Dipyrone and aminopyrine are effective scavengers of reactive nitrogen species

Reactive nitrogen species (RNS), namely nitric oxide (NO*) and peroxynitrite (ONOO-) are produced in the inflammatory sites and may contribute to the deleterious effects of inflammation. The aim of the present study was to evaluate the putative scavenging effect of a particular group of non-steroidal anti-inflammatory drugs (NSAIDs), the pyrazolone derivatives dipyrone, aminopyrine, isopropylantipyrine, and antipyrine against RNS, using in vitro non-cellular screening systems. The results obtained showed that dipyrone and aminopyrine were highly potent scavengers of NO* and ONOO- while antipyrine exerted little effect and isopropylantipyrine no effect whatsoever against these two RNS and that, in the presence of bicarbonate, the scavenging potencies of both dipyrone and aminopyrine were slightly decreased. It could thus be inferred that the observed scavenging effects may be of therapeutic benefit for patients under anti-inflammatory treatment with dipyrone and aminopyrine in the case of overproduction of RNS. On the other hand, the possible depletion of physiological NO* concentrations, namely at the gastrointestinal tract as well as the formation of reactive derivatives of aminopyrine and/or dipyrone, resulting from their reaction with RNS, may otherwise be harmful for these patients.     

Dipyrone and aminopyrine are effective scavengers of reactive nitrogen species

Abstract

Reactive nitrogen species (RNS), namely nitric oxide (NO^?) and peroxynitrite (ONOO^?) are produced in the inflammatory sites and may contribute to the deleterious effects of inflammation. The aim of the present study was to evaluate the putative scavenging effect of a particular group of non-steroidal anti-inflammatory drugs (NSAIDs), the pyrazolone derivatives dipyrone, aminopyrine, isopropylantipyrine, and antipyrine against RNS, using in vitro non-cellular screening systems. The results obtained showed that dipyrone and aminopyrine were highly potent scavengers of NO^? and ONOO^? while antipyrine exerted little effect and isopropylantipyrine no effect whatsoever against these two RNS and that, in the presence of bicarbonate, the scavenging potencies of both dipyrone and aminopyrine were slightly decreased. It could thus be inferred that the observed scavenging effects may be of therapeutic benefit for patients under anti-inflammatory treatment with dipyrone and aminopyrine in the case of overproduction of RNS. On the other hand, the possible depletion of physiological NO^? concentrations, namely at the gastrointestinal tract as well as the formation of reactive derivatives of aminopyrine and/or dipyrone, resulting from their reaction with RNS, may otherwise be harmful for these patients.     

Hydrogen peroxide scavenging activity by non-steroidal anti-inflammatory drugs

Hydrogen peroxide (H2O2) has been shown to be formed during inflammatory processes and is implicated in its pathophysiology. Thus, a putative scavenging activity against this reactive oxygen species (ROS) by anti-inflammatory drugs may be of great therapeutical value. The present study was undertaken to evaluate the scavenging activity for H2O2 by several non-steroidal anti-inflammatory drugs (NSAIDs), namely indomethacin, acemetacin, etodolac, tolmetin, ketorolac, oxaprozin, sulindac and its metabolites sulindac sulfide and sulindac sulfone. The H2O2 scavenging assay was performed by measuring H2O2-elicited lucigenin chemiluminescence using a microplate reader. The specificity of the method was confirmed by the use of catalase, which completely prevented the H2O2-induced lucigenin chemiluminescence. The endogenous antioxidants melatonin and reduced glutathione (GSH) were used as positive controls. The obtained results demonstrated that all the studied NSAIDs display H2O2 scavenging activity, although in different extents. The ranking order of potency found was sulindac sulfone > sulindac sulfide > GSH > sulindac > indomethacin > acemetacin > etodolac > oxaprozin > ketorolac approximately melatonin > tolmetin.     

