Nicorandil inhibits neutrophil recruitment in ...

European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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Immunopharmacology and inflammation

Nicorandil inhibits neutrophil recruitment in carrageenan-induced experimental pleurisy in mice Tamires C. Matsui a, Giovanna M.E. Coura a, Ivo S.F. Melo a, Carla R.A. Batista a, Paulo Sérgio A. Augusto a, Adriana M. Godin a, Débora P. Araújo b, Isabela C. César a, Lucas S. Ribeiro c, Danielle G. Souza c, André Klein d, Ângelo de Fátima b, Renes R. Machado a, Márcio M. Coelho a,n a Departamento de Produtos Farmacêuticos, Faculdade de Farmácia, Universidade Federal de Minas Gerais, Avenida Antônio Carlos, 6627, Pampulha, CEP 31270-901 Belo Horizonte, MG, Brazil b Departamento de Química, Instituto de Ciências Exatas, Universidade Federal de Minas Gerais, Avenida Antônio Carlos, 6627, Pampulha, CEP 31270-901 Belo Horizonte, MG, Brazil c Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Avenida Antônio Carlos, 6627, Pampulha, CEP 31270-901 Belo Horizonte, MG, Brazil d Departamento de Farmacologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Avenida Antônio Carlos, 6627, Pampulha, CEP 31270-901 Belo Horizonte, MG, Brazil

art ic l e i nf o

a b s t r a c t

Article history: Received 11 September 2015 Received in revised form 16 November 2015 Accepted 18 November 2015

Nicorandil is a drug characterized by the coupling of a nitric oxide (NO) donor to nicotinamide. We have previously demonstrated that nicotinamide exhibits activity in different models of pain and inflammation. Now, we investigated the effects induced by per os (p.o.) administration of nicorandil (25, 50 or 100 mg/Kg) on neutrophil recruitment in a carrageenan-induced model of pleurisy in mice. Effects induced by nicorandil (100 mg/kg) were compared with those induced by equimolar doses of nicotinamide (58 mg/kg) and N-(2-hydroxyethyl)-nicotinamide (NHN; 79 mg/kg). We also investigated whether effects on the production of inflammatory mediators play a role in the activity of nicorandil. P.o. nicorandil, 0.5 h before and 1 h after the i.pl. injection of carrageenan, reduced neutrophil recruitment. However, equimolar doses of nicotinamide or NHN failed to induce such effect. Single treatment (previous or late) with nicorandil (100 mg/Kg, p.o.) also reduced neutrophils recruitment, although to a lesser extent when compared to the double treatment. Nicorandil reduced the concentrations of interleukin-1β, CXCL-1 and prostaglandin E2 in the pleural exudate. Concluding, we demonstrated the activity of nicorandil in a model of pleurisy induced by carrageenan. This activity was characterized by reduction of the neutrophil accumulation and inhibition of production of inflammatory mediators. The effects induced by nicorandil on the leukocytes recruitment and production of inflammatory mediators contribute to a better understanding of its clinical benefits and indicate that these benefits may be due to its vasodilating and anti-inflammatory activities. & 2015 Elsevier B.V. All rights reserved.

Keywords: Nicorandil Nicotinamide Inflammation Neutrophils Cytokines Prostaglandin E2

1. Introduction Neutrophils are circulating polymorphonuclear leukocytes that knowingly play a major role during acute inflammation (Ley et al., 2007; Sadik et al., 2011). Many chemoattractants, including cytokines, chemokines and eicosanoids, released at the sites of injury induce the recruitment of neutrophils from the blood vessels (Kobayashi, 2008; Samuelsson, 1983; Goodman et al., 1993; Perretti et al., 1993; Vieira et al., 2009; Lemos et al., 2009). At the site n

Corresponding author. E-mail address: [email protected] (M.M. Coelho).

of injury, neutrophils contribute to tissue damage and killing of pathogens by releasing many inflammatory proteins, such as proteases and lysosomal enzymes, and reactive oxygen species. There is increasing evidence that neutrophils may also contribute to chronic inflammatory conditions, adaptative immune responses, tissue repair and resolution of inflammation (Kolaczkowska and Kubes, 2013). As neutrophils play key roles in acute and chronic inflammation, identification of molecules that inhibit its recruitment to the inflammatory site may result in new approaches to treat patients with inflammatory diseases. We have previously demonstrated that both nicotinic acid (Ferreira et al., 2013) and nicotinamide (Ferreira et al., 2012), two members of the vitamin B family,

http://dx.doi.org/10.1016/j.ejphar.2015.11.034 0014-2999/& 2015 Elsevier B.V. All rights reserved.

Please cite this article as: Matsui, T.C., et al., Nicorandil inhibits neutrophil recruitment in carrageenan-induced experimental pleurisy in mice. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.11.034i

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T.C. Matsui et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

Fig. 1. Neutrophils (A) and total leukocytes (B) recruitment to the pleural cavity 2, 4 and 6 h after i.pl. injection of carrageenan (Cg, 200 μg) or vehicle (PBS, 0.1 ml). *** significantly different from PBS-treated group (Po 0.01). n¼ 6.

Fig. 2. Effects induced by nicorandil, nicotinamide or NHN on neutrophil (A and C) and total leukocytes (B and D) recruitment induced by i.pl. injection of carrageenan (Cg, 200 μg). Mice were treated p.o. with nicorandil (NIL; 25, 50 or 100 mg/kg), nicotinamide (NIC; 58 mg/kg), NHN (79 mg/kg) or vehicle (0.5% CMC) 0.5 before and 1 h after the i. pl. injection of carrageenan. Cell recruitment was evaluated 4 h after injection of carrageenan. ### significantly different from CMC þ PBS (P o0.001). *, ** and *** significantly different from CMC þCg (P o0.05, P o 0.01 and P o0.001, respectively). n¼ 6.

markedly inhibit the neutrophil recruitment in experimental models of pleurisy induced by carrageenan, chemokine (C–X–C motif) ligand (CXCL) 8, and leukotriene B4 (LTB4) in mice. An expected step in this line of investigation is the evaluation of the activity of nicorandil in these experimental models. Nicorandil is a vasodilatory drug that has been approved for the treatment of patients with angina pectoris (El-Moselby et al., 2009; Frampton et al., 1992). Multiple reasons justify the interest in evaluating the effect induced by nicorandil on neutrophil recruitment. First, nicorandil is a molecule characterized by the coupling of a nitric oxide (NO) donor to nicotinamide and many studies have shown that the coupling of NO donors to anti-inflammatory drugs increase their therapeutic efficacy and safety (Keeble and Moore, 2002; Fiorucci et al., 2000; Miller and Megson, 2007).

Second, nicorandil activates guanylyl cyclase (Schmidt et al., 1985; Lefer and Lefer, 1988), an enzyme that catalyzes the conversion of GTP to cGMP, a cyclic nucleotide that may play a role in neutrophil recruitment in different experimental models of inflammation (Freitas et al., 2006; Iseri et al., 2009). Third, we have demonstrated that nicorandil inhibits the inflammatory phase of the nociceptive response induced by formaldehyde at doses much lower than those of nicotinamide (Dutra et al., 2013). Thus, in the present study, we investigated the effect induced by nicorandil on neutrophil recruitment in a model of pleurisy induced by carrageenan in mice. To evaluate mechanisms mediating the effect induced by nicorandil, we compared its activity with those of nicotinamide and N-(2-hydroxyethyl)-nicotinamide (NHN), as these two last molecules may be released during



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Fig. 3. Effects induced by nicorandil on neutrophils (A) and total leukocytes (B) recruitment induced by i.pl. injection of carrageenan (Cg, 200 μg). Mice were treated p.o. with nicorandil (NIL; 25, 50 or 100 mg/kg) or vehicle (0.5% CMC) 0.5 h before the i.pl. injection of carrageenan. Cell recruitment was evaluated 4 h after injection of carrageenan. ### significantly different from CMCþ PBS (Po 0.001). * and ** significantly different from CMCþ Cg (Po 0.05 and P o 0.01, respectively). n ¼6.

Fig. 4. Effects induced by nicorandil on neutrophils (A) and total leukocytes (B) recruitment induced by i.pl. injection of carrageenan (Cg, 200 μg). Mice were treated p.o. with nicorandil (NIL; 25, 50 or 100 mg/kg) or vehicle (0.5% CMC) 1 h after the i.pl. injection of carrageenan. Cell recruitment was evaluated 4 h after injection of carrageenan. ### significantly different from CMC þPBS (P o 0.001). * significantly different from CMC þ Cg (P o0.05). n¼ 6.

2. Materials and methods 2.1. Drugs and vehicles Nicorandil and NHN, both with purity 499.0%, were synthesized at the Department of Chemistry, Federal University of Minas Gerais. Nicotinamide and λ-carrageenan were purchased from Sigma, USA. The suspensions of nicorandil, nicotinamide and NHN were prepared in carboxymethylcellulose (CMC; 0.5% w/v in saline) and administered per os (p.o.) in a volume of 8 ml/kg. Suspension of carrageenan (0.2%) was prepared in phosphate buffered saline (PBS) and administered via the intrapleural (i.pl.) route in a volume of 0.1 ml. All solutions or suspensions were prepared immediately before each experiment. 2.2. Animals Fig. 5. Concentrations of nicorandil and NHN in the pleural exudate after treatment of mice with nicorandil. Nicorandil (25, 50 and 100 mg/kg, p.o.) was administered 0.5 h before and 1 h after i.pl. injection of carrageenan (200 μg). Pleural exudate was collected 4 h after injection of carrageenan. n¼ 6.

biotransformation of nicorandil (César et al., 2011; Frydman et al., 1989). In addition, we also evaluated whether inhibition of cytokines and eicosanoids production mediates the effect induced by nicorandil on neutrophil recruitment.

Female Swiss mice (25–30 g) were used throughout the study and had free access to food and water. The animals were kept in a room with 12 h light–dark cycle and temperature of 27 °C, which corresponds to the thermoneutral zone for rodents, for at least 3 days before the experiment to allow acclimatization. This study was approved by the Ethics Committee in Animal Experimentation of the Federal University of Minas Gerais (Protocol 106/14) and all experiments were conducted according to the ethical guidelines for investigation of experimental pain in conscious animals



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Fig. 6. Concentrations of IL-1β (A), CXCL-1 (C) and IL-10 (E) in the pleural exudate collected at 2, 4 and 6 h after i.pl. injection of carrageenan (Cg, 200 g) or vehicle (PBS, 0.1 ml). Effects induced by nicorandil (NIL; 25, 50 or 100 mg/kg, p.o.) or vehicle (0.5% CMC; p.o.) on the concentrations of IL-1β (B), CXCL-1 (D) and IL-10 (F) in the pleural exudate collected 2 h after i.pl. injection of carrageenan. Mice were treated with nicorandil or vehicle 0.5 h before and 1 h after the i.pl. injection of Cg. (A, C and E) *** significantly different from PBS (P o 0.001). (B, D and F) ## and ### significantly different from CMC þPBS (P o 0.01 and Po 0.001, respectively). * and *** significantly different from CMC þCg (P o0.05 and Po 0.001, respectively). n ¼6.

(Zimmermann, 1983). 2.3. Leukocyte recruitment to the pleural cavity induced by carrageenan The leukocyte recruitment to the pleural cavity was evaluated according to Klein et al. (2001). Briefly, carrageenan (200 g/0.1 ml) or vehicle (PBS/0.1 ml) were injected via the i.pl. route and the animals were euthanized in a CO2 chamber 2, 4 or 6 h later. The cells present in the cavity were harvested by injecting 2 ml of PBS followed by collection of the exudate. Total cell counts were

performed in a modified Neubauer chamber using Turk's stain. Differential cell counts were performed on cytospin preparations, which were stained with May–Grunwald and Giemsa to identify cell types according to standard morphological criteria. 2.4. Effects induced by nicorandil, nicotinamide or NHN on the leukocyte recruitment induced by carrageenan To investigate the effect induced by nicorandil on the leukocyte recruitment induced by carrageenan (200 μg/0.1 ml, i.pl.), mice were treated p.o. with nicorandil (25, 50 or 100 mg/kg),



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the concentrations of cytokines and PGE2, respectively. The samples were immediately frozen and stored at 70 °C until the assays were performed. The samples, diluted in PBS (1:3), were used to determine IL-1β, IL-10, CXCL-1 and PGE2 concentrations by ELISA, following the instructions supplied by the manufacturer (R&D Systems, Minneapolis, USA). The results were expressed as pg/ml. 2.7. Statistical analysis Data were presented as mean 7standard error of the mean (S. E.M.). Groups were compared by one-way ANOVA and differences between groups were assessed with the Newman–Keuls post-test. A P value o0.05 was considered significant and statistical analysis was conducted using GraphPad Prism 5.0 for Windows.

