Dipyrone and Aminopyrine Are Effective Scavengers of Reactive Nitrogen Species

Research article

Dipyrone and aminopyrine are effective scavengers of reactive nitrogen species

David Costa1, Abel Vieira2, Eduarda Fernandes1

1REQUIMTE, Departamento de Química-Física, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal 2REQUIMTE, CQFB, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal

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.

Keywords: Dipyrone, aminopyrine, isopropylantipyrine, antipyrine, reactive nitrogen species, nitric oxide, peroxynitrite

INTRODUCTION antipyrine being first prepared in 1883.1 Chemically, these compounds are pyrazolone derivatives. Isopropyl- Dipyrone (metamizol), aminopyrine (4-dimethylaminoan- antipyrine was obtained from antipyrine by introducing tipyrine, aminophenazone), isopropylantipyrine (propy- an isopropyl group on C-4 in order to increase its phenazone) and antipyrine (phenazone) (Fig. 1) are potency. This modification improved the antipyretic and non-steroidal anti-inflammatory drugs (NSAIDs) pertain- analgesic properties while maintaining the anti-inflam- ing to the first group of compounds used as analgesic, matory activity.1 The introduction of a dimethylamino antipyretic and anti-inflammatory therapeutic drugs,1–3 with group on C-4 of the antipyrine molecule resulted in aminopyrine (4-dimethylaminoantipyrine).1 A disadvan- tage of aminopyrine is its relative insolubility in water. Received 17 January 2006 The search for more soluble compounds led to the production Revised 27 May 2006 of the sodium salt of antipyrinyl methylaminomethane- Accepted 28 May 2006 sulphonic acid (dipyrone).1 The pharmacological activi- Correspondence to: Eduarda Fernandes PhD, REQUIMTE, ties of pyrazolones are not fully understood but it is Departamento de Química-Física, Faculdade de Farmácia, known that they involve the inhibition of cyclo- Universidade do Porto, Rua Aníbal Cunha 164, 4099-030 Porto, Portugal oxygenase isoenzymes, platelet thromboxane synthesis 4–6 Tel: +351222078968; Fax: +351222004427; and prostanoid synthesis. Notably, other putative ther- E-mail: [email protected] apeutic effects have been found for this family of

Redox Report, Vol. 11, No. 3, 2006 ©W. S. Maney & Son Ltd DOI 10.1179/135100006X116637 Dipyrone and aminopyrine are effective scavengers of reactive nitrogen species 137

•– with O2 ), the induction of cyclooxygenase as well as angio- genic and inflammatory cytokines, activation of matrix met- alloprotease and induction of chondrocyte apoptosis.12 ONOO– itself is a relatively long-lived cytotoxicant with strong oxidizing properties towards various cellular con- stituents, including sulphydryls, lipids, amino acids and nucleotides.13 Therefore, considering the possible importance of RNS in the pathophysiology of inflammation, indicative of bene- ficial effects by RNS scavengers, the aim of the present study was to evaluate the putative inhibitory effect of the pyrazolones dipyrone, aminopyrine, isopropylantipyrine and antipyrine against RNS, using in vitro non-cellular screening systems.

Fig. 1. Chemical structures of dipyrone, aminopyrine, isopropylantipyrine MATERIALS AND METHODS and antipyrine. Materials

