Molecular hybrids of substituted phenylcarbamoylpiperidine and 1,2,4-triazole methylacetamide as potent 15-LOX inhibitors: Design, synthesis, DFT calculations and molecular docking studies.

Inflammation is a multifaceted phenomenon triggered by potentially active mediators acutely released arachidonic acid metabolites partially in lipoxygenase (LOX) pathway which are primarily accountable for causing several diseases in humans. It is widely believed that an inhibitor of the LOX pathway represents a rational approach for designing more potent antiinflammatory leads with druggable super safety profiles. In our continual efforts in search for anti-LOX molecules, the present work was to design a new series of N-alkyl/aralkyl/aryl derivatives (7a-o) of 4-phenyl-5-(1-phenylcarbamoylpiperidine)-4H-1,2,4-triazole-3-thiol which was commenced in seriate formation of phenylcarbamoyl derivative (1), hydrazide (2), semicarbazide (3) and 4-phenyl-5-(1-phenylcarbamoylpiperidine)-4H-1,2,4-triazole-3-thiol (4). The aimed compounds were obtained by reacting 4-phenyl-5-(1-phenylcarbamoylpiperidine)-4H-1,2,4-triazole-3-thiol with assorted N-alkyl/aralkyl/aryl electrophiles. All compounds were characterized by FTIR, 1H-, 13C-NMR spectroscopy, EI-MS and HR-EI-MS spectrometry and screened against soybean 15-LOX for their inhibitory potential using chemiluminescence method. All the compounds except 7m and 7h inhibited the said enzyme remarkably. Compounds 7c,7l, 7j and 7a displayed potent inhibitions ranging from IC50 1.92 ± 0.13 µM to 7.65 ± 0.12 µM. Other analogues 7g, 7o, 7e, 7b, 7d, 7k and 7n revealed excellent inhibitory values ranging from IC50 12.45 ± 0.38 µM to 24.81 ± 0.47 µM. All these compounds did not reveal DPPH radical scavenging activity. Compounds 7i-o maintained > 90 % human blood mononuclear cells (MNCs) viability at 0.125 mM as assayed by MTT whilst others were found toxic. Pharmacokinetic profiles predicted good oral bioavailability and drug-likeness properties of the active scaffolds. SAR investigations showed that phenyl substituted analogue on amide side decreased inhibitory activity due to inductive and mesomeric effects while the mono-alkyl substituted analogues were more active than disubstituted ones and ortho substituted analogues were more potent than meta substituted ones. MD simulation predicted the stability of the 7c ligand and receptor complex as shown by their relative RMSD (root mean square deviation) values. Molecular docking studies displayed hydrogen bonding between the compounds and the enzyme with Arg378 which was common in 7n, 7g, 7h and baicalein. In 7a and quercetin, hydrogen bonding was established through Asn375. RMSD values exhibited good inhibitory profiles in the order quercetin (0.73 Å) < 7 g < baicalein < 7a < 7n < 7 h (1.81 Å) and the binding free energies followed similar pattern. Density functional theory (DFT) data established good correlation between the active compounds and significant activity was associated with more stabilized LUMO (lowest unoccupied molecular orbitals) orbitals. Nevertheless, the present studies declare active analogues like 7c, 7 l, 7a, 7j as leads. Work is ongoing in derivatizing active molecules to explore more effective leads as 15-LOX inhibitors as antiinflammatory agents.

Targeting new N-furfurylated 4-chlorophenyl-1,2,4-triazolepropionamide hybrids as potential 15-lipoxygenase inhibitors supported with in vitro and in silico studies.