Fig. 7. Effect induced by nicorandil (NIL; 25, 50 or 100 mg/kg, p.o.) or vehicle (0.5% CMC, p.o.) on the concentration of PGE2 in the pleural exudate collected 4 h after i. pl. injection of carrageenan (Cg, 200 g) or vehicle (PBS, 0.1 ml). Mice were treated with nicorandil or vehicle 0.5 h before and 1 h after the i.pl. injection of Cg. ### significantly different from CMC þ PBS (P o 0.001). * and ** significantly different from CMC þ Cg (P o 0.05 and Po 0.01, respectively). n¼ 6.

nicotinamide (58 mg/kg), NHN (79 mg/kg) or vehicle (CMC 0.5%, 8 ml/kg) twice, 0.5 h before and 1 h after injection of the inflammatory stimulus. In additional experiments, the animals were only pretreated (0.5 h) or post-treated (1 h) with nicorandil (25, 50 or 100 mg/kg) or vehicle (CMC 0.5% w/v, 8 ml/kg). The doses of nicotinamide (58 mg/kg) and NHN (79 mg/kg) were equimolar to the highest dose of nicorandil (100 mg/kg). The number of infiltrating leukocytes was evaluated 4 h after the injection of carrageenan. 2.5. Quantification of nicorandil and NHN in the pleural exudate by liquid chromatography–electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) Nicorandil was administered p.o. at the doses of 25, 50 or 100 mg/kg, 0.5 h before and 1 h after the i.pl. injection of carrageenan. The animals were euthanized in a CO2 chamber 4 h after i. pl. injection of the inflammatory stimulus. After injecting 2 ml of PBS into the pleural cavity, a sample of the pleural exudate from each animal was collected in a tube and immediately centrifuged at 48,294 G for 10 min. Then, the supernatant was transferred to plastic tubes and stored at 70 °C until analyses. The method used for the quantification of nicorandil and NHN in pleural exudate samples was adapted from that developed and validated by César et al. (2011) for plasma samples. The mobile phase consisted of methanol and aqueous ammonium acetate containing 2 mM formic acid 0.03% (v/v) (33:67 v/v) in the flow rate of 1 ml/min. The injection volume was 50 μl. The linear ranges of concentrations were: 25–75 μg/ml (nicorandil) and 25–50 μg/ml (NHN). 2.6. Effects induced by nicorandil on the production of IL-1β, IL-10, CXCL-1 and PGE2 induced by carrageenan Supernatants obtained from pleural exudates collected at different times after i.pl. injection of carrageenan or PBS were used to determine IL-1β, IL-10, CXCL-1 and PGE2 concentrations. Initially, we evaluated the concentrations of the cytokines at 2, 4 and 6 h after carrageenan or PBS injection to determine the best moment to collect the pleural exudate samples in the next experiments. After that, mice were treated with nicorandil (100 mg/kg), 0.5 h before and 1 h after the i.pl. injection of carrageenan (200 μg/ cavity). The pleural exudate samples were collected as previously described 2 h or 4 h after injection of carrageenan to determine