NSAIDs, related to the prevention of deleterious effects All chemicals and reagents were of analytical grade. mediated by reactive oxygen species (ROS). ROS are Dipyrone, aminopyrine, antipyrine, dihydrorhodamine generated during inflammation, following the activation 123 (DHR 123), diethylenetriaminepentaacetic acid of mast cells, macrophages, eosinophils and neutrophils (DTPA), 4,5-diaminofluorescein (DAF-2), sodium nitro- and may subsequently provoke or exacerbate damage at prusside dihydrate, ebselen, carboxy-PTIO and manganese the inflammatory site.7,8 It was demonstrated that dioxide, were obtained from Sigma Chemical Co. (St aminopyrine and dipyrone prevent phorbol myristate Louis, MO, USA). Potassium chloride was obtained from acetate (PMA)-induced neutrophil burst with a high Fluka Chemie GmbH (Steinheim, Germany). Hydrogen degree of efficiency, while isopropylantipyrine exerted peroxide (30% solution), sodium bicarbonate, sodium little effect and antipyrine no effect whatsoever.9 It was nitrite, sodium hydroxide, hydrochloric acid and sodium also observed that dipyrone and aminopyrine are highly chloride were obtained from Merck (Darmstadt, Germany). potent scavengers of the hydroxyl radical (HO•) and Isopropylantipyrine was kindly supplied by Novartis hypochlorous acid (HOCl) while, in accordance with the International Pharmaceutical Ltd, Ireland and ONOO– was neutrophil burst results, isopropylantipyrine showed lit- obtained by synthesis (see below). tle effect and antipyrine no effect whatsoever against these two ROS.9 None of the studied pyrazolones was •– – capable of scavenging the superoxide radical (O2 ) or Synthesis of ONOO hydrogen peroxide (H2O2) and dipyrone was shown to be the most reactive against the peroxyl radical (ROO•).9 Synthesis of ONOO– was essentially performed as Significantly, aminopyrine is a strong myeloperoxidase described before.14 Briefly, an acidic solution (0.7 M inhibitor whereas antipyrine showed no effect against HCl) of 0.6 M H2O2 was mixed with 0.66 M NaNO2 on this enzyme.10 Thus, the inhibition of the neutrophil ice for 1 s and the reaction quenched with ice-cold 3 M oxidative burst by pyrazolone derivatives together with NaOH. Residual H2O2 was removed by mixing with the reported ROS scavenging effects may contribute to granular MnO2 pre-washed with 3 M NaOH. The stock their therapeutic efficacy. ONOO– solution was filtered, then frozen (–20°C) and However, the picture remained incomplete since reac- the top layer of the solution collected for the experiment. tive nitrogen species (RNS), namely nitric oxide (NO•) and ONOO– concentration was determined by measuring peroxynitrite (ONOO–) are also produced in the inflamma- absorbance at 302 nm (ε = 1670 M–1cm–1). The typical yield tory sites and may contribute to the deleterious effects of of freshly prepared ONOO– ranged from 60–80 mM. inflammation. Indeed, NO• is produced by a family of isoen- Higher concentrations (> 200 mM) of ONOO– can be zymes, termed nitric oxide synthases (NOSs) which are typi- obtained by freeze fractionation. However, in the present cally induced during inflammation.11 Possible study, only freshly prepared ONOO– solution was used in pro-inflammatory effects of NO• include augmentation of an effort to minimise nitrite ion contamination. Prior to vascular permeability of inflamed tissues, the generation of each study, the concentration of the ONOO– stock was other destructive free radicals (namely ONOO– by reaction determined spectrophotometrically in 0.1 M NaOH. 138 Costa, Vieira, Fernandes

Assay for measurement of ONOO– scavenging activity mixtures contained the following reagents at the indicated final concentrations (in a final volume of 1.5 ml): ONOO– scavenging activity was measured by fluorime- DAF-2 (3 mM), tested compounds dissolved in DMSO, try, through monitoring the oxidation of non-fluorescent at various concentrations (0–2 µM for dipyrone, 0–25 dihydrorhodamine 123 (DHR 123) to the fluorescent mM for aminopyrine and 0–5 mM for antipyrine and rhodamine 123 by ONOO– according to a described pro- isopropylantipyrine) and sodium nitroprusside (20 mM). cedure15 with modifications.16 A stock solution of 2.89 The reaction mixture was incubated for 10 min at 37°C mM DHR 123 in dimethylformamide was purged with under a tungsten light. Light is of prime importance for nitrogen and stored at –20°C. Working solutions of DHR the sustained release of NO• by sodium nitroprusside.20 123 diluted from the stock solution were placed on ice The fluorescence signal induced by DAF-2 reacting with under darkness immediately before the determinations. NO• was measured using a spectrofluorimeter (LS-50B,

