2, cells were first incubated with acetylsalicylic acid to block COX-1, then stimulation of COX-2 was performed using 8 mcg/ml lipopolysaccharide (LPS) for 3 h at 37 degrees C with 50 microM arachidonic acid. The IC(50) values for reference compounds were reproducible, taking into consideration the passage through the plasmatic membrane and the transduction signal, while avoiding the problems of binding with plasma proteins and using identical cells, unlike other models.     

小檗碱对p38MAPK通路及COX-2mRNA表达的作用

目的研究中药小檗碱是否通过p38MAPK信号转导途径抑制人外周血单核细胞COX-2mRNA及蛋白表达。方法取人外周静脉血分离及培养单核细胞,分为对照组、脂多糖(LPS)组、LPS+小檗碱25μmol/L组、LPS+小檗碱50μmol/L组、LPS+小檗碱100μmol/L组。分别在培养后0.5、6、12、24h提取细胞,行RT-PCR法测定COX-2mRNA水平,行Westernblot法测定p38MAPK、p-p38MAPK及COX-2蛋白水平。同时加入选择性p38MAPK抑制剂,分别测定COX-2mRNA及蛋白水平。结果与对照组相比,LPS组COX-2mRNA及蛋白表达明显增强(P<0.01)。与LPS组相比,小檗碱组COX-2mRNA及蛋白表达明显抑制(P<0.05),且随着浓度增加,抑制作用更明显,在给药后12h,小檗碱对COX-2抑制作用最强,但是与LPS组相比,小檗碱组p38MAPK活性水平无明显统计学差异(P>0.05)。加入p38MAPK抑制剂之后,COX-2mRNA及蛋白水平降低明显(P<0.05)。结论小檗碱能抑制人外周血单核细胞COX-2mRNA及蛋白水平,并呈浓度依赖性,p38MAPK与人外周血COX-2表达有关,而小檗碱对p38MAPK活性蛋白表达无明显抑制作用。     

ERK信号转导途径在小檗碱抑制脂多糖诱导的COX一2表达中的作用

目的探讨小檗碱是否抑制脂多糖诱导的COX-2mRNA及蛋白表达,以及小檗碱是否通过ERK信号转导途径抑制cOx-2的表达。方法取健康志愿者外周血,分离及培养单核细胞,分为五组,分别为对照组;脂多糖（Lipopolysaccharide,LPS）组;LPS＋小檗碱25μmol／L组;LPS＋小檗碱50μmol/L组;LPS＋小檗碱100μmoL／L组。分别在培养后30min,6h,12h,24h提取细胞,行RT—PCR法测定COX-2mRNA水平,行westemblot法测定ERK、P—ERK及COX-2蛋白水平。同时加入选择性ERK抑制剂,分别测定C0X-2mRNA及蛋白水平。结果与对照组相比,LPS组COX-2mRNA及蛋白表达明显增强（P〈0．01）。与LPS组相比,小檗碱组COX-2mRNA及蛋白表达明显抑制（P〈0．05）,且随着浓度增加,抑制作用更明显,在给药后12h,小檗碱对COX-2抑制作用最强。与LPS组相比,小檗碱组ERK活性水平有明显统计学差异（P〈0．05）。加入ERK抑制剂之后,COX-2mRNA及蛋白水平降低明显（P〈0．05）。结论小檗碱能抑制人外周血单核细胞cOx-2mRNA及蛋白水平,其作用程度呈浓度依赖性,小檗碱对ERK活性蛋白表达有明显抑制作用。小檗碱可能通过ERK信号转导途径抑制人外周血单核细胞C0x_2mRNA及蛋白表达。     

Design,synthesis and preliminary evaluation of the anti-inflammatory of the specific selective targeting druggable enzymome cyclooxygenase-2 (COX-2) small molecule

Context: Development of a reliable and selective anti-inflammatory agent of cyclooxygenase-2 (COX-2), induced or up-regulated by inflammatory/injury stimulus such as IL-1β, TNF-α and LPS in the various types of organs, tissues and cells, with low side effects is a long-standing medicinal chemistry problem with significant social implications.

Objective: To target druggable enzymome COX-2 by exploiting NSAIDs and genipin (GEP) in anti-inflammatory infection.

Materials and methods: The compound aspirin GEP ester (AGE) was designed by computer-assisted screening, synthesized in the esterification of the acylate derivative and the methylate derivative with Et₃N, and evaluated with 20, 40 and 60?mg/kg from days 18 to 24 after immunization in collagen-induced arthritis (CIA) rats by the sequential enzymatic experiments, western-blot analysis and pathological observation methods.