Inhibition of human neutrophil oxidative burst by pyrazolone derivatives

The risk of agranulocytosis associated with the use of pyrazolone drugs at therapeutical doses and for short periods of time has been considered to be very low. However, little or no attention at all has been devoted to the possible hindrance of neutrophil burst and scavenging of neutrophil-generated reactive oxygen species (ROS) by these compounds. Such an effect could be beneficial in the case of overactivation of neutrophils but could also be highly detrimental if the number of circulating neutrophils is already decreased. Thus, the aim of the present study was to evaluate the putative inhibitory effect of the pyrazolones dipyrone, aminopyrine, isopropylantipyrine, and antipyrine against human neutrophil burst and their scavenging activity against O2.-, H2O2, HO., ROO., and HOCl. The obtained results showed that dipyrone and aminopyrine prevent phorbol-12-myristate-13-acetate-induced neutrophil burst with high efficiency, while isopropylantipyrine had little effect and antipyrine had no effect at all. Dipyrone and aminopyrine were highly potent scavengers of HO. and HOCl, while, in accordance with the neutrophil burst results, isopropylantipyrine had little effect and antipyrine had no effect at all against these two ROS. None of the studied pyrazolones was capable of scavenging O2.- or H2O2, while dipyrone was shown to be the most reactive against ROO..     

In vitro scavenging activity for reactive oxygen and nitrogen species by nonsteroidal anti-inflammatory indole, pyrrole, and oxazole derivative drugs

This study was undertaken to evaluate the scavenging activity for reactive oxygen species (ROS) and reactive nitrogen species (RNS) by several nonsteroidal anti-inflammatory drugs (NSAIDs), namely indole derivatives (indomethacin, acemetacin, etodolac), pyrrole derivatives (tolmetin and ketorolac), and an oxazole derivative (oxaprozin). The inhibition of prostaglandin synthesis constitutes the primary mechanism of the anti-inflammatory action of these drugs. Nevertheless, it has been suggested that the anti-inflammatory activity of NSAIDs may be also partly due to their ability to scavenge ROS and RNS and to inhibit the respiratory burst of neutrophils triggered by various activator agents. Thus, the scavenging activity of these NSAIDs was evaluated against an array of ROS (O(2)(-), HO, HOCl, and ROO) and RNS (NO and ONOO(-)) using noncellular in vitro systems. The results obtained demonstrated that tolmetin, ketorolac, and oxaprozin were not active against O(2)(-), while acemetacin, indomethacin, and etodolac exhibited concentration-dependent effects. Oxaprozin was also the least active scavenger for HO, among all the tested NSAIDs shown to be active. The scavenging effect for HOCl was not observed for any of the tested NSAIDs. The ROO was effectively scavenged by etodolac, with the other tested NSAIDs being much less active. NO and ONOO(-) were scavenged by all the tested NSAIDs. These effects may strongly contribute to the anti-inflammatory therapy benefits that may be attained with some of the studied NSAIDs.     

The metabolism of sulindac enhances its scavenging activity against reactive oxygen and nitrogen species

Sulindac is a sulfoxide prodrug that, in vivo, is converted to the metabolites sulindac sulfide and sulindac sulfone. It is therapeutically used as an anti-inflammatory and analgesic in the symptomatic treatment of acute and chronic rheumatoid arthritis, osteoarthritis, and ankylosing spondylitis. In addition to its anti-inflammatory properties, sulindac and its metabolites have been shown to have an important role in the prevention of colonic carcinogenesis. Although the inhibition of prostaglandin synthesis constitutes the primary mechanism of action of sulindac, it is well known that reactive oxygen species (ROS) and reactive nitrogen species (RNS) are implicated in the pathophysiology of inflammation and cancer. Thus, the aim of this study was to evaluate the scavenging activity of sulindac and its sulfone and sulfide metabolites for an array of ROS (HO*, O2(*-), and HOCl) and RNS (*NO and ONOO-) using in vitro systems. The results we obtained demonstrate that the metabolism of sulindac increases its scavenging activity for all RNS and ROS studied, notably with regard to the scavenging of HOCl. These effects may strongly contribute to the anti-inflammatory and anticarcinogenic efficacy that has been shown for sulindac.     