Buffer (90 mM NaCl, 50 mM Na3PO4 and 5 mM KCl, Perkin-Elmer) with excitation and emission wavelengths pH 7.4) was purged with nitrogen and placed on ice of 495 nm and 521 nm, respectively, and excitation and before use. At the outset of the experiments, 100 µM emission slit widths of 8 nm. Effects are expressed as the DTPA was added to the buffer. Reaction mixtures con- percentage inhibition of the NO•-induced DAF-2 oxida- tained the following reagents at the indicated final con- tion. Carboxy-PTIO was used as a positive control. Each centrations (in a final volume of 300 µl): DHR 123 (5 study corresponds to four experiments, performed in µM), tested compounds dissolved in DMSO, at various triplicate. The following formula was used for calculat- concentrations (0–10 µM for dipyrone and aminopyrine ing NO• scavenging activity: and 0–5 mM for antipyrine and isopropylantipyrine) and •NO scavenging activity (%) = ONOO– (600 nM). The mixtures were incubated in a microplate reader (Synergy HT, BIO-TEK) for 5 min at Fsample – Fblank 37°C. The fluorescence signal induced by DHR 123 100 – × 100% Eq. 2 – reacting with ONOO was measured using the Fcontrol – Fblank microplate reader with excitation and emission wave- where F is fluorescence intensity in arbitrary units. lengths of 485 ± 20 nm and 528 ± 20 nm, respectively. Ebselen was used as a positive control. Effects are – expressed as the percentage inhibition of the ONOO - RESULTS induced DHR 123 oxidation. In a parallel set of experiments, the assays were performed in the presence of 25 NO• scavenging activity mM NaHCO3 in order to simulate the physiological con- • ditions with high CO2 concentrations in vivo. This evalu- Figure 2 shows the results obtained from the NO scav- ation is important because, under physiological enging assay for dipyrone and aminopyrine. These com- – • conditions, the reaction of ONOO with CO2 predomi- pounds were strong inhibitors of the NO -elicited nates, with the rate constant of reaction of CO2 with oxidation of DAF-2 to triazolofluorescein in a concen- – × 4 –1 –1 17 ONOO being very rapid (k2 = 3–5.8 10 M s ). tration-dependent manner with dipyrone demonstrating Thus, the reactivity of the putative scavengers for itself to be the most active, while antipyrine and iso- ONOO– should be able to match or exceed that of bicarbonate. Each study corresponds to four experiments, Table 1. Scavenging activities (IC50; mean ± SEM) of dipyrone, performed in triplicate. The following formula was used aminopyrine, isopropylantipyrine, antipyrine, carboxy-PTIO, and for calculating ONOO– scavenging activity: ebselen against NO• and ONOO– (in the absence or presence of NaHCO ) ONOO– scavenging activity (%) = 3 NO• ONOO– Fsample – Fblank 100 – × 100% Eq. 1 Absence of Presence of Tested NaHCO 25 mM NaHCO Fcontrol – Fblank 3 3 compound IC (µM) IC (µM) IC (µM) where F is fluorescence intensity in arbitrary units. 50 50 50 Dipyrone 0.54 ± 0.04 1.8 ± 0.2 4.8 ± 0.7 Aminopyrine 4.3 ± 0.8 1.5 ± 0.1 3.5 ± 0.6 Assay for measurement of NO• scavenging activity Isopropylantipyrine > 5000 NA NA Antipyrine > 5000 > 5000 > 5000 Carboxy-PTIO 1.8 ± 0.4 – – • NO scavenging activity was measured by fluorimetry, Ebselen – 2.5 ± 0.1 16 ± 1 through monitoring the NO•-induced oxidation of 4,5- diaminofluorescein (DAF-2) to triazolofluorescein as NA, no activity was found within the tested concentrations previously described18 with modifications.19 Reaction (up to 5 mM). Dipyrone and aminopyrine are effective scavengers of reactive nitrogen species 139 A B

Fig. 2.NO• scavenging activity of dipyrone and aminopyrine. Each point represents the values obtained from four experiments, performed in triplicate (mean ± SEM). propylantipyrine were only shown to exert weak activity aminopyrine were slightly decreased. In the absence of against this RNS (data not shown). The resulting IC50 NaHCO3, the resulting IC50 values were 1.8 ± 0.2, 1.5 ± 0.1 values were 0.54 ± 0.04, 4.3 ± 0.8, > 5000, > 5000 µM and > 5000 µM (mean ± SEM) for dipyrone, aminopyrine (mean ± SEM) for dipyrone, aminopyrine, isopropylan- and antipyrine, respectively (Table 1). In the presence of tipyrine and antipyrine, respectively (Table 1). The posi- NaHCO3, the resulting IC50 values were 4.8 ± 0.7, 3.5 ± 0.6 µ tive carboxy-PTIO control gave rise to an IC50 of 1.8 ± and > 5000 M (mean ± SEM) for dipyrone, aminopyrine 0.4 µM (mean ± SEM; Table 1). and antipyrine, respectively (Table 1). The positive control µ ebselen gave rise to an IC50 of 2.5 ± 0.1 and 16 ± 1 M (mean ± SEM) in both the absence and presence of 25 mM – ONOO scavenging activity NaHCO3, respectively (Table 1).