Results: AGE exhibited higher binding affinity with COX-1 and displayed the lowest estimated free energy with COX-2 than other ligands built by hanging NSAIDs with GEP, and was characterized by ¹H NMR, ¹³C NMR and HRMS. AGE was competed against COX-2 with molecule-dependent potencies and selectivity (IC₅₀: 0.36?mM; selectivity index: 275) in the sequential enzymatic experiments and decreased the expression of COX-2 in peripheral blood lymphocytes of CIA rats. AGE (40 and 60?mg/kg) could significantly relieve the secondary hind paw swelling and arthritis index, along with observing AGE attenuated histopathological changes of fibroblast like synovial tissue (FLST) and mesenteric lymph node lymphocytes (MLNL) in CIA rats.

Discussion and conclusion: AGE pharmacophore reported herein may be an effective strategy to develop a novel anti-inflammatory agent and potential inhibitor of COX-2.     

Design, synthesis, and structure-activity relationship studies of 3,4,6-triphenylpyran-2-ones as selective cyclooxygenase-2 inhibitors

A group of regioisomeric 3,4,6-triphenylpyran-2-ones with a MeSO(2) pharmacophore at the para-position of either a C-3 phenyl or a C-4 phenyl substituent on the central six-membered pyran-2-one ring were prepared and evaluated in vitro for their abilities to inhibit the isozymes COX-1 and COX-2. Structure-activity relationship (SAR) data, acquired by substituent modification at the para-position of the C-6 phenyl ring attached to the central pyranone, showed that 6-(4-methoxyphenyl)-3-(4-methanesulfonylphenyl)-4-phenylpyran-2-one (12e) was the most potent and selective COX-2 inhibitor (COX-2 IC(50) = 0.02 microM; COX-1 IC(50) > 100 microM) with a high COX-2 selectivity index (SI > 5000) relative to the reference drugs celecoxib (COX-2 IC(50) = 0.07 microM; SI = 474) and rofecoxib (COX-2 IC(50) = 0.50 microM; SI > 200). 6-(4-Methoxyphenyl)-3-(4-methanesulfonylphenyl)-4-phenylpyran-2-one (12e) was a more potent oral antiinflammatory agent (ID(50) = 5.6 mg/kg) than celecoxib (ID(50) = 10.8 mg/kg) in a carrageenan-induced rat paw edema assay. In a 4% NaCl-induced abdominal constriction assay, a 5 mg/kg oral dose of 12e exhibited good analgesic activity at different time intervals producing 37.5 and 69% inhibition of writhing at 30 and 60 min, respectively. In contrast, the corresponding 6-(4-methoxyphenyl)-4-(4-methanesulfonylphenyl)-3-phenylpyran-2-one regiosiomer (12o) was a less potent and selective COX-2 inhibitor (COX-2 IC(50) = 0.45 microM; SI = 70). A molecular modeling study for 12e indicated that the p-OMe substituent on the C-6 phenyl ring interacts with the COX-2 binding site amino acids Ile(345), Val(349), Leu(359), Leu(531), and Met(535) and that the OMe substituent may be responsible for proper orientation of the C-3 p-SO(2)Me-phenyl ring within the COX-2 secondary pocket (Gln(192), Arg(513), and Phe(518)). These results show that the COX-2 selectivity and potency of 3,4,6-triphenylpyranone regioisomers can be modulated by appropriate placement of the p-SO(2)Me pharmacophore on either the C-3 or C-4 phenyl moiety. In addition, electronic properties at the para-position of a C-6 phenyl substituent on the central pyranone ring govern COX-2 inhibitory potency and selectivity by controlling the orientation of the p-SO(2)Me pharmacophore within the COX-2 secondary pocket.     

Design, synthesis and biological evaluation of new 5,5-diarylhydantoin derivatives as selective cyclooxygenase-2 inhibitors

A new group of 5,5-diarylhydantoin derivatives bearing a methylsulfonyl COX-2 pharmacophore at the para position of the C-5 phenyl ring were designed and synthesized as selective COX-2 inhibitors. In vitro COX-1/COX-2 inhibition structure-activity relationships identified 5-[4-(methylsulfonyl)phenyl]-5-phenyl-hydantoin (4) as a highly potent and selective COX-2 inhibitor (COX-2 IC(50) = 0.077 μM; selectivity index > 1298). It was more selective than the reference drug celecoxib (COX-2 IC(50) = 0.060 μM; selectivity index = 405). A molecular modeling study where 4 was docked in the binding site of COX-2 indicated that the p-MeSO(2) COX-2 pharmacophore group on the C-5 phenyl ring is oriented in the vicinity of the COX-2 secondary pocket. The results of this study showed that the type of substituent on the N-3 hydantoin ring substituent is important for COX-2 inhibitory activity.     

