Research article

Singlet oxygen scavenging activity of non- steroidal anti-inflammatory drugs

David Costa, Ana Gomes, José L.F.C. Lima, Eduarda Fernandes

REQUIMTE, Departamento de Química-Física, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal

1 It has long been known that singlet oxygen ( O2) is generated during inflammatory processes. 1 Once formed in substantial amounts, O2 may have an important role in mediating the destruction 1 of infectious agents during host defense. On the other hand, O2 is capable of damaging almost all biological molecules and is particularly genotoxic, which gives a special relevance to the scavenging of this ROS throughout anti-inflammatory treatments. Considering that the use of non- steroidal anti-inflammatory drugs (NSAIDs) constitutes a first approach in the treatment of persistent inflammatory processes (due to their ability to inhibit cyclooxygenase), a putative 1 scavenging activity of NSAIDs for O2 would also represent a significant component of their 1 therapeutic effect. The aim of the present study was to evaluate the scavenging activity for O2 by several chemical families of NSAIDs. The results suggested that the pyrazole derivatives (dipyrone 1 and aminopyrine) are, by far, the most potent scavengers of O2 (much more potent compared to the other tested NSAIDs), displaying IC 50 -values in the low micromolar range. There was a lack of activity for most of the arylpropionic acid derivatives tested, with only naproxen and indoprofen displaying residual activities, as for the oxazole derivative, oxaprozin. On the other hand, the pyrrole derivatives (tolmetin and ketorolac), the indolacetic acid derivatives (indomethacin, and etodolac), as well as sulindac and its metabolites (sulindac sulfide and sulindac sulfone) displayed scavenging activity in the high micromolar range. Thus, the scavenging effect observed for dipyrone and aminopyrine will almost certainly contribute to their healing effect in the treatment of prolonged or chronic inflammation, while that of the other studied NSAIDs may have a lower contribution, though these assumptions still require further in vivo validation.

Keywords: NSAIDs, singlet oxygen, endoperoxide of disodium 3,3 ′-(1,4-naphthalene)bispropionate (NDPO 2), dihydrorhodamine 123, microplate screening assay

Introduction infection, ischemia or injury. It is well known that a few mediators play a prominent role in the In biological tissues, inflammatory processes involve inflammatory cascade, namely arachidonic acid- complex physiological responses, resulting from derived lipid compounds ( i.e . eicosanoids) and interactions between cells and soluble factors, to cytokines. On the other hand, several lines of evidence indicate that reactive oxygen species (ROS) and reactive nitrogen species (RNS) play a pivotal role in Correspondence to: Eduarda Fernandes PhD, REQUIMTE, Departa- cellular damage often observed in many inflammatory mento de Química-Física, Faculdade de Farmácia, Universidade do 1–3 Porto, Rua Aníbal Cunha, 164 4099-030 Porto, Portugal. conditions. Accordingly, it has been shown that Tel: +35 1 222 078968; Fax: +35 1 222 078961; several synthetic and/or natural antioxidants are E-mail: [email protected] 4 Received 25 October 2007, revised manuscript accepted 19 February 2008 endowed with potent anti-inflammatory activity.

© 2008 W. S. Maney and Son Ltd Redox Report 2008 Vol 13 No 4 153 DOI 10.1179/135100008X308876 Costa et al. Singlet oxygen scavenging activity of non-steroidal anti-inflammatory drugs

1 Figure 1 ROS and RNS produced in the event of inflammatory processes, with emphasis to singlet oxygen ( O2). Attraction of neutrophils to inflammatory sites and subsequent activation of NADPH oxidase results in the formation of •– •– superoxide radicals (O 2 ). O 2 is converted to hydrogen peroxide (H 2O2) spontaneously or through catalysis of •– superoxide dismutase (SOD) from ingested microorganisms. The interactions of H 2O2 with O 2 or with trace levels of • transition metals can lead to the formation of hydroxyl radicals (HO ). H 2O2 can react with hypochlorous acid (HOCl), • – 1 nitric oxide ( NO) or peroxynitrite (ONOO ) to yield O2. Myeloperoxidase (MPO), a hydrogen peroxide oxidoreductase that is specifically found in mammalian granulocytic leukocytes, including neutrophils, monocytes, basophils, and