3. Results 3.1. Effects induced by nicorandil, nicotinamide or NHN on the leukocyte recruitment As shown in Fig. 1, an accumulation of leukocytes was observed 2, 4 and 6 h after i.pl. injection of carrageenan. Neutrophils represented approximately 90% of the accumulated cells. As the accumulation of neutrophils was more consistent 4 h after injection of the inflammatory stimulus, we collected samples of pleural exudate at this time in the subsequent experiments to evaluate the effects induced by nicorandil, nicotinamide or NHN. Treatment with nicorandil (25, 50 or 100 mg/kg), 0.5 h before and 1 h after the injection of carrageenan, markedly inhibited the neutrophil recruitment (Fig. 2). However, nicotinamide (58 mg/kg; Fig. 2) and NHN (79 mg/kg; Fig. 2), at doses equimolar to the highest dose of nicorandil (100 mg/kg), failed to inhibit the neutrophil recruitment induced by carrageenan. The activity of nicoradil was also demonstrated after treatment with a single dose. As shown in Fig. 3, previous (0.5 h) administration of nicorandil (50 or 100 mg/ kg, p.o.) reduced the neutrophil recruitment induced by carrageenan. The neutrophil recruitment was inhibited even when nicorandil (100 mg/kg, p.o.) was administered 1 h after the injection of carrageenan (Fig. 4). 3.2. Concentrations of nicorandil and NHN in the pleural exudate after treatment with nicorandil After p.o. administration of nicorandil (25, 50 and 100 mg/kg, p. o., 0,5 h before and 1 h after carrageenan), its concentrations in the pleural exudate samples increased in a dose-dependent manner. NHN was also detected in the samples, but its concentrations were much lower than those of nicorandil (Fig. 5). 3.3. Effects induced by nicorandil on the production of IL-1β, IL-10, CXCL-1 and PGE2 Concentrations of cytokines in the pleural exudate increased 2 h (IL-1β, CXCL-1, IL-10), 4 h (IL-1β, IL-10) and 6 h (IL-1β) after injection of carrageenan (Fig. 6A, C and E). Nicorandil (25, 50 or 100 mg/kg), administered 0.5 h before and 1 h after the injection of carrageenan, markedly reduced the concentrations of IL-1β (Fig. 6B) and CXCL-1 (Fig. 6D), but did not alter the concentration of IL-10 (Fig. 6F), in the pleural exudate collected 2 h after injection of the inflammatory stimulus. Concentration of PGE2 in the pleural exudate also increased 4 h after injection of carrageenan. Nicorandil (25, 50 or 100 mg/kg), administered 0.5 h before and 1 h after the injection of carrageenan, markedly reduced the concentrations of PGE2 (Fig. 7).



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4. Discussion We have previously demonstrated that nicotinamide markedly inhibits the neutrophil recruitment in experimental models of pleurisy induced by carrageenan, CXCL-8 and LTB4 in mice (Ferreira et al., 2012). Now, we demonstrate that nicorandil, a nicotinamide derivative with a nitrate group, also inhibits the neutrophil recruitment in an experimental model of pleurisy and exhibits higher potency than nicotinamide. The model of pleurisy induced by carrageenan is widely used to evaluate the leukocyte recruitment, particularly neutrophils, and the production of mediators that play important roles in acute inflammation (Fröde et al., 2001, 2002; Luchese et al., 2012). The highest leukocyte accumulation observed 4 h after i.pl. injection of carrageenan and characterized predominantly by neutrophils, as demonstrated in the present study, support results of other studies (Saleh et al., 1996; Sampaio et al., 2000; Ferreira et al., 2012, 2013). The neutrophil recruitment induced by carrageenan was markedly inhibited by a double treatment with nicorandil. This protocol was initially chosen considering the treatment schedule with nicotinamide used in our previous study (Ferreira et al., 2012) and also the pharmacokinetic profile of nicorandil (Dutra et al., 2013). The great activity of nicorandil is further demonstrated by its ability to reduce neutrophil recruitment after a single administration, even when the administration occurred after injection of carrageenan. As nicotinamide and NHN may be formed during biotransformation of nicorandil (Frydman et al., 1989; César et al., 2011), we also investigated the effects induced by the two first molecules. However, nicotinamide and NHN, at doses equimolar to 100 mg/kg of nicorandil, did not affect the neutrophil recruitment induced by carrageenan. We have previously shown that nicotinamide exhibits activity in this experimental model of inflammation, but at doses much higher than those of nicorandil (Ferreira et al., 2012). The lack of activity of NHN in this model and also in the model of nociceptive response induced by formaldehyde (Dutra et al., 2013) may be associated with its lower concentrations in the plasma (Dutra et al., 2013) and in the pleural exudate (present study) after administration of nicorandil. Whether higher doses of NHN induce antinociceptive and anti-inflammatory effects is a matter of future investigation. The results of the present study represent the first demonstration of the effect induced by nicorandil on leukocyte recruitment in a model of acute inflammation induced by an exogenous stimulus. The studies that provided the closest results investigated the effect induced by nicorandil on the neutrophil recruitment in experimental models of ischemia, in line with its main clinical use, the treatment of patients with ischemic heart diseases. In these studies, it was demonstrated that nicorandil reduces the neutrophil recruitment to pig myocardium (Galiè et al., 1995) or newborn rat kidney (Zhang et al., 2013) in models of ischemia– reperfusion injury. Next, we evaluated the effect induced by nicorandil on the production of some inflammatory mediators that play important roles in neutrophil recruitment. The rationale underlying this evaluation comes from studies demonstrating that nicorandil inhibit biochemical pathways with crucial role to the development of the inflammatory response. Nicorandil attenuates NF-kappaB activation and the production of tumor necrosis factor-α (TNF-α), IL-6 and IL-8 in blood samples from patients with coronary artery bypass surgery (Kawamura et al., 2005) and inhibits the inflammasome activation and Toll-like receptor-4 signal transduction induced by oxygen–glucose deprivation in rodent primary microglia culture (Zhao et al., 2014). In the present study, we demonstrated that nicorandil markedly reduced the concentrations of IL-1β, CXCL-1 and PGE2 in the pleural exudate. Although the molecular targets to which