Antiproliferative effects of nitrosulindac on human colon adenocarcinoma cell lines

Nonsteroidal anti-inflammatory drugs (NSAIDs) reduce the incidence of colon cancer, but their use is limited by toxicity in the gastrointestinal tract. The coupling of a nitric oxide-releasing moiety to NSAIDs strongly reduces these side effects. We demonstrated that the NO-releasing sulindac (nitrosulindac) has much more potent effects on colon adenocarcinoma cell lines compared to sulindac. Moreover, it could inhibit the growth of cells in soft agar experiments, demonstrating the antineoplastic activity at low concentration of nitrosulindac. However, this reduction in the growth of colon cancer cells seemed to be independent of the classical apoptosis pathway and could be explained by a cytostatic effect. Nitrosulindac caused a light perturbation of the cell cycle parameters not linked to a modification of the levels of p21 or the proliferating cell nuclear antigen. Moreover, neither sulindac, nor nitrosulindac, were able to inhibit the NF-kappa B pathway. These data suggested that nitrosulindac could be a better solution compared to other NSAIDs in the treatment of colon cancer.     

Growth compensatory role of sulindac sulfide-induced thrombospondin-1 linked with ERK1/2 and RhoA GTPase signaling pathways

Previously, we reported that non-steroidal anti-inflammatory drugs (NSAIDs) suppress cellular invasion which was mediated by thrombospondin-1 (TSP-1). As the extending study of the previous observation, we investigated the effect of NSAID-induced TSP-1 on the cellular growth and its related signaling transduction of the TSP-1 production. Among diverse NSAIDs, sulindac sulfide was most potent of inducing the human TSP-1 protein expression. Functionally, induced TSP-1 expression was associated with the growth-compensatory action of NSAID. TSP-1 expression was also elevated by mitogenic signals of ERK1/2 and RhoA GTPase pathway which had also growth-promotive capability after sulindac sulfide treatment. These findings suggest the possible mechanism through which tumor cells can survive the chemopreventive action of NSAIDs or the normal epithelium can reconstitute after NSAID-mediated ulceration in a compensatory way.     

Synthetic inhibitors of human granulocyte elastase, Part 4. Inhibition of human granulocyte elastase and cathepsin G by non-steroidal anti-inflammatory drugs (NSAID)s

A series of non-steroidal anti-inflammatory drugs (NSAIDs) and some of their metabolites were evaluated for their ability to inhibit the proteolytic activity of human granulocyte elastase (HGE) and cathepsin G (HGC-G). The enzyme activity was monitored using specific synthetic chromogenic substrates. The results obtained indicated that phenylbutazone, sulindac, piroxicam and gold sodium thiomalate significantly inhibited HGE (Ki less than 0.5 mM), while only sulindac, diflunisal and gold sodium thiomalate were effective inhibitors of HGC-G (Ki less than 0.4 mM). Studies on metabolites of some of the NSAID tested were found to be superior inhibitors of both HGE and HGC-G than the parent molecules. Moreover, of the 18 compounds examined, the major metabolite of sulindac, sulindac sulphide was the most potent inhibitor of HGE (Ki = 0.01 mM) and HGC-G (Ki = 0.15 mM).     