eosinophils, produce HOCl from H 2O2 and chloride ions. Besides its well-established reactivity with H 2O2, HOCl also reacts with lipid hydroperoxides (LOOH; generated during interaction of ROS and RNS with cell membranes, and 1 through catalysis by lipoxygenases and cyclooxygenases) to yield O2. LOOH also reacts with transition metals, resulting in the formation of lipid alcoxyl (LO •) and/or lipid peroxyl radicals (LOO •). It is now well established that the α 1 self-reaction of LOO, containing -hydrogen, generates O2. The inducible nitric oxide synthase enzymes (iNOS) also become activated, resulting in the production of continuous near-micromolar amounts of •NO for long periods. – • •– ONOO is formed from the diffusion-controlled reaction between NO and O 2 . Under physiological pH conditions, – • • – – ONOO is in equilibrium with ONOOH, yielding HO , nitrogen dioxide radical ( NO 2) and nitrate (NO 3 ). ONOO can also – – react with carbon dioxide (CO 2) to form the nitrosoperoxycarbonate anion (ONOOCO 2 ). ONOOCO 2 decomposition – •– • •– then leads to the formation of CO 2 and NO 3 , NO 2 and the carbonate radical anion (CO 3 ). Both HO and CO 3 are known to react with hydroperoxides (ROOH) to generate peroxyl radicals (ROO •), which add to those formed during lipid peroxidation. As stated above, termination reactions involving primary or secondary ROO • generates 1 predominantly O2

Furthermore, it has been extensively demonstrated important role in the inflammatory process. 13–18 Figure 1 1 that several non-steroidal anti-inflammatory drugs summarizes the production of ROS, including O2, and (NSAIDs) are effective scavengers of ROS and RNS, 5–12 RNS during inflammatory processes as reported which probably contributes to their final therapeutic elsewhere. 12,18–21 Beyond its possible contribution to tissue 1 activity. In this respect, the most studied reactive species healing during acute inflammation, O2-mediated tissue •– have been the ROS superoxide radical (O 2 ), hydrogen damage in sustained or chronic inflammatory states, • peroxide (H 2O2), hydroxyl radical (HO ), peroxyl radical suggests that a potential effect against this ROS would be (ROO •) and hypochlorous acid (HOCl), and the RNS important for the therapeutic effect of anti-inflammatory nitric oxide ( •NO) and peroxynitrite anion (ONOO –). drugs. Notwithstanding this rationale, the potential 1 1 Nevertheless, another ROS, singlet oxygen ( O2), in scavenging activity of NSAIDs for O2 remains to be addition to its well-known formation during photo- evaluated. The aim of the present study was to evaluate the 1 sensitization processes, has also been shown to have an scavenging activity for O2 by several chemical families

154 Redox Reort 2008 Vol 13 No 4 Costa et al. Singlet oxygen scavenging activity of non-steroidal anti-inflammatory drugs of NSAIDs – the pyrazole derivatives antipyrine, ketorolac and tolmetin, the indolacetic acid derivatives propy phenazone , dipyrone, and amino pyrine, sulindac etodolac, indomethacin, and acemetacin, as well as the and its metabolites sulindac sulfide and sulindac sulfone, arylpropionic acid derivatives indoprofen, naproxen, the oxazole derivative oxaprozin, the pyrrole derivatives ketoprofen, fenoprofen, flurbiprofen, ibuprofen, and

Figure 2 Chemical structures of the studied NSAIDs: the pyrazole derivatives antipyrine, propyphenazone, dipyrone, and aminopyrine, sulindac and its metabolites sulindac sulfide and sulindac sulfone, the oxazole derivative oxaprozin, the pyrrole derivatives ketorolac and tolmetin, the indolacetic acid derivatives etodolac, indomethacin, and acemetacin, as well as the arylpropionic acid derivatives indoprofen, naproxen, ketoprofen, fenoprofen, flurbiprofen, ibuprofen, and fenbufen