Figure 3 shows the results obtained from the ONOO–- scavenging assay for dipyrone and aminopyrine in both DISCUSSION the absence and presence of 25 mM NaHCO3. In the absence of NaHCO3, these compounds were shown to be The results obtained from the present study showed that strong inhibitors of the ONOO–-elicited oxidation of dipyrone and aminopyrine were highly potent scav- DHR 123 in a concentration-dependent manner and at a engers of NO• and ONOO–, while antipyrine had little rather similar potency. Isopropylantipyrine was devoid effect and isopropylantipyrine no effect whatsoever of any activity, while antipyrine was shown to be only a against these two RNS. In the presence of NaHCO3, the weak inhibitor (data not shown). In the presence of inhibitor activities of both dipyrone and aminopyrine were

NaHCO3, the inhibitor activities of both dipyrone and slightly decreased. It has been reported that physiological A B

– Fig. 3. ONOO scavenging activity of dipyrone and aminopyrine in the absence of NaHCO3, and in the presence of 25 mM NaHCO3. Each point represents the values obtained from four experiments, performed in triplicate (mean ± SEM). 140 Costa, Vieira, Fernandes

– • concentrations of CO2 can modulate ONOO reactivity patient. At physiological levels, NO is mainly involved due to the rapid nature of the reaction between these two in homeostatic biochemical and physiological processes 17 – compounds. In fact, CO2, by competing with ONOO , such as signal transduction, neurotransmission, smooth significantly weakens the ability of several phenolic muscle relaxation, peristalsis, gastroprotective effects, compounds such as caffeic acid, o- and p-coumaric acid, inhibition of platelet aggregation, blood pressure modu- gallic acid and ferulic acid21 as well as Trolox, glu- lation, immune system control and learning and mem- tathione and uric acid.17 The effects observed in the pre- ory.26–28 Therefore, a possible variation of NO• sent study are in line with those findings. physiological levels by dipyrone and aminopyrine could It is interesting to note that the secondary amine pre- potentially affect these physiological processes. The sent in the chemical structure of aminopyrine (but absent contribution of NO• to maintaining blood pressure is par- from the chemical structure of isopropylantipyrine and adigmatic. However, from looking at the literature it can antipyrine; Fig. 1) has previously been reported to be be assumed that these NSAIDs do not affect or even lead highly reactive with pro-oxidant compounds, namely to a small decrease in blood pressure.29,30 This apparent 9,22,23 ROS. Notably, the IC50 values observed for the stud- contradiction may be due to other dipyrone- and ied RNS were similar to those obtained before for HO• aminopyrine-related effects such as the scavenging and HOCl.9 Therefore, it is very much likely that its effect of these compounds against ROS, ONOO– or their reactivity with RNS is similar to that of ROS. As noted neutrophil burst inhibitory activities.9 ROS are generated by Halliwell and Gutteridge,24 an antioxidant is ‘any in the vasculature mainly by NAD(P)H oxidase in a substance that, when present at low concentrations com- mechanism that is angiotensin II-dependent. Activation •– pared with those of an oxidisable substrate, significantly of this enzyme leads to the production of O2 and a delays or prevents the oxidation of that substrate’. This decreased availability of NO• while increasing the levels implies that the scavenging activities for RNS and ROS of tissue-damaging ONOO– by virtue of the reaction • •– 31 for any antioxidant should be tested in vivo, in the pres- between NO and O2 . NAD(P)H-dependent ROS for- ence of endogenous antioxidants. Although this point mation, could also contribute to vascular injury by sus- still requires experimental clarification, under sustained taining NAD(P)H oxidase activation, promoting overproduction of ROS and RNS, endogenous antioxi- inflammatory gene expression, extracellular matrix re- dants may decrease substantially or become depleted, organization and growth (hypertrophy/hyperplasia) of thereby increasing the importance of exogenous antioxi- vascular smooth muscle cells.31 It seems, therefore, from dants. Significantly, aminopyrine was also previously the literature-derived data, that the ONOO– and ROS shown to exhibit a strong inhibitor effect against scavenging effects of dipyrone and aminopyrine proba- • myeloperoxidase within similar concentrations (IC50 of bly prevail over NO depleting effects. 