Idiosyncratic NSAID drug induced oxidative stress

Many idiosyncratic non-steroidal anti-inflammatory drugs (NSAIDs) cause GI, liver and bone marrow toxicity in some patients which results in GI bleeding/ulceration/fulminant hepatic failure/hepatitis or agranulocytosis/aplastic anemia. The toxic mechanisms proposed have been reviewed. Evidence is presented showing that idiosyncratic NSAID drugs form prooxidant radicals when metabolised by peroxidases known to be present in these tissues. Thus GSH, NADH and/or ascorbate were cooxidised by catalytic amounts of NSAIDs and hydrogen peroxide in the presence of peroxidase. During GSH and NADH cooxidation, oxygen uptake and activation occurred. Furthermore the formation of NSAID oxidation products was prevented during the cooxidation indicating that the cooxidation involved redox cycling of the first formed NSAID radical product. The order of prooxidant catalytic effectiveness of fenamate and arylacetic acid NSAIDs was mefenamic acid>tolfenamic acid>flufenamic acid, meclofenamic acid or diclofenac. Diphenylamine, a common moiety to all of these NSAIDs was a more active prooxidant for NADH and ascorbate cooxidation than these NSAIDs which suggests that oxidation of the NSAID diphenylamine moiety to a cation and/or nitroxide radical was responsible for the NSAID prooxidant activity. The order of catalytic effectiveness found for sulfonamide derivatives was sulfaphenazole>sulfisoxazolez.Gt;dapsone>sulfanilic acid>procainamide>sulfamethoxazole>sulfadiazine>sulfadimethoxine whereas sulfanilamide, sulfapyridine or nimesulide had no prooxidant activity. Although indomethacin had little prooxidant activity, its major in vivo metabolite, N-deschlorobenzoyl indomethacin had significant prooxidant activity. Aminoantipyrine the major in vivo metabolite of aminopyrine or dipyrone was also more prooxidant than the parent drugs. It is hypothesized that the NSAID radicals and/or the resulting oxidative stress initiates the cytotoxic processes leading to idiosyncratic toxicity.     

Synthesis and biological evaluation of 2,5-disubstituted 1,3,4-oxadiazole derivatives with both COX and LOX inhibitory activity

Dual cyclooxygenase/lipoxygenase (COX/LOX) inhibitors constitute a valuable alternative to classical nonsteroidal anti-inflammatory drugs (NSAIDs) and selective COX-2 inhibitors for the treatment of inflammatory diseases. A series of 3-(5-phenyl/phenylamino-[1,3,4]oxadiazol-2-yl)-chromen-2-one and N-[5-(2-oxo-2H-chromen-3-yl)-[1,3,4]oxadiazol-2-yl]-benzamide derivatives were synthesized and screened for anti-inflammatory, analgesic activity. All the derivatives prepared are active in inhibiting oedema induced by carrageenan. Compound 4e was found more potent with 89% of inhibition followed by compound 4b (86%). Compounds with >70% of anti-inflammatory activity were tested for analgesic, ulcerogenic, and lipid peroxidation profile. Selected compounds were also evaluated for inhibition of COXs (COX-1 and COX-2) and LOXs (LOX-5, LOX-12, and LOX-15). Compound 4e was comparatively selective for COX-2, LOX-5, and LOX-15. Study revealed that these derivatives were more effective than ibuprofen with reduced side effects. It can be suggested that these derivatives could be used to develop more potent and safer NSAIDs.     

Opioidergic effects of nonopioid analgesics on the central nervous system

1. The analgesic effect of nonsteroidal anti-inflammatory drugs (NSAIDs) is partly due to the fact that they act upon the periaqueductal gray matter (PAG) and the rostral ventromedial medulla of the brain stem and thus activate the descending pain-control system, which inhibits nociceptive transmission at the spinal dorsal horn.2. The analgesic action of dipyrone (metamizol) and of lysine-acetylsalicylate (LASA), two well-known NSAIDs, whether microinjected into the PAG or given systemically, can be reverted by naloxone. Repeated administration of dipyrone or LASA induces tolerance to their antinociceptive effect, with cross-tolerance to morphine, and a withdrawal syndrome upon naloxone administration. Dipyrone tolerance can be reverted by proglumide, a cholecystokinin antagonist.3. These findings reveal a close association between the central action of NSAIDs and endogenous opioids.     

NSAIDs do not require the presence of a carboxylic acid to exert their anti-inflammatory effect – why do we keep using it?

The carboxylic acid group (–COOH) present in classical NSAIDs is partly responsible for the gastric toxicity associated with the administration of these drugs. This concept has been extensively proven using NSAID prodrugs. However, the screening of NSAIDs with no carboxylic acid at all has been neglected. The goal of this work was to determine if new NSAID derivatives devoid of acidic moieties would retain the anti-inflammatory activity of the parent compound, without causing gastric toxicity. To test this concept, we replaced the carboxylic acid group in ibuprofen, flurbiprofen, and naproxen with three ammonium moieties. We tested the resulting water-soluble NSAID derivatives for anti-inflammatory and ulcerogenic activity in vitro and in vivo. In this regard, we observed that all non-acidic NSAIDs exerted a potent anti-inflammatory activity, suggesting that the acid group in commercial 2-phenylpropionic acid NSAIDs not be an essential requirement for anti-inflammatory activity. These data provide complementary evidence supporting the discontinuation of ulcerogenic acidic NSAIDs.     