Redox Report 2008 Vol 13 No 4 155 Costa et al. Singlet oxygen scavenging activity of non-steroidal anti-inflammatory drugs

fenbufen (Fig. 2). For this purpose, a previously developed specially N,N-dimethyl-4-nitrosoaniline, proceeds with 22 1 non-cellular screening system was used, in which O2 was much faster rates than by the direct oxidation through 1 22 generated by thermal dissociation of the water-soluble light-excited dyes or by O2. Reaction mixtures in the endoperoxide of disodium 3,3 ′-(1,4-naphthalene) sample wells contained the following reagents at the bispropionate (NDPO 2) and was detected by monitoring indicated final concentrations (in a final volume of the 1O -induced oxidation of non-fluorescent dihydro - 250 l): DHR (50 M), histidine (10 mM), the tested 2 µ µ rhodamine 123 (DHR) to fluorescent rhodamine 123 NSAIDs at various concentrations (dissolved in 100 (RH). This system allowed a rapid and thorough mM phosphate buffer, pH 7.4, or DMSO) and 1 comparison of O2 scavenging potencies among the NDPO 2 (1 mM). The assays were performed at 37°C. studied NSAIDs. No interference was found for DMSO under these assay conditions. The fluorescence signal was collected Materials and methods after a 30 min incubation period at the emission wavelength 528 nm with excitation at 485 nm. The Materials effects are expressed as the percentage inhibition of 1 All the chemicals and reagents were of analytical the O2-induced oxidation of DHR. Each study grade. Indomethacin, acemetacin, etodolac, tolmetin, corresponds to four experiments, performed in ketorolac, sulindac, flurbiprofen, fenoprofen, fenbufen, triplicate. The following equation was used for 1 ketoprofen, naproxen, indoprofen, ibuprofen, dipyrone, calculating O2 scavenging activity, where F is the aminopyrine, antipyrine, dihydrorhodamine 123 (DHR), fluorescence intensity (arbitrary units): ascorbic acid, and dimethylformamide were obtained 1O scavenging activity (%) from Sigma-Aldrich (St Louis, MO, USA). Oxaprozin 2 F – F was a gift from Helsinn Healthcare SA, Switzerland. = 100 –––sam–p–le –––b–lan–k – x 100 Eq. 1 F – F Sulindac sulfide and sulindac sulfone were a gift from control blank Merck Research Laboratories, USA. Propyphenazone was a gift from Novartis International Pharmaceutical Results Ltd (Ireland). Histidine was obtained from Fluka 1O scavenging activity for pyrazole derivatives Chemie GmbH (Steinheim, Germany). NDPO 2 was 2 obtained by synthesis as reported previously. 22 All other The pyrazole NSAIDs dipyrone and aminopyrine 1 reagents were obtained from Merck (Darmstadt, were shown to be potent scavengers of O2 in a Germany).

Generation of 1O by thermodissociation of NDPO 2 2 1 O2 was generated by the thermal dissociation of water- soluble endoperoxide (NDPO ) to 3,3 -(1,4-naph - 2 ′ thalene)bispropionic acid (NDP) at 37°C, as described before. 22

1O scavenging assay 2 1 The O2 scavenging activity assay was performed by 1 monitoring the O2-induced oxidation of non- fluorescent DHR to fluorescent RH. The fluorimetric determinations were performed in a microplate reader (BIO-TEK, Synergy HT). A stock solution of 2.89 mM DHR in dimethylformamide was purged with nitrogen and stored at –20°C. Working solutions of DHR,

histidine and NDPO 2 were prepared immediately before the assays, with phosphate buffer 100 mM, pH 7.4 placed on ice and protected from light. The use of histidine is of utmost importance for the sensitivity of the assay. This 1 1 amino-acid reacts with O2 to form a trans -annular Figure 3 O2 scavenging activity of dipyrone and amino- 22 1 peroxide of the histidine imidazole ring moiety. pyrine as determined by their ability to prevent O2- Significantly, the reactivity of trans -annular endo - elicited oxidation of dihydro rhodamine 123. Each peroxides of imidazole compounds with 1O probes, point represents the values obtained from four 2 experi ments, performed in triplicate (mean ± SEM)

156 Redox Reort 2008 Vol 13 No 4 Costa et al. Singlet oxygen scavenging activity of non-steroidal anti-inflammatory drugs

1 Table 1 O2 scavenging activity (IC 50 , mean ± SEM) of the studied NSAIDs

NSAIDs IC50 (mM) Indolacetic acid derivatives Indomethacin 3.0 ± 0.1 Acemetacin > 5 Etodolac 3.4 ± 0.1 Pyrrole derivatives Tolmetin 2.4 ± 0.3 Ketorolac 3.0 ± 0.3 Oxazole derivative Oxaprozin > 5 Sulindac and metabolites Sulindac 1.9 ± 0.1 Sulindac sulfide 1.0 ± 0.06 Sulindac sulfone 1.7 ± 0.08 Pyrazole derivatives Aminopyrine 0.056 ± 0.002 Dipyrone 0.018 ± 0.002 Propyphenazone na Antipyrine na Arylpropionic acid derivatives Ibuprofen na Flurbiprofen na 1 Fenoprofen na Figure 4 O2 scavenging activity of sulindac, sulindac sulfone and Fenbufen na sulindac sulfide, as determined by their ability to prevent 1 Ketoprofen na O2-elicited oxidation of dihydrorhodamine 123. Each Naproxen na point represents the values obtained from four Indoprofen > 5 experiments, performed in triplicate (mean ± SEM) Positive control Ascorbic acid 0.0003 ± 0.0002 0.1 and 3.4 ± 0.1 mM, respectively (Table 1). Acemetacin na, no activity was found up to the concentration of 5 mM. 1 only displayed residual scavenging activity for O2 up to the concentration of 5 mM (38 ± 3% of inhibition concentration-dependent manner, dipyrone being the at 5 mM). most potent (Fig. 3). IC 50 -values were 18.3 ± 1.8 and 56.3 ± 2.3 µM, respectively (Table 1). Neither antipyrine nor propyphenazone displayed any scavenging activity 1 for O2, up to the concentration of 5 mM. 1O scavenging activity for sulindac and sulindac 2 metabolites Sulindac and its two metabolites sulindac sulfide and sulindac sulfone were shown to be efficient scavengers 1 of O2 in a concentration-dependent manner, sulindac sulfide being the most potent (Fig. 4). IC 50 -values were 1.9 ± 0.1, 1.0 ± 0.06 and 1.7 ± 0.08 mM, respectively (Table 1).

1O scavenging activity for pyrrole derivatives 2 The pyrrole NSAIDs tolmetin and ketorolac were 1 shown to be efficient scavengers of O2 with similar potencies (Fig. 5). IC 50 -values were 2.4 ± 0.3 and 3.0 ± 0.3 mM, respectively (Table 1).

1O scavenging activity for indolacetic acid derivatives 1 2 Figure 5 O2 scavenging activity of tolmetin, ketorolac, oxaprozin, The indolacetic acid NSAIDs indomethacin and indomethacin and etodolac, as determined by their ability 1 etodolac were shown to be efficient scavengers of 1O to prevent O2-elicited oxidation of dihydro rhodamine 2 123. Each point represents the values obtained from four with similar potencies (Fig. 5). IC -values were 3.0 ± 50 experiments, performed in triplicate (mean ± SEM)

Redox Report 2008 Vol 13 No 4 157 Costa et al. Singlet oxygen scavenging activity of non-steroidal anti-inflammatory drugs