0.72 ± 0.09 µM)10 which re-inforces the antioxidant pro- In the gastrointestinal (GI) tract, NO• participates in file of this drug. Dipyrone reactivity may also be the modulation of the smooth musculature tone, such as explained by the presence of this secondary amine in its the regulation of intestinal peristaltism, gastric emptying structure. However, when administered orally, dipyrone and antral motor activity. It also regulates acid and gas- may itself be considered a pro-drug since it undergoes tric mucus secretion, alkali production and is involved in non-enzymatic hydrolysis in the stomach to form 4- the maintenance of mucosal blood flow. Under physio- methylaminoantipyrine, which is rapidly and almost logical conditions, NO• acts as an endogenous mediator, completely absorbed.25 The maximum therapeutic con- modulating both the repair and integrity of the tissues centrations of 4-methylaminoantipyrine reached after and exhibits gastroprotective properties against various administration of a single oral dose of dipyrone is up to types of aggressive agents. However, high NO• concentra- 36 µM while that of aminopyrine is up to 160 µM.24 tions are related to numerous pathological GI tract Since the RNS scavenging effects observed in vitro for processes including peptic ulcer, chronic gastritis, gastroin- dipyrone and aminopyrine were well within these thera- testinal cancer, bacterial gastroenteritis, celiac or chronic peutic concentrations, it is highly likely that during ther- inflammatory bowel diseases (for a review, see Martin et apy with these NSAIDs, NO• and ONOO– may be al.28). Thus, NSAIDs with NO•-depleting activities may yet effectively scavenged in vivo. again have ambivalent damaging/protecting effects in the As explained in the introduction, these RNS may con- GI tract. Of note is that long-term NSAIDs use commonly tribute to the pathophysiology of inflammation and, as associated with gastric erosions as well as a 2–5-fold such, it could be inferred that the scavenging effects increase in relative risk and 30% attributable risk of ulcer observed for dipyrone and aminopyrine in the present perforation, upper gastrointestinal bleeding and death.32 study are of enormous benefit for the patient under treat- NSAIDs may also damage the small and large bowel. In reg- ment. However, these data should be interpreted with a ular users of NSAIDs, an 8–10% prevalence of small intesti- degree of caution since the possible depletion of physio- nal ulceration has been reported as well as an increased logical NO• concentrations may also be harmful to the frequency of small and large bowel perforation.32 Dipyrone and aminopyrine are effective scavengers of reactive nitrogen species 141

Significantly, apart from NO•, prostaglandins synthe- observed scavenging effects are of therapeutic benefit sised by cyclooxygenase-1 (COX-1) and cyclooxyge- for the patient under treatment. However, the possible nase-2 (COX-2) contribute strongly to the gastric depletion of physiological NO• concentrations, namely defence system.33 Both COX-1 and COX-2 contribute to at the GI tract together with the formation of reactive gastric mucosal defence. In normal gastric mucosa, the derivatives of aminopyrine and/or dipyrone may also be combined inhibition of both COX-1 and COX-2 is nec- harmful for the patient. Thus, more experimental data essary to induce lesion formation while in the face of are required to evaluate the possible risk/benefit pending injury, such as the presence of exogenous acid obtained by the RNS scavenging effects of dipyrone and in the gastric lumen, isolated inhibition of COX-1 is suf- aminopyrine. ficient to damage the mucosa.32 Specific inhibition of COX-2 does not induce gastric injury either in normal mucosa or in the presence of exogenous intraluminal ACKNOWLEDGEMENTS acid. However, severe injury develops during suppres- sion of COX-2 when the function of one of the other fac- The authors acknowledge the financial support given by tors involved in the complex system of mucosal defence REQUIMTE (project REQEVA). David Costa thanks such as NO• or afferent neurons is impaired,32 which may FCT and FSE for a PhD grant (SFRH/BD/10483/2002). be the case during inflammation therapy with dipyrone and aminopyrine. 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