Lipoxygenases (LOXs) are a group of enzymes that catalyze the oxidation of polyunsaturated fatty acids and initiate the biosynthesis of secondary metabolites that are involved to control inflammation. In search of new and more potent LOX inhibitors, a series of new 3-(5-(4-chlorophenyl)-4-(2-furylmethyl)-1,2,4-triazole hybrids was prepared and screened for its LOX inhibitory potential. 4-Chlorobenzoic acid (a) was metamorphosed into N-furfuryl-5-(4-chlorophenyl)-4-(2-furylmethyl)-1,2,4-triazole (4) via intermediates like benzoate (1), hydrazide (2) and semicarbazide (3). Finally, triazole (4) was fused with propionamides (6a-o) and transformed it into the aimed derivatives (7a-o). The structural interpretations of the prepared derivatives (7a-o) were accomplished via FTIR, 1H-, 13C-NMR spectroscopy, EI-MS and HR-EI-MS spectrometry. The inhibitory potency of the compounds against soybean 15-LOX was determined by in vitro assay using chemiluminescence method. Compounds 7a and 7f exhibited potent LOX inhibitory profiles with IC50 21.83 ± 0.56 and 25.72 ± 0.51 µM, whereas 7d and 7e showed comparable inhibitory potential with IC50 values of 34.52 ± 0.39 and 39.12 ± 0.46 µM, respectively. Compounds 7a, 7f, 7d and 7e exhibited 65.58 ± 1.4%, 54.72 ± 1.3%, 58.52 ± 1.2% and 63.56 ± 1.4% blood mononuclear cells viability, respectively. Density functional theory and molecular docking studies further strengthened the studies of the synthesized compounds and these derivatives perceived to be potential 'lead' compounds in drug discovery as anti-LOX.Communicated by Ramaswamy H. Sarma.

Assessing p-tolyloxy-1,3,4-oxadiazole acetamides as lipoxygenase inhibitors assisted by in vitro and in silico studies.

The underlying correlation between the inflammation, innate immunity and cancer is extensively familiar and linked through a process mediated by three enzymes; cyclooxygenase (COX), lipoxygenase (LOX) and cytochrome P450 (CYP450). The ever increase in the reported side effects of the antiinflammatory drugs against the targeted enzymes and the resistance developed afterwards compels the researchers to synthesize new effective molecules with safer profile. On the basis of these facts, our ongoing research on 1,3,4-oxadiazole derivatives deals with the synthesis of a new series of N-alkyl/aralky/aryl derivatives of 5-((p-tolyloxymethyl)-4H-1,3,4-oxadiazole-2-ylthio)acetamide (6a-o) which were developed by the sequential conversion of p-tolyloxyacetic acid (a) into ester (1) hydrazide (2) and 5-(p-tolyloxymethyl)-4H-1,3,4-oxadiazole-2-thiol (3). The designed compounds (6a-o) were acquired by the reaction of 1,3,4-oxadiazole (3) with numerous electrophiles (5a-o) in KOH. The synthesized analogues (6a-o) were characterized by FTIR, 1H-, 13C NMR spectroscopy, EI-MS and HR-EI-MS spectrometry, and were further assessed for their inhibitory potential against the soybean 15-LOX enzyme. The results showed excellent inhibitory potential of the compounds against the said enzyme, specifically 6o, 6b, 6n and 6e with inhibitory values (IC50 ± SEM) of 21.5 ± 0.76, 24.3 ± 0.45, 29.1 ± 0.65 and 31.3 ± 0.78 µM, respectively. These compounds displayed < 55 % blood mononuclear cells (MNCs) cellular viability as measured by MTT assay at 0.25 mM concentration. Other compounds demonstrated moderate inhibitory activities with IC50 values in the range of 33.2 ± 0.78 to 96.3 ± 0.73 µM and exhibited little cellular viability against MNCs except 6i, 6j, 6 m and 6 k that showed 61-79 % cellular viability. It was observed that most of the compounds (6o, 6b, 6n, 6e) were found more toxic towards MNCs at studied concentration of 0.25 mM. SAR studies revealed that the positions and nature of substituents accompanying phenyl ring have great influence on 15-LOX inhibitory activity. In the most active compound 6o, the amino acids Asp768 and Val126 were involved in hydrogen bonding, Thr529 was linked with π-anion interaction and π-sulphur interaction was displayed with Tyr525 and two π-alkyl interactions were formed with the benzene ring and amino acid residues Pro530 and Arg533. The in silico pharmacokinetics profiles and density functional theory calculations of the compounds further supported the in vitro findings. Further work on the synthesis of more oxadiazole derivatives is in progress in search for potential 'leads' for the drug discovery as LOX inhibitors.

4-Chlorophenyl-N-furfuryl-1,2,4-triazole Methylacetamides as Significant 15-Lipoxygenase Inhibitors: an Efficient Approach for Finding Lead Anti-inflammatory Compounds.

Lipoxygenases (LOXs) are a class of enzymes that catalyze the production of pro-inflammatory mediators, such as leukotrienes and lipoxins, via an arachidonic acid cascade as soon as they are released from the membrane phospholipids after tissue injury. In continuation of our efforts in search for new LOX inhibitors, a series of chlorophenyl-furfuryl-based 1,2,4-triazole derivatives were prepared and evaluated for their 15-LOX inhibitory activities. A simple precursor, 4-chlorobenzoic acid (a), was consecutively transformed into benzoate (1), hydrazide (2), semicarbazide (3), and N-furfuryl 5-(4-chlorobenzyl)-4H-1,2,4-triazole (4), which when further merged with electrophiles (6a-o) resulted in end products (7a-o). The structural elucidations of the newly synthesized compounds (7a-o) were carried out by Fourier transform infrared, 1H-, 13C NMR spectroscopy, EI-MS, and HR-EI-MS spectrometry. The inhibitive capability of compounds (7a-o) on soybean 15-LOX was performed in vitro using the chemiluminescence method. The compounds 7k, 7o, 7m, 7b, and 7i demonstrated potent activities (IC50 17.43 ± 0.38, 19.35 ± 0.71, 23.59 ± 0.68, 26.35 ± 0.62, and 27.53 ± 0.82 µM, respectively). These compounds revealed 79.5 to 98.8% cellular viability as measured by the MTT assay at 0.25 mM concentration. The structure-activity relationship (SAR) studies showed that the positions and the nature of substituents bonded to the phenyl ring are important in the determination of 15-LOX inhibitory activities. ADME, in silico, and density functional theory studies supported the evidence as yet another class of triazoles with potential lead properties in search for anti-LOX compounds with a safe gastrointestinal safety profile for various inflammatory diseases. Further work is in progress on the synthesis of more derivatives in search for anti-inflammatory agents.

The effect of 24-week belimumab treatment withdrawal followed by treatment restart in patients with SLE: an open-label, non-randomised 52-week study.

BACKGROUND: Treatment goals for patients with systemic lupus erythematosus (SLE) include minimising disease activity and reducing the risk of flares. Although belimumab is effective at reducing disease activity and risk of severe flares, it was previously unknown what the clinical effects were upon treatment discontinuation. The objective of this study was to assess the impact of temporary withdrawal of intravenous (IV) belimumab in patients with SLE. METHODS: This multicentre, open-label, non-randomised, 52-week study (GSK Study BEL116027; NCT02119156) recruited patients with SLE and stable low disease activity, of whom those on belimumab 10 mg/kg IV plus standard therapy either discontinued belimumab for 24 weeks and then restarted belimumab 10 mg/kg IV every 4 weeks (q4w) for 28 weeks (treatment holiday [TH] group), or continued on belimumab 10 mg/kg IV plus standard therapy q4w for 52 weeks (treatment continuation [TC] group). The primary endpoint was median time to first Safety of Estrogens in Lupus Erythematosus National Assessment-SLE Disease Activity Index (SELENA-SLEDAI) Flare Index flare. Secondary and other endpoints included rate of any flare, time to severe flare, time to renal flare and rebound (SELENA-SLEDAI score exceeding parent study baseline). Data on rebound phenomenon in patients with any disease level of SLE who had permanently withdrawn from further belimumab treatment (long-term discontinuation group [LTD]) were also assessed. Safety was assessed. RESULTS: The primary endpoint was not evaluable in the TH (n = 12) and TC (n = 29) groups as fewer than half of patients flared. Unadjusted flare rates per patient-year were 1.0 during treatment discontinuation and 0.3 during treatment restart (0.6 overall) in the TH group and 0.6 in the TC group; there were no severe or renal flares. No TH patients rebounded; 2 (6.9%) TC patients rebounded; 2 (5.1%) patients in the LTD group rebounded. There were no new safety signals. CONCLUSIONS: Twenty-four-week belimumab discontinuation did not appear to increase the risk of flares or rebound in patients with low SLE disease activity; flare rates were low in both groups. Further studies may help to fully determine the effect of belimumab discontinuation. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02119156 . Registered on April 21, 2014.

Exploring phenylcarbamoylazinane-1,2,4-triazole thioethers as lipoxygenase inhibitors supported with in vitro, in silico and cytotoxic studies.

Searching small molecules as an auspicious approach to develop new anti-inflammatory drugs is a challenge for the researchers especially by modifying active pharmacophoric groups in the targeted molecules. In the current work, a series of new S-alkyl/aralky derivatives (8a-h; 9a-h) of 2-(4-ethyl/phenyl-5-(1-phenylcarbamoylpiperidine)-4H-1,2,4-triazol-3-ylthio)ether were synthesized and assessed for their inhibitory action against the 15-lipoxygenase from soybean (15-sLOX). The basic precursor ethyl piperidine-4-carboxylate (a) was consecutively changed into phenylcarbamoyl derivative (1), hydrazide (2), semicarbazides (3/4) and N-ethyl/phenyl-5-(1-phenylcarbamoylpiperidine)-1,2,4-triazoles (5/6), which further in association with electrophiles (7a-h) promoted to the final products (8a-h; 9a-h). The synthesized derivatives were characterized by FT-IR, 1H-, 13C NMR spectroscopy, EI-MS, and HR-EI-MS spectrometry. Amongst these, 8a, 8c, and 9c, expressed potent inhibitory profiles against the 15-sLOX enzyme with IC50 values of 12.52 ± 0.35 to 35.64 ± 0.29 µM, followed by the compounds 9b, 9g, 9d, 9a, 8b, 8e, 8d, 8g, 8h, 8f and 9h with IC50 values in the range of 43.78 ± 0.43 to 108.65 ± 0.38 µM. All compounds exhibited variable cellular viability levels by MTT assay. Flow cytometric data demonstrated that 8f, 8g, 8h have maximal lymphocyte cellular viability and all compounds affected cells in the late apoptosis phase. In silico ADMET studies supported the drug-likeness of most of the molecules. These studies were supported by molecular docking against 15-sLOX, human 5-LOX (5-hLOX) and human 15-LOX (5-hLOX); that inhibitors of 15-sLOX docked-in the active pocket of either 5-hLOX or 15-hLOX and docking score remained constant for all three enzymes within a narrow range (-6.8 to -9.7) as did it for standard quercetin (-8.4 to -9.0). The most dominant bonding interactions were π-π, π-anion, and π-alkyl type along with the hydrogen bonding. The data collected altogether demonstrates the better possibility of some of these compounds as good LOX inhibitors in search for 'lead' as anti-inflammatory agents in the process of drug discovery and development.

Identification of phenylcarbamoylazinane-1,3,4-oxadiazole amides as lipoxygenase inhibitors with expression analysis and in silico studies.

In search for new anti-inflammatory agents that inhibit the enzymes of arachidonic acid pathway as the drug targets, the present article describes the screening of 1,3,4-oxadiazole analogues against lipoxygenase (LOX) enzyme. The work is based on the synthesis of new N-alkyl/aralky/aryl derivatives (6a-o) of 2-(4-phenyl-5-(1-phenylcarbamoylpiperidine)-4H-1,3,4-oxadiazol-3-ylthio)acetamide which were obtained by the reaction of 1,3,4-oxadiazole (3) with various electrophiles (5a-o), in KOH. The synthesized analogues showed potent to moderate inhibitory activity against the soybean 15-LOX enzyme; especially 6g, 6b, 6a and 6l displayed the potent inhibitory potential with IC50 values 7.15 ± 0.26, 9.32 ± 0.42, 15.83 ± 0.45 & 18.37 ± 0.53 µM, respectively, while excellent to moderate inhibitory profiles with IC50 values in the range of 26.13-98.21 µM were observed from the compounds 6k, 6m, 6j, 6o, 6h, 6f, 6n and 6c. Most of the active compounds exhibited considerable cell viability against blood mononuclear cells (MNCs) at 0.25 mM by MTT assay except 6f, 6h, 6k and 6m which showed around 50% cell viability. Flow cytometry studies of the selected compounds 6a, 6j and 6n revealed that these caused 79.5-88.51% early apoptotic changes in MNCs compared with 4.26% for control quercetin at their respective IC50 values. The relative expression of 5-LOX gene was monitored in MNCs after treatment with these three molecules and all down-regulated the enzyme activity. In silico ADME and molecular docking studies further supported these studies of oxadiazole derivatives and considered it as potential 'lead' compounds in drug discovery and development.

Probing phenylcarbamoylazinane-1,2,4-triazole amides derivatives as lipoxygenase inhibitors along with cytotoxic, ADME and molecular docking studies.

Hunting small molecules as anti-inflammatory agents/drugs is an expanding and successful approach to treat several inflammatory diseases such as cancer, asthma, arthritis, and psoriasis. Besides other methods, inflammatory diseases can be treated by lipoxygenase inhibitors, which have a profound influence on the development and progression of inflammation. In the present study, a series of new N-alkyl/aralky/aryl derivatives (7a-o) of 2-(4-phenyl-5-(1-phenylcarbamoyl)piperidine-4H-1,2,4-triazol-3-ylthio)acetamide was synthesized and screened for their inhibitory potential against the enzyme 15-lipoxygenase. The simple precursor ethyl piperidine-4-carboxylate (a) was successively converted into phenylcarbamoyl derivative (1), hydrazide (2), semicarbazide (3) and N-phenylated 5-(1-phenylcarbamoyl)piperidine-1,2,4-triazole (4), then in combination with electrophiles (6a-o) through further multistep synthesis, final products (7a-o) were generated. All the synthesized compounds were characterized by FTIR, 1H, 13C NMR spectroscopy, EIMS, and HREIMS spectrometry. Almost all the synthesized compounds showed excellent inhibitory potential against the tested enzyme. Compounds 7c, 7f, 7d, and 7g displayed potent inhibitory potential (IC50 9.25 ± 0.26 to 21.82 ± 0.35 µM), followed by the compounds 7n, 7h, 7e, 7a, 7b, 7l, and 7o with IC50 values in the range of 24.56 ± 0.45 to 46.91 ± 0.57 µM. Compounds 7c, 7f, 7d exhibited 71.5 to 83.5% cellular viability by MTT assay compared with standard curcumin (76.9%) when assayed at 0.125 mM concentration. In silico ADME studies supported the drug-likeness of most of the molecules. In vitro inhibition studies were substantiated by molecular docking wherein the phenyl group attached to the triazole ring was making a π-δ interaction with Leu607. This work reveals the possibility of a synthetic approach of compounds in relation to lipoxygenase inhibition as potential lead compounds in drug discovery.

NADPH oxidase 4 mediates upregulation of type 4 phosphodiesterases in human endothelial cells.

The protective actions of prostacyclin (PGI(2) ) are mediated by cyclic AMP (cAMP) which is reduced by type 4 phosphodiesterases (PDE4) which hydrolyze cAMP. Superoxide (O2(-)) from NADPH oxidase (Nox) is associated with impaired PGI(2) bioactivity. The objective of this study, therefore, was to study the relationship between Nox and PDE4 expression in human umbilical vein endothelial cells (HUVECs). HUVECs were incubated with the thromboxane A(2) analog, U46619, 8-isoprostane F(2α) (8IP), or tumor necrosing factor alpha (TNFα) [±iloprost (a PGI(2) analog)] and the expression of PDE4A, B, C, and D and splice variants thereof assessed using Western blotting and qPCR and mRNA silencing of Nox4 and Nox5. Effects on cell replication and angiogenesis were also studied. U46619, 8IP, and TNFα increased the expression of Nox 4 and Nox 5 and all PDE4 isoforms as well as cell replication and tubule formation (index of angiogenesis), effects inhibited by mRNA silencing of Nox4 (but not Nox5) and iloprost and rolipram. These data demonstrate that upregulation of Nox4 leads to an upregulation of PDE4A, B, and D and increased hydrolysis of cAMP which in turn augments cell replication and angiogenesis. This mechanism may be central to vasculopathies associated with endothelial dysfunction since the PGI(2)-cAMP signaling axis plays a key role in mediating functions that include hemostasis and angiogenesis.

NADPH oxidase 1 mediates upregulation of thromboxane A2 synthase in human vascular smooth muscle cells: inhibition with iloprost.