1 cancer, and dementia. Importantly, O2 is capable of damaging almost all biological molecules and is particularly genotoxic, 26 which gives a special relevance to the scavenging of this ROS through anti- inflammatory treatments. Considering that the use of NSAIDs constitutes a first approach in the treatment of persistent inflammatory processes, due to their ability to inhibit cyclooxygenase, a putative scavenging 1 activity of NSAIDs for O2 would also be of high importance for their therapeutic effect. From the NSAIDs assayed in the present non-cellular in vitro screening system, the results indicate that the pyrazole derivatives dipyrone and aminopyrine are, by far, the 1 most potent scavengers of O2 (much more potent compared to the other tested NSAIDs), displaying IC -values in the low micromolar range, near ascorbic 1 50 Figure 6 O2 scavenging activity of naproxen and indoprofen, 1 acid values. Certainly, such potent activity will have an as determined by their ability to prevent O2-elicited impact on biological tissues where 1O is being oxidation of dihydrorhodamine 123. Each point 2 represents the values obtained from four experi- produced at a high rate. It is noteworthy that, at a ments, performed in triplicate (mean ± SEM) similar concentration, these two compounds were previously reported to inhibit the oxidative burst in 1O scavenging activity for the oxazole derivative human neutrophils and to scavenge several other ROS 2 oxaprozin (HOCl, HO •, ROO •) and RNS ( •NO and ONOO –), 10,11 The oxazole NSAID oxaprozin only displayed residual which corroborates the thesis that their antioxidant 1 scavenging activity for O2 up to the con centration of 5 activity plays a crucial role in the respective anti- mM (30 ± 2% of inhibition at 5 mM; Fig. 5). inflammatory activity. Dipyrone is itself a pro-drug that undergoes non-enzymatic hydrolysis in the 1O scavenging activity for arylpropionic acid 2 stomach to form aminopyrine, which is rapidly and derivatives almost completely absorbed, 27,28 meaning that only the The arylpropionic acid NSAIDs naproxen and antioxidant potency of the latter will be mandatory indoprofen only displayed residual scavenging activity for the systemic therapeutic activity. The other tested for 1O up to the concentration of 5 mM (Fig. 6). The 2 pyrazole derivatives, antipyrine and propyphenazone, other NSAIDs from this family, flurbiprofen, were devoid of any activity up to 5 mM concentration, fenoprofen, fenbufen, ketoprofen, and ibuprofen, were which is also in accordance to previous results for devoid of any activity. other ROS and RNS. 10,11 Accordingly, it may be 1 considered that O2 reacts with the secondary amine, Discussion present in the chemical structure of dipyrone and aminopyrine but absent in antipyrine and 1 It has long been known that O2 is generated during propyphenazone, in a similar way to what was already 29 1 inflammatory processes by means of the demonstrated for HOCl. If true, the presumed O2- – 23,24 myeloperoxidase/H 2O2/Cl system, through the induced transformation of aminopyrine into the 16 •+ reaction between H 2O2 and HOCl. This ROS may characteristic blue cation radical (APS ), deserves also be formed in inflammatory processes as a by- further attention, since prolonged exposure to APS •+ product resulting from the spontaneous dismutation may lead to agranulocytosis. 29,30 Considering the other •– 25 1 of O 2 . Once formed, in substantial amounts, O2 studied NSAIDs, there was an observed lack of may have an important role in mediating the activity for most of the arylpropionic acid derivatives, destruction of infectious agents during host defense. 13 with naproxen and indoprofen only displaying On the other hand, it is common knowledge that a residual activities, as happened for the oxazole sustained production of ROS in prolonged or chronic derivative oxaprozin. On the other hand, the pyrrole inflammation can lead to severe damage to derivatives tolmetin and ketorolac, and the indolacetic surrounding tissues, with consequences like acid derivatives indomethacin and etodolac, displayed cardiovascular disease, multiple sclerosis, diabetes, scavenging activity in the high micromolar range. The

158 Redox Reort 2008 Vol 13 No 4 Costa et al. Singlet oxygen scavenging activity of non-steroidal anti-inflammatory drugs

1 Acknowledgements lack of O2 scavenging activity of acemetacin compared to indomethacin is somewhat striking. Nevertheless, in vivo , acemetacin is rapidly converted The authors acknowledge the financial support into indomethacin, 31 minimizing this way the given by FCT and FEDER for the project differences between these two compounds. Similarly, POCI/QUI/59284/2004. David Costa and Ana sulindac is converted, in vivo , to the metabolites Gomes thank FCT and FSE for PhD study grants sulindac sulfide and sulindac sulfone. 32,33 Sulindac (SFRH/BD/10483/2002 and SFRH/BD/23299/2005, sulfide, but not sulindac sulfone, blocks prostaglandin respectively). synthesis by non-selective inhibition of cyclo- 34 oxygenase-1 and cyclooxygenase-2. Sulindac sulfide References 1 is the most active O2 scavenger among these three compounds, which gives credibility to a possible 1. Kapoor M, Clarkson AN, Sutherland BA, Appleton I. The role of 1 antioxidants in models of inflammation: emphasis on L-arginine contribution of O2 scavenging activity for the final and arachidonic acid metabolism. Inflammopharmacology 2005; therapeutic activity of sulindac. It may be argued that 12 : 505–519. the effects described for these NSAIDs occur at 2. Ferrè N, Clária J. New insights into the regulation of liver inflammation and oxidative stress. Mini Rev Med Chem 2006; 6: relatively high concentrations compared to those in 1321–1330. plasma and synovial fluid of healthy humans. 35,36 3. Halliwell B. Phagocyte-derived reactive species: salvation or suicide? Trends Biochem Sci 2006; 31 : 509–515. However, the lower part of the corresponding activity 4. Geronikaki AA, Gavalas AM. Antioxidants and inflammatory curves still lies within therapeutic concentrations. disease: synthetic and natural antioxidants with anti-inflammatory activity. Comb Chem High Throughput Screen 2006; 9: 425–442. Considering that these NSAIDs are also scavengers of 5. Aruoma OI, Halliwell B. The iron-binding and hydroxyl radical other ROS and RNS, as well as inhibitors of scavenging action of anti-inflammatory drugs. Xenobiotica 1988; 18 : 459–470. neutrophil oxidative burst under these con - 6. Parij N, Nagy AM, Neve J. Linear and non linear competition centrations, 5–12 the sum of these activities will certainly plots in the deoxyribose assay for determination of rate constants have an impact on their final antioxidant effect. In for reaction of nonsteroidal antiinflammatory drugs with hydroxyl radicals. Free Radic Res 1995; 23 : 571–579. addition, in inflamed tissues some NSAIDs were 7. Fernandes E, Toste SA, Lima JL, Reis S. The metabolism of reported to accumulate leading, for example, to sulindac enhances its scavenging activity against reactive oxygen and nitrogen species. Free Radic Biol Med 2003; 35 : 1008–1017. concentrations in the synovial fluid about 3–8 times 8. Fernandes E, Costa D, Toste SA, Lima JL, Reis S. In vitro higher than those in control joints. 37 Furthermore, the scavenging activity for reactive oxygen and nitrogen species by nonsteroidal anti-inflammatory indole, pyrrole, and oxazole scavenging effect of antioxidants is highly dependent derivative drugs. Free Radic Biol Med 2004; 37 : 1890–1895. on the amount of reactive species being produced, 9. Costa D, Gomes A, Reis S, Lima JL, Fernandes E. Hydrogen 38 peroxide scavenging activity by non-steroidal anti-inflammatory giving rise to completely different IC 50 -values. Thus, drugs. Life Sci 2005; 76 : 2841–2848. it is possible that much lower concentrations of 10. Costa D, Moutinho L, Lima JL, Fernandes E. Antioxidant activity NSAIDs are active in vivo . Indeed, it has already been and inhibition of human neutrophil oxidative burst mediated by arylpropionic acid non steroidal anti-inflammatory drugs. Biol shown that indomethacin is a strong antioxidant in Pharm Bull 2006; 29 : 1659–1670. vivo ,39 despite the high concentrations required in vitro 11. Costa D, Vieira A, Fernandes E. Dipyrone and aminopyrine are effective scavengers of reactive nitrogen species. Redox Rep 2006; for this NSAID in this and previous assays for 11 : 136–142. scavenging ROS and RNS. 12. Costa D, Marques AP, Reis RL, Lima JL, Fernandes E. Inhibition of human neutrophil oxidative burst by pyrazolone derivatives. Free Radic Biol Med 2006; 40 : 632–640. 13. Wentworth Jr P, McDunn JE, Wentworth AD et al . Evidence for Conclusions antibody-catalyzed ozone formation in bacterial killing and inflammation. Science 2002; 298 : 2195–2199. 14. Wentworth Jr P, Nieva J, Takeuchi C et al . Evidence for ozone The results obtained in the present study showed that formation in human atherosclerotic arteries. Science 2003; 302 : the pyrazole derivatives dipyrone and aminopyrine are 1053–1056. 15. Fubini B, Hubbard A. Reactive oxygen species (ROS) and reactive 1 potent scavengers of O2 within therapeutic nitrogen species (RNS) generation by silica in inflammation and concentrations. Some of the other studied NSAIDs fibrosis. Free Radic Biol Med 2003; 34 : 1507–1516. 16. Schiller J, Fuchs B, Arnhold J, Arnold K. Contribution of reactive 1 also display O2 scavenging activity, though at much oxygen species to cartilage degradation in rheumatic diseases: higher concentrations. Thus, the scavenging effect molecular pathways, diagnosis and potential therapeutic strategies. Curr Med Chem 2003; 10 : 2123–2145. observed for pyrazole derivatives will almost certainly 17. Symons AM, King LJ. Inflammation, reactive oxygen species and contribute to their healing effect in the treatment of cytochrome P450. Inflammopharmacology 2003; 11 : 75–86. 18. Miyamoto S, Ronsein GE, Prado FM et al . Biological prolonged or chronic inflammation, while that of the hydroperoxide and singlet molecular oxygen generation. IUBMB other studied NSAIDs may have a more limited Life 2007; 59 : 323–331. contribution, though these assumptions still require 19. Gomes A, Fernandes E, Lima JLFC. Use of fluorescence probes for detection of reactive nitrogen species: a review. J Fluoresc 2006, further in vivo validation. 16 : 119–139.