nicorandil interacts to induce such effects are not known, it may be suggested that inhibition of the inflammasome, as demonstrated by Zhao et al. (2014), may play an important role. The inflammasomes are intracellular multi-protein complexes that activates inflammatory caspases, which proteolytically activates inflammatory cytokines. The inhibition of the inflammasomes may result in the reduced production of inflammatory cytokines, such as IL-1β and IL-18 (Lamkanfi and Dixit, 2014; Guo et al., 2015) and other inflammatory mediators whose production may be stimulated by these cytokines. The reduction of IL-1β, CXCL-1 and PGE2 in the pleural exudate of animals treated with nicorandil may underlie its marked effect on neutrophil recruitment. It has been shown that IL-1β (Perretti et al., 1993; Goodman et al., 1993; Matsukawa et al., 1997), CXCL-1 (Vieira et al., 2009; De Filippo et al., 2013) and PGE2 (Lemos et al., 2009) play important roles in leukocyte recruitment in different models of inflammation. Although IL-10 has been shown to inhibit neutrophil recruitment in a model of pleurisy induced by methylated bovine serum albumin in mice (Fine et al., 2003), nicorandil failed to increase the concentrations of this cytokine in the pleural exudate. Thus, the effect induced by nicorandil in our experimental model may not be attributed to an increase in the production of IL-10. The demonstration of the inhibitory effect induced by nicorandil on neutrophil recruitment and production of inflammatory mediators may contribute to better understand important activities of this drug, some of clinical relevance. Benefits provided by nicorandil that may be related to the findings of the present study include protection of the myocardium in ischemic conditions (Kawamura et al., 2005), protection of the liver against the consequences of excessive fat accumulation (Elshazly, 2015), reduction of oxidative stress induced by cardiac surgery (Galiñanes et al., 2008) and reduction of lesions in models of ischemia–reperfusion injury (Galiè et al., 1995; Zhang et al., 2013). In all these conditions, an excessive inflammatory response results in increased tissue lesion, thus contributing to negative prognosis and delayed recovery.

5. Conclusion In conclusion, we demonstrated the marked activity of nicorandil in a model of pleurisy induced by carrageenan. This activity was characterized by reduction of neutrophil accumulation in the pleural exudate, associated with inhibition of production of inflammatory cytokines and eicosanoids. The effects induced by nicorandil on the leukocytes recruitment and production of inflammatory mediators contribute to a better understanding of its clinical benefits and indicate that these benefits may be due to its vasodilating and anti-inflammatory activities.

Acknowledgments We thank Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG, Proc. Nr. APQ-02466-14), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Programa Institucional de Auxílio à Pesquisa de Doutores RecémContratados da PRPq/Universidade Federal de Minas Gerais for financial support. This study was supported by a scholarship from PROBIC/FAPEMIG (I.S.F. Melo). References César, I.C., Bastos, L.F., Godin, A.M., Coelho, M.M., Araujo, D.P., de Fátima, Â., Guidine, P.A., Pianetti, G.A., 2011. Simultaneous quantitation of nicorandil and its


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