Thromboxane A(2) (TXA(2)) upregulates and activates NADPH oxidase (Nox) both of which are associated with cardiovascular disease. The aim of this study, therefore, was to investigate the relationship between thromboxane A(2) synthase (TXAS) status and Nox in human vascular smooth muscle cells (hVSMCs), in particular, whether superoxide (O(2)(▪-)) derived from Nox influences TXAS expression and activity. hVSMCs were incubated with TNFα: (10 ng/ml), TXA(2) mimetic U46619 (100 nM), 8-isoprostane F(2α) (8-IP; 100 nM) and hypoxia. Expression of TXAS was assessed using western blotting and quantitative PCR. The role of Nox1 and Nox4 was studied using apocynin and mRNA silencing. The effect of the thromboxane receptor antagonist picotamide and of iloprost, a prostacyclin (PGI(2)) analogue was also studied. TNF-α, U46619 and 8-IP and hypoxia all augmented TXAS expression as well as TXA(2) formation, effects inhibited by apocynin. Nox-1 (but not Nox4) gene silencing inhibited the increase in TXAS expression and activity. Both picotamide and iloprost inhibited the upregulation of TXAS as well as TXA(2) formation induced by TNF-α, U46619 and 8-isoprostane F(2α) and hypoxia. It is concluded that upregulation of TXA(2) synthase expression and activity in human VSMCs is mediated by an a priori upregulation of Nox1 and represents a self amplifying cascade. The inhibition of this effect with iloprost consolidates that PGI(2) plays a protective anti-oxidative role in the vasculature and that picotamide and like drugs may be effective in reducing the incidence of cardiovascular disease associated with an oxidative aetiology.

Exogenous hydrogen sulfide inhibits superoxide formation, NOX-1 expression and Rac1 activity in human vascular smooth muscle cells.

The activity of NADPH oxidase (NOX) is blocked by nitric oxide (NO). Hydrogen sulfide (H(2)S) is also produced by blood vessels. It is reasonable to suggest that H(2)S may have similar actions to NO on NOX. In order to test this hypothesis, the effect of sodium hydrosulfide (NaHS) on O(2)(-) formation, the expression of NOX-1 (a catalytic subunit of NOX) and Rac(1) activity (essential for full NOX activity) in isolated vascular smooth muscle cells (hVSMCs) was investigated. hVSMCs were incubated with the thromboxane A(2) analogue U46619 +/- NaHS for 1 or 16 h, and O(2)(-) formation, NOX-1 expression and Rac(1) activity were assessed. The possible interaction between H(2)S and NO was also studied by using an NO synthase inhibitor, L-NAME, and an NO donor, DETA-NONOate. The role of K(ATP) channels was studied by using glibenclamide. NaHS inhibited O(2)(-) formation following incubation of 1 h (IC(50), 30 nM) and 16 h (IC(50), 20 nM), blocked NOX-1 expression and inhibited Rac(1) activity. These inhibitory effects of NaHS were mediated by the cAMP-protein-kinase-A axis. Exogenous H(2)S prevents NOX-driven intravascular oxidative stress through an a priori inhibition of Rac(1) and downregulation of NOX-1 protein expression, an effect mediated by activation of the adenylylcyclase-cAMP-protein-kinase-G system by H(2)S.

Superoxide auto-augments superoxide formation and upregulates gp91(phox) expression in porcine pulmonary artery endothelial cells: inhibition by iloprost.

Central to the aetiology of Acute Respiratory Distress Syndrome (ARDS) is superoxide, the principal source of which is nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase). To test whether superoxide may influence NADPH oxidase expression directly, the effect of incubation of superoxide with porcine pulmonary arterial endothelial cells on the expression of gp91(phox) (a catalytic subunit of NADPH oxidase) and superoxide formation was investigated. Since iloprost has been purported to be potentially effective in treating ARDS, the effect of iloprost on superoxide-mediated effects was also studied. Pulmonary artery endothelial cells were incubated with xanthine/xanthine oxidase which generates superoxide, or tumour necrosis factor alpha (TNFalpha) or thromboxane A(2) analogue, U46619 (+/- superoxide dismutase [SOD] or catalase or iloprost) for 16 h. Cells were then washed and superoxide formation assessed spectrophometrically and gp91(phox) expression using Western blotting. The role of NADPH oxidase was also studied in the above settings using apocynin, an NADPH oxidase inhibitor. Superoxide, TNFalpha and U46619 elicited an increase in the formation of superoxide and induced gp91(phox) expression in pulmonary artery endothelial cells following a 16 h incubation an effect blocked by the continual presence of SOD and apocynin but not catalase. Apocynin completely inhibited superoxide formation induced with xanthine/xanthine oxidase after the 16 h incubation. Rotenone and allopurinol were without effect. Iloprost inhibited the formation of superoxide and gp91(phox) expression. These data demonstrate that superoxide upregulates gp91(phox) expression in pulmonary artery endothelial cells and thus augments superoxide formation, an effect blocked by iloprost. This constitutes a novel mechanism by which vascular superoxide creates a self-perpetuating cascade that may be of importance to the etiology of ARDS and other vasculopathies.

Nicotinamide adenine dinucleotide phosphate oxidase: a promiscuous therapeutic target for cardiovascular drugs?

The increased expression and activity of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex has emerged as a major common factor in the etiology of all forms of cardiovascular diseases since the upregulation of intravascular NADPH oxidase results in the formation of superoxide (O(2)(-)), which in turn promotes vasculopathy. An ever-increasing number of drugs commonly used in cardiovascular medicine have been shown to influence NADPH oxidase expression and activity. These include nitric oxide donors, nitroaspirin, eicosanoids, phosphodiesterase inhibitors, corticosteroids, antioxidants, and specific inhibitors. The objective of this review is to discuss these drugs in relation to the mechanisms underlying their effects on NADPH oxidase activity and the expression and therapeutic implications of these effects.

Sildenafil inhibits the formation of superoxide and the expression of gp47 NAD[P]H oxidase induced by the thromboxane A2 mimetic, U46619, in corpus cavernosal smooth muscle cells.

OBJECTIVE: To assess the effect of sildenafil on superoxide formation and p47(phox) (the active subunit of NADPH oxidase) expression in cultured corpus cavernosal smooth muscle cells (CVSMCs). MATERIALS AND METHODS: CVSMCs derived from rabbit penis were incubated with U46619 (thromboxane A2 analogue) with or without sildenafil for 1 or 16 h at 37 degrees C. Superoxide dismutase-inhibitable superoxide formation was assessed using the reduction of ferricytochrome c measured spectrophotometrically, and gp47(phox) assessed using Western blot analysis. The role of NAD[P]H oxidase and cGMP was further studied by using specific inhibitors of each. RESULTS: Superoxide formation was significantly greater in cells incubated with U46619 after 1 and 16 h incubation than in controls, an effect blocked by NADP(H) oxidase inhibitors. These effects of U46619 were inhibited by sildenafil (1 and 10 nmol/L), which in turn were negated by the guanylyl cyclase inhibitor, ODQ; 10 nmol/L sildenafil inhibited p47phox expression induced by U46619. CONCLUSIONS: Sildenafil is a potent inhibitor of superoxide formation in CVSMCs. This effect is mediated through the inhibition of PDE-5 which in turn augments the inhibitory action of the NO-cGMP axis on NAD[P]H oxidase expression and activity. This mechanism constitutes a new pharmacological action of sildenafil, consolidates the potential role of superoxide in ED, and indicates that thromboxane A(2) may be an important mediator of intrapenile oxidative stress.

Sildenafil citrate and sildenafil nitrate (NCX 911) are potent inhibitors of superoxide formation and gp91phox expression in porcine pulmonary artery endothelial cells.