Redox Report 2008 Vol 13 No 4 159 Costa et al. Singlet oxygen scavenging activity of non-steroidal anti-inflammatory drugs

20. Russell GA. Deuterium-isotope effects in the autoxidation of 30. Kalyanaraman B, Sohnle PG. Generation of free radical aralkyl hydrocarbons-mechanism of the interaction of peroxy intermediates from foreign compounds by neutrophil-derived radicals. J Am Chem Soc 1957; 79 : 3871–3877. oxidants. J Clin Invest 1985; 75 : 1618–1622. 21. Dedon PC, Tannenbaum SR. Reactive nitrogen species in the 31. Jones RW, Collins AJ, Notarianni LJ, Sedman E. The comparative chemical biology of inflammation. Arch Biochem Biophys 2004; pharmacokinetics of acemetacin in young subjects and elderly 423 : 12–22. patients. Br J Clin Pharmacol 1991; 31 : 543–545. 22. Costa D, Fernandes E, Santos JL, Pinto DC, Silva AM, Lima JL. 32. Duggan DE, Hare LE, Ditzler CA, Lei BW, Kwan KC. The New noncellular fluorescence microplate screening assay for disposition of sulindac. Clin Pharmacol Ther 1977; 21 : 326–335. scavenging activity against singlet oxygen. Anal Bioanal Chem 33. Duggan DE, Hooke KF, Hwang SS. Kinetics of the tissue 2007; 387 : 2071–2081. distributions of sulindac and metabolites. Relevance to sites and 23. Steinbeck MJ, Khan AU, Karnovsky MJ. Intracellular singlet rates of bioactivation. Drug Metab Dispos 1980; 8: 241–246. oxygen generation by phagocytosing neutrophils in response to 34. Duggan DE, Hooke KF, Risley EA, Shen TY, Arman CG. particles coated with a chemical trap. J Biol Chem 1992; 267 : Identification of the biologically active form of sulindac. J 13425–13433. Pharmacol Exp Ther 1977; 201 : 8–13. 24. Steinbeck MJ, Khan AU, Karnovsky MJ. Extracellular production 35. Netter P, Bannwarth B, Royer-Morrot MJ. Recent findings on the of singlet oxygen by stimulated macrophages quantified using 9,10- pharmacokinetics of non-steroidal anti-inflammatory drugs in diphenylanthracene and perylene in a polystyrene film. J Biol synovial fluid. Clin Pharmacokinet 1989; 17 : 145–162. Chem 1993; 268 : 15649–15654. 36. Urquhart E. A comparison of synovial fluid concentrations of 25. Mayeda EA, Bard AJ. Singlet oxygen. The suppression of its non-steroidal antiinflammatory drugs with their in vitro activity. production in dismutation of superoxide ion by superoxide Agents Actions 1991; 32 : 261–265. dismutase. J Am Chem Soc 1974; 96 : 4023–4024. 37. Graf P, Glatt M, Brune K. Acidic nonsteroid anti-inflammatory 26. Epe B. Genotoxicity of singlet oxygen. Chem Biol Interact 1991; drugs accumulating in inflamed tissue. Experientia 1975; 31 : 80 : 239–260. 951–953. 27. Bentur Y, Cohen O. Dipyrone overdose. J Toxicol Clin Toxicol 38. Sousa T, Fernandes E, Nunes C et al . Scavenging of nitric oxide by 2004; 42 : 261–265. an antagonist of adenosine receptors. J Pharm Pharmacol 2005; 57 : 28. Volz M, Kellner HM. Kinetics and metabolism of pyrazolones 399–404. (propyphenazone, aminopyrine and dipyrone). Br J Clin 39. Stetinova V, Smetanova L, Grossmann V, Anzenbacher P. In vitro Pharmacol 1980; 10 : 299S–308S. and in vivo assessment of the antioxidant activity of melatonin and 29. Sayo H, Saito M. The mechanism of myeloperoxidase-catalysed related indole derivatives. Gen Physiol Biophys 2002; 21 : 153–162. oxidation of aminopyrine. Xenobiotica 1990; 9: 957–965.

160 Redox Reort 2008 Vol 13 No 4