Petasites hybridus extracts in vitro inhibit COX-2 and PGE2 release by direct interaction with the enzyme and by preventing p42/44 MAP kinase activation in rat primary microglial cells

Rhizomes of butterbur, Petasites hybridus L. (Asteraceae), have been used since ancient times for the treatment of inflammatory diseases. In the present study, the effects of lipophilic extracts from rhizomes of Petasites hybridus on the formation and release of prostaglandin E2 were investigated. The extracts had different contents of petasin and isopetasin: A: 2.1 % and 0.4 %, B: 0.2 % and 0.1 %, C: 12.1 % and 6.1 % and D: 21.9 % and 9.4 %, respectively. Direct inhibition of cyclooxygenase (COX) -1 and -2 isoenzymes and inhibition of the expression of COX-2 and p42/44 MAP kinase in rat primary microglial cells were tested. All extracts were found to be only weak direct inhibitors of COX-1 (IC50> 400 microg/mL). However, most extracts revealed a strong inhibitory activity against the inducible isoform COX-2 ( A: IC50=30.4 microg/mL; B: IC50=60.6 microg/mL; C: IC50=22.6 microg/mL; D: IC50=20.0 microg/mL). This activity was not correlated to the content of petasin and isopetasin. Pure petasin and isopetasin neither inhibited COX-1 nor COX-2 (IC50 > 400 microM for both compounds and enzymes). Petasites extracts dose-dependently inhibited LPS-induced and thus COX-2-mediated PGE2 release in primary rat microglial cells (A: IC50= 2.4 microg/mL; C: IC50=5.8 microg/mL and D: IC50=4.6 microg/mL). Also this effect was independent from the petasin and isopetasin content. COX-2 synthesis in microglia was totally blocked with 5 microg/mL of C whereas COX-1 synthesis was not influenced. C and D did not affect the LPS-induced activation of p38 MAPK and IkappaBalpha, but they prevented the LPS-induced activation of p42/44 MAPK. Therefore, these Petasites hybridus extracts can be regarded as natural selective inhibitors of COX-2 and its expression, an effect which is independent from the petasin content.     

Effect of SK&F 86002 on cytokine production by human monocytes

Human monocytes respond to a variety of stimuli in vitro by producing a number of physiologically important macromolecules including the cytokines. SK&F 86002, a dual inhibitor of the arachidonate metabolism, has been shown to inhibit LPS induced IL-1 production in human monocytes. We examined its effect on the production of other cytokines which are coordinately expressed as a result of LPS stimulation such as tumor necrosis factor alpha (TNF), alpha interferon (IFN-A), interferon beta-2 (IL-6) and granulocyte colony stimulating factor (g-CSF). The IC50 of SK&F 86002 for the TNF production was 5-8 microM, and greater than 20 microM for the other three cytokines. These IC50s were significantly higher than that previously reported for IL-1 production (1-2 microM). Taken together these data indicate that the inhibitory effect of SK&F 86002 on IL-1 production is selective and the production of cytokines in drug treated monocytes can be differentially affected.     

Isolation of (S)-(+)-naproxene from Musa acuminata. Inhibitory effect of naproxene and its 7-methoxy isomer on constitutive COX-1 and inducible COX-2

The isolation and characterisation of (S)-(+)-6-methoxy-alpha-methyl-2-naphthaleneacetic acid, a well known synthetic non-steroidal anti-inflammatory drug (naproxene), from a natural source is described for the first time. We evaluated the ability of naproxene and its 7-methoxy isomer to abrogate constitutive COX-1 and inducible COX-2 activity in human A549 cells. Naproxene inhibited COX-1 (IC50 = 3.42 microM) and COX-2 (IC50 = 1.53 microM), whereas the 7-methoxy isomer had no appreciable effect on COX-1 (IC50 > 100 microM) but also abrogated the activity of COX-2 enzyme (IC50 = 14.42 microM).     

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.     

Comparison of the influence of NSAIDs with different COX-selectivity on histamine release from mast cells isolated from naïve and sensitized rats

Mast cell stimulation leads to an early response with histamine release and prostaglandin (PGD₂) production but attempts to link these two events have been contradictory. In IgE-mediated mast cell activation, a late-phase PGD₂-production is caused by increased cyclooxygenase-2 (COX-2) expression whereas a COX-2 involvement in the early response is uncertain. The present study compares the influence of four COX-inhibitors (NSAIDs) on the histamine release of mast cells from naïve and actively sensitized rats. NSAIDs of different COX-1 vs. COX-2 selectivity were used, i.e. acetylsalicylic acid (ASA), piroxicam, meloxicam, and NS-398, a selective COX-2-inhibitor. All could inhibit antigen-induced histamine release, with 64%, 34%, 27% and 85% inhibition by ASA (5 mM), piroxicam (100 μM), meloxicam (100 μM) and NS-398 (100 μM), respectively. Similar inhibition was found with compound 48/80 without calcium added to the medium whereas compound 48/80 with calcium was affected less by ASA and NS-398 and unaffected by the oxicams. Only small differences between the two kinds of mast cells were found, except with NS-398 which was a significantly more effective inhibitor of naïve than sensitized cells when exposed to compound 48/80 with calcium present. The results do not show any consistent relationship between the influence of the NSAIDs and their COX-2-selectivity. The high NSAID-concentrations required for inhibition cast doubt about an involvement of COX-inhibition and indicate additional or other targets. The results seem to exclude toxic effects on mast cell energy production but are consistent with an interference with the calcium disposition.