Acute respiratory distress syndrome (ARDS) is associated with increased superoxide (O(2)(*-)) formation in the pulmonary vasculature and negation of the bioavailability of nitric oxide (NO). Since NO inhibits NADPH oxidase expression through a cyclic GMP-mediated mechanism, sildenafil, a type V phosphodiesterase inhibitor, may be therapeutically effective in ARDS through an augmentation of NO-mediated inhibition of NADPH oxidase. Therefore, the effect of sildenafil citrate and NO-donating sildenafil (NCX 911) on O(2)(*-) formation and gp91(phox) (active catalytic subunit of NADPH oxidase) expression was investigated in cultured porcine pulmonary artery endothelial cells (PAECs). PAECs were incubated with 10 nM TXA(2) analogue, 9,11-dideoxy-9alpha,11alpha-methanoepoxy-prostaglandin F(2alpha) (U46619) (+/-sildenafil or NCX 911), for 16 h and O(2)(*-) formation measured spectrophometrically and gp91(phox) using Western blotting. The role of the NO-cGMP axis was studied using morpholinosydnonimine hydrochloride (SIN-1), the diethylamine/NO complex (DETA-NONOate), the guanylyl cyclase inhibitor, 1H-{1,2,4}oxadiazolo{4,3-a}quinoxalin-1-one (ODQ), and the protein kinase G inhibitor, 8-bromoguanosine-3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-8-Br-cGMPS). NO release was studied using a fluorescence assay and O(2)(*-)-NO interactions by measuring nitrites. After a 16-h incubation with 10 nM U46619, both NCX 911 and sildenafil elicited a concentration-dependent inhibition of O(2)(*-) formation and gp91(phox) expression, NCX 911 being more potent (IC(50); 0.26 nM) than sildenafil citrate (IC(50); 1.85 nM). These inhibitory effects were reversed by 1 microM ODQ and 10 microM Rp-8-Br-cGMPS. NCX 911 stimulated the formation of cGMP in PAECs and generated NO in a cell-free system to a greater degree than sildenafil citrate. The inhibitory effect of sildenafil was augmented by 1 muM SIN-1 and blocked partially by the eNOS inhibitor 10 microM N(5)-(1-iminoethyl)-ornithine (L-NIO). Acutely, sildenafil and NCX 911 also inhibited O(2)(*-) formation, again blocked by 1 microM ODQ. NCX 911 reacted with O(2)(*-) generated by xanthine oxidase, an effect that was inhibited by superoxide dismutase (500 U ml(-1)). Since O(2)(*-) formation plays contributory role in ARDS, both sildenafil citrate and NCX 911 may be indicated for treating ARDS through suppression of NADPH oxidase expression and therefore of O(2)(*-) formation and preservation of NO bioavailability.

Prednisolone augments superoxide formation in porcine pulmonary artery endothelial cells through differential effects on the expression of nitric oxide synthase and NADPH oxidase.

1. Prednisolone, a potent anti-inflammatory drug, has proved ineffective in treating acute respiratory distress syndrome (ARDS). ARDS is associated with superoxide (O(2)(*-)) generation, which negates nitric oxide (NO). NO also downregulates NADPH oxidase and inhibits O(2)(*-) formation. A possible reason for the lack of effect of prednisolone may due to an inhibition of eNOS expression. In order to test this proposal, the effect of prednisolone on O(2)(*-) formation and the expression of gp91(phox) (catalytic subunit of NADPH oxidase) and eNOS in pig pulmonary artery (PA) segments and PA endothelial cells (PAECs) and PA vascular smooth muscle cells (PAVSMCs) was investigated. 2. PA segments and cells were incubated with prednisolone and tumour necrosis factor-alpha (TNF-alpha) for 16 h. O(2)(*-) formation was measured spectrophometrically and gp91(phox) and eNOS expression by Western blotting. The role of the NO-cGMP axis was studied using morpholinosydnonimine hydrochloride, the diethylamine/NO complex (DETA-NONOate), the guanylyl cyclase inhibitor, 1H-{1,2,4}oxadiazolo{4,3-a}quinoxalin-1-one (ODQ) and the stable cGMP analogues, 8-bromo cGMP and 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP). NO release was studied using a fluorescence assay and O(2)(*-)-NO interactions with a nitrite/nitrate assay. 3. Prednisolone elicited significant increase in O(2)(*-) formation in intact PA segments and PAECs, but not PAVSMCs, in a concentration-dependent manner. In endothelium-denuded segments, prednisolone slightly enhanced O(2)(*-) release. TNF-alpha further increased prednisolone-enhanced O(2)(*-) formation in intact PA segments and PAECs. NADPH oxidase inhibitor, apocynin, inhibited O(2)(*-) formation. Increased O(2)(*-) release and gp91(phox) expression in PAECs elicited by prednisolone was blocked by SIN-1 (3-morpholinosydnonimine hydrochloride), DETA-NONOate, 8-pCPT-cGMP and 8-bromo cGMP. The effects of SIN-1 on gp91(phox) expression were reversed by ODQ. Finally, eNOS protein expression was significantly reduced by prednisolone. 4. Prednisolone increases O(2)(*-) in porcine PAECs through a downregulation of endogenous eNOS expression. Since the NO-cGMP axis inhibits gp91(phox) expression, the resultant decrease in endogenous NO formation then augments NADPH oxidase activity, which in turn results in increased O(2)(*-) formation. Since O(2)(*-) promotes inflammation, this mechanism may explain why prednisolone is ineffective in treating ARDS. Therapeutically, the coadministration of an NO donor may render prednisolone more effective in treating ARDS.