Nonsteroidal anti-inflammatory drugs and their analogues as inhibitors of aldo-keto reductase AKR1C3: new lead compounds for the development of anticancer agents

Nonsteroidal anti-inflammatory drugs (NSAIDs) like indomethacin, flufenamic acid, and related compounds have been recently identified as potent inhibitors of AKR1C3. We report that some other NSAIDs (diclofenac and naproxen) also inhibit AKR1C3, with the IC(50) values in the low micromolar range. In order to obtain more information about the structure-activity relationship and to identify new leads, a series of compounds designed on the basis of NSAIDs were synthesized and screened on AKR1C3. The most active compounds were 2-[(2,2-diphenylacetyl)amino]benzoic acid 4 (IC(50)=11microM) and 3-phenoxybenzoic acid 10 (IC(50)=0.68microM). These compounds represent promising starting points for the development of new anticancer agents.     

Antimalarial histone deacetylase inhibitors containing cinnamate or NSAID components

Malaria is the most lethal parasite-mediated tropical infectious disease, killing 1-2 million people each year. An emerging drug target is the enzyme Plasmodium falciparum histone deacetylase 1 (PfHDAC1). We report 26 compounds designed to bind the zinc and exterior surface around the entrance to the active site of PfHDAC1, 16 displaying potent in vitro antimalarial activity (IC(50)<100 nM) against P. falciparum. Selected compounds were shown to cause hyperacetylation of P. falciparum histones and be >10-fold more cytotoxic towards P. falciparum than a normal human cell type (NFF). Twenty-two inhibitors feature cinnamic acid derivatives or non-steroidal anti-inflammatory drugs (NSAIDs) as HDAC-binding components. A homology model of PfHDAC1 enzyme gives new insights to interactions likely made by some of these inhibitors. Results support PfHDAC1 as a promising new antimalarial drug target.     

Synthesis and biological evaluation of 2,5-disubstituted 1,3,4-oxadiazole derivatives with both COX and LOX inhibitory activity

Dual cyclooxygenase/lipoxygenase (COX/LOX) inhibitors constitute a valuable alternative to classical nonsteroidal anti-inflammatory drugs (NSAIDs) and selective COX-2 inhibitors for the treatment of inflammatory diseases. A series of 3-(5-phenyl/phenylamino-[1,3,4]oxadiazol-2-yl)-chromen-2-one and N-[5-(2-oxo-2H-chromen-3-yl)-[1,3,4]oxadiazol-2-yl]-benzamide derivatives were synthesized and screened for anti-inflammatory, analgesic activity. All the derivatives prepared are active in inhibiting oedema induced by carrageenan. Compound 4e was found more potent with 89% of inhibition followed by compound 4b (86%). Compounds with >70% of anti-inflammatory activity were tested for analgesic, ulcerogenic, and lipid peroxidation profile. Selected compounds were also evaluated for inhibition of COXs (COX-1 and COX-2) and LOXs (LOX-5, LOX-12, and LOX-15). Compound 4e was comparatively selective for COX-2, LOX-5, and LOX-15. Study revealed that these derivatives were more effective than ibuprofen with reduced side effects. It can be suggested that these derivatives could be used to develop more potent and safer NSAIDs.     

A Fas-associated death domain protein-dependent mechanism mediates the apoptotic action of non-steroidal anti-inflammatory drugs in the human leukemic Jurkat cell line

Non-steroidal anti-inflammatory drugs (NSAIDs) are inhibitors of cyclooxygenase-1 and -2 and are useful for prevention and cure of cancers, especially colon and rectal cancers. The NSAIDs indomethacin and sulindac sulfide have been shown to induce apoptosis of colon epithelial cancer cells by a Bax-dependent mechanism that involves mitochondria-mediated activation of a caspase-9-dependent pathway. In this report, we demonstrate that indomethacin and sulindac sulfide induce apoptosis of human leukemic Jurkat cells by a mechanism that requires the Fas-associated Death Domain Protein-mediated activation of a caspase-8-dependent pathway. Therefore, NSAIDs induce apoptosis by different mechanisms depending on the cell type.     

Sulindac sulfide is a noncompetitive gamma-secretase inhibitor that preferentially reduces Abeta 42 generation

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been known to reduce risk for Alzheimer's disease. In addition to the anti-inflammatory effects of NSAIDs to block cylooxygenase, it has been shown recently that a subset of NSAIDs selectively inhibits the secretion of highly amyloidogenic Abeta42 from cultured cells, although the molecular target(s) of NSAIDs in reducing the activity of gamma-secretase for Abeta42 generation (gamma(42)-secretase) still remain unknown. Here we show that sulindac sulfide (SSide) directly acts on gamma-secretase and preferentially inhibits the gamma(42)-secretase activity derived from the 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate-solubilized membrane fractions of HeLa cells, in an in vitro gamma-secretase assay using recombinant amyloid beta precursor protein C100 as a substrate. SSide also inhibits activities for the generation of Abeta40 as well as for Notch intracellular domain at higher concentrations. Notably, SSide displayed linear noncompetitive inhibition profiles for gamma(42)-secretase in vitro. Our data suggest that SSide is a direct inhibitor of gamma-secretase that preferentially affects the gamma(42)-secretase activity.     

Design and synthesis of 3,4-methylenedioxy-6-nitrophenoxyacetylhydrazone derivatives obtained from natural safrole: new lead-agents with analgesic and antipyretic properties

In this work, we reported the synthesis and evaluation of the analgesic, anti-inflammatory, and antipyretic properties of new 2-(6-nitro-benzo[1,3]dioxol-5-yloxy)-acetylhydrazone derivatives (3), designed exploring molecular hybridization and isosteric replacement approaches between nimesulide (1) and carbanalogue NAH series (2) developed at LASSBio. Target compounds were synthesized in very good yields exploiting abundant Brazilian natural product safrole (4) as starting material. The evaluation of the antinociceptive properties of this series led us to discover a new potent prototype of analgesic and antipyretic agent, that is, NAH derivative 3c, named LASSBio-891, which showed to be more potent than dipyrone used as standard.     

Induction of multidrug resistance proteins MRP1 and MRP3 and gamma-glutamylcysteine synthetase gene expression by nonsteroidal anti-inflammatory drugs in human colon cancer cells

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been demonstrated to suppress colorectal tumorigenesis. NSAIDs have also been used to treat inflammatory illnesses. However, the underlying mechanisms of action by NSAIDs have not been completely elucidated. In this study, we reported that among the six members of the multidrug resistance protein gene (MRP1 to MRP6) family which encode membrane transporters for a diverse group of antitumor agents, expression of MRP1 and MRP3 but not the others in human colorectal cancer cell lines was induced by sulindac. This induction profile is consistent with the results using prooxidants which produce reactive oxygen species (ROS) and generate oxidative stress as previously reported. Moreover, treatment of colorectal cancer cells with sulindac induced ROS. Suppression of ROS formation by antioxidant N-acetylcysteine (NAC) downregulated the induction of MRP1 and MRP3 expression. Expression of another oxidative stress-sensitive gene, gamma-glutamylcysteine synthetase heavy subunit gene (gamma-GCSh), which encodes the rate-limiting enzyme in glutathione biosynthesis, was also induced by sulindac. However, the suppression of sulindac-induced gamma-GCSh expression by NAC was less sensitive compared with that of MRP1 and MRP3. We also demonstrated that induction of MRP3 and gamma-GCSh was independent of intracellular COX-2 levels. These results, collectively, suggest a ROS-related, COX-2-independent mechanism for the induction of drug resistance gene expression that bears important implications to the roles of NSAIDs in colorectal carcinogenesis and inflammatory response.