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Nicorandil inhibits mechanical allodynia induced by paclitaxel by activating opioidergic and serotonergic mechanisms
Author’s Accepted Manuscript Nicorandil inhibits mechanical allodynia induced by paclitaxel by activating opioidergic and serotonergic mechanisms Marcela I. Morais, Felipe F. Rodrigues, Sarah O.A.M. Costa, Franciele A. Goulart, Fábio C. Costa, Ivo S.F. Melo, Paulo S.A. Augusto, Marcela M.G.B. Dutra, Ângelo de Fátima, Márcio M. Coelho, Renes R. Machado
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S0014-2999(18)30089-X https://doi.org/10.1016/j.ejphar.2018.02.014 EJP71667
To appear in: European Journal of Pharmacology Received date: 19 December 2017 Revised date: 7 February 2018 Accepted date: 9 February 2018 Cite this article as: Marcela I. Morais, Felipe F. Rodrigues, Sarah O.A.M. Costa, Franciele A. Goulart, Fábio C. Costa, Ivo S.F. Melo, Paulo S.A. Augusto, Marcela M.G.B. Dutra, Ângelo de Fátima, Márcio M. Coelho and Renes R. Machado, Nicorandil inhibits mechanical allodynia induced by paclitaxel by activating opioidergic and serotonergic mechanisms, European Journal of Pharmacology, https://doi.org/10.1016/j.ejphar.2018.02.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Nicorandil inhibits mechanical allodynia induced by paclitaxel by activating opioidergic and serotonergic mechanisms
Marcela I. Morais1, Felipe F. Rodrigues1, Sarah O. A. M. Costa1, Franciele A. Goulart1, Fábio C. Costa1, Ivo S. F. Melo1, Paulo S. A. Augusto1, Marcela M. G. B. Dutra1,3, Ângelo de Fátima2, Márcio M. Coelho1, Renes R. Machado1,*
1
Departamento de Produtos Farmacêuticos, Faculdade de Farmácia, Universidade
Federal de Minas Gerais, Avenida Antônio Carlos, 6627, CEP 31270-901, Belo Horizonte, MG, Brazil 2
Departamento de Química, Instituto de Ciências Exatas, Universidade Federal de
Minas Gerais, Avenida Antônio Carlos, 6627, CEP 31270-901, Belo Horizonte, MG, Brazil 3
Centro Universitário Newton Paiva, Avenida Silva Lobo, 1730, CEP 30460-000, Belo
Horizonte, MG, Brazil.
*
Corresponding author: Departamento de Produtos Farmacêuticos, Faculdade de
Farmácia, Universidade Federal de Minas Gerais, Avenida Antônio Carlos, 6627, 31270-901, Belo Horizonte, MG, Brazil. Tel.: + 55 31 3409 6965, fax: + 55 31 3409 6935; [email protected] ABSTRACT
Recently, we demonstrated that nicorandil exhibits activities in models of inflammatory and nociceptive pain. In the present study, we extended this investigation by evaluating the effects of nicorandil in models of neuropathic pain induced by paclitaxel or nerve injury in mice. Four intraperitoneal (i.p.) injections of paclitaxel (2 mg/kg.day, cumulative dose 8 mg/kg) or chronic constriction injury (CCI) of the sciatic nerve induced a long lasting mechanical allodynia. Per os (p.o.) administration of two doses of
nicorandil (50, 100 and 150 mg/kg) on the 14th day after the first paclitaxel injection attenuated the mechanical allodynia. Equimolar doses of nicotinamide (86.7 mg/kg, p.o.) or nicotinic acid (87.7 mg/kg, p.o.) were devoid of effect. Mechanical allodynia induced by CCI was also attenuated by p.o. administration of two doses of nicorandil (150 mg/kg) on the 14th day after nerve injury. Nicorandil (50, 100 and 150 mg/kg, p.o.) did not affect motor activity. The antinociceptive activity of nicorandil in the model of mechanical allodynia induced by paclitaxel was partially attenuated by naltrexone (5 and 10 mg/kg, i.p.) or cyproheptadine (5 and 10 mg/kg, i.p.), but not by glibenclamide (20 and 40 mg/kg, p.o.). Concluding, nicorandil exhibits activity in experimental models of neuropathic pain when mechanical allodynia is fully established. Activation of opioidergic and serotonergic pathways mediates the antinociceptive activity of nicorandil. It is unlikely that this activity requires biotransformation to nicotinamide or nicotinic acid. Nicorandil should be further evaluated aiming to identify a new alternative in the pharmacological management of neuropathic pain.
Keywords: Pain; Neuropathic pain; Nicorandil; Paclitaxel; Chronic constriction injury.
1. Introduction
Neuropathic pain is triggered by injuries or diseases affecting the central and peripheral nervous systems. This modality of chronic pain represents a manifestation of maladaptive plasticity in the nervous system and the affected individuals may report spontaneous lancinating, shooting and/or burning pain, tingling sensation and amplified responses to noxious (hyperalgesia) and innocuous (allodynia) stimuli (Woolf, 2004; Costigan et al., 2009; Jensen et al., 2011).
Although the commonly used pharmacotherapy of neuropathic pain includes many drugs such as antiepileptics, antidepressants (tricyclic antidepressants, selective serotonin reuptake inhibitors, serotonin-norepinephrine reuptake inhibitors), opioid analgesics and local anesthetics, shortcomings are well recognized as many patients do not experience sufficient relief (Baron et al., 2010). The reduced efficacy of the available drugs may be associated with the heterogeneity of causes, symptoms and underlying mechanisms of neuropathic pain (Beniczky et al., 2005). Acknowledging the shortcomings of the current available pharmacotherapy of neuropathic pain prompts investigation of other alternatives. Among the alternatives that have been evaluated aiming to identify new therapies that promote the relief of neuropathic pain is drug repositioning. This involves the identification of new functions of clinical approved drugs thus allowing the development of new and safe pharmacotherapies in shorter period of time and with lower costs (Ashburn and Thor, 2004). Nicorandil, a nicotinamide nitrate derivative (2-nicotinamide ethyl nitrate), is a vasodilator drug that has been approved is some countries for the treatment of patients with angina pectoris (Frampton et al., 1992; El-Moselby et al., 2009). It has been suggested that release of nitric oxide (NO), activation of guanylyl cyclase and opening of ATP-dependent potassium channels may contribute to the vasodilator activity of nicorandil (Kukovetz et al., 1992; Yasuda et al., 2001; Barbato, 2005). Although the vasodilation may represent an important activity contributing to the pain relief in patients with myocardial ischemia (Abrams, 1985; Fung, 1993), it has been acknowledged that NO donors may also exhibit an intrinsic analgesic activity. Central or peripheral administration of NO donors exhibit antinociceptive and anti-hyperalgesic effects in different experimental models of pain that are not associated with ischemia (Duarte et al., 1990; Ferreira et al., 1992; Soares et al., 2000; Sousa and Prado, 2001). Recently, we demonstrated that nicorandil, as other NO donors, also exhibits activities in models of inflammatory and nociceptive pain (Dutra et al., 2013; Cesar et al., 2014; Dutra et al., 2015). Activation of guanylyl cyclase (Dutra et al., 2013) and opioid receptors (Dutra et al., 2015) are suggested as mechanisms that could mediate the antinociceptive activity of nicorandil in these models. In the present study, we extended the investigation on the antinociceptive activity of nicorandil by evaluating its effects in the models of neuropathic pain induced by paclitaxel or nerve injury in mice and also evaluated the possible mechanisms involved in the antinociceptive activity of this drug.
2. Materials and methods
2.1. Animals
Male Swiss mice (25–30 g) were kept at home cage with food and water ad libitum except when the protocol required per os (p.o.) administration. In this case, the animals were subjected to food restriction of at least 10 h before the experiment. Three days before the experiments, 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. The Ethics Committee on Animal Experimentation of the Federal University of Minas Gerais (Protocol 339/2015) approved the study and ethical guidelines for investigation of experimental pain in conscious animals were followed (Zimmermann, 1983).
2.2. Drugs
Nicorandil (purity >99.0%) was synthesized at the Department of Chemistry, Federal University of Minas Gerais. Nicotinamide, nicotinic acid, naltrexone, glibenclamide, cyproheptadine, carboxymethylcellulose (CMC), paclitaxel (SigmaAldrich, USA), phenobarbital (Sanofi-Aventis, Brazil), ketamine and xylazine (Ceva Sante Animale, Brazil) were obtained commercially. Solutions of paclitaxel, phenobarbital, naltrexone, glibenclamide and cyproheptadine were prepared in sterile saline. Suspensions of nicorandil, nicotinamide and nicotinic acid were prepared in CMC (0.5% w/v in sterile saline). All solutions or suspensions were prepared immediately before each experiment.
2.3. Mechanical allodynia induced by paclitaxel
Paclitaxel was used to induce a sensitization state thus mimicking a model of neuropathic pain induced by an antineoplastic (Smith et al., 2004). Paclitaxel (2 mg/kg.day, cumulative dose 8 mg/kg) or saline (2 ml/kg; control group) were administered intraperitoneally (i.p.) on four alternate days (days 1, 3, 5 and 7).
2.4. Mechanical allodynia induced by chronic constriction injury (CCI) of the sciatic nerve
The method described by Bennet and Xie (1988), with adaptations to mice, was used. Briefly, in mice anesthetized with ketamine (90 mg/kg, i.p.) plus xylazine (9 mg/kg, i.p.), three ligatures of 5.0 chromic gut (Brasuture, Brazil) were tightly tied around the sciatic nerve at the level of the midthigh. In sham-operated mice, the same surgical procedure was followed, except the ligatures of the nerve.
2.5. Evaluation of the mechanical allodynia
An electronic von Frey apparatus (Model EFF 301, Insight, Brazil) was used to evaluate mechanical allodynia. After acclimatization of the animals to the experimental apparatus (1 h/day during 2 days), the basal paw withdrawal threshold of each animal was determined (mean of three measurements). The animals were divided into the experimental groups in such a way that the mean withdrawal thresholds of the different groups were similar. The paw withdrawal threshold of each animal was measured every 2 days during 14 days after the first injection of paclitaxel or CCI.
2.6. Evaluation of the effects induced by nicorandil, nicotinamide or nicotinic acid on the mechanical allodynia induced by paclitaxel
Initially, nicorandil (50, 100 and 150 mg/kg) or vehicle (CMC 0.5%, 8 ml/kg) were administered p.o. on the 14th day after the first paclitaxel injection. The animals received two doses of nicorandil or CMC at an interval of 2 h (0 h and 2 h). Two doses of nicorandil were used as its effect is not long lasting (Dutra et al., 2015). Mechanical allodynia was evaluated 1, 3, 5 and 7 h after the first dose of nicorandil. In another protocol, the effects induced by nicorandil were compared to those induced by nicotinamide and nicotinic acid. The animals received two doses of nicorandil (150 mg/kg) or two equimolar doses of nicotinamide (86.7 mg/kg) or nicotinic acid (87.7 mg/kg), at an interval of 2 h, on the 14th day after the first paclitaxel injection. Mechanical allodynia was evaluated 1, 3, 5 and 7 h after the first dose of nicorandil, nicotinamide or nicotinic acid.
2.7. Evaluation of the effects induced by nicorandil on the mechanical allodynia induced by CCI
Nicorandil (150 mg/kg) or vehicle (CMC 0.5%, 8 ml/kg) were administered p.o. on the 14th day after CCI. The animals received two doses of nicorandil at an interval of 2 h (0 h and 2 h). Mechanical allodynia was evaluated 1, 3, 5 and 7 h after the first dose of nicorandil.
2.8. Evaluation of the effects induced by naltrexone, cyproheptadine or glibenclamide on the antiallodynic activity of nicorandil
Nicorandil (150 mg/kg) or vehicle (CMC 0.5%, 8 ml/kg) were administered p.o. on the 14th day after the first paclitaxel injection. The animals received two doses of nicorandil at an interval of 2 h (0 h and 2 h). To evaluate the mechanisms mediating the antiallodynic activity of nicorandil, naltrexone (5 or 10 mg/kg, i.p., -15 min), cyproheptadine (5 or 10 mg/kg, i.p., -15 min), glibenclamide (20 or 40 mg/kg, p.o., -30 min) or vehicle (saline 2 ml/kg, i.p., -15 min or saline 8 ml/kg, p.o., -30 min) were administered before the first dose of nicorandil (150 mg/kg, p.o.). Mechanical allodynia was evaluated 1, 3, 5 and 7 h after the first dose of nicorandil.
2.9. Evaluation of the effects induced by nicorandil on the motor activity
The motor activity of the animals was evaluated on a rota-rod apparatus. Two days before the evaluation, the animals were trained on the apparatus. To evaluate the motor activity, the animals were placed on the rota-rod (14 rpm) and the time they spent on it was determined. The cut-off time was 120 s. After obtaining baseline values, the animals received two doses of nicorandil (50, 100 or 150 mg/kg, p.o) or vehicle (CMC 0.5%; 8 ml/kg) at an interval of 2 h. Another group of animals was treated with a single dose of phenobarbital (50 mg/kg, p.o.; positive control). Motor activity was evaluated 1, 3, 5 and 7 h after the first dose of nicorandil or phenobarbital.
2.10. Statistical analysis
Results were expressed as mean ± standard error of the mean. Results were analyzed by using two-way ANOVA followed by Bonferroni post test (temporal course of the mechanical allodynia induced by paclitaxel or CCI) or one-way ANOVA followed by Dunnett's test (area under the curve of the mechanical allodynia induced by paclitaxel and motor activity). A P < 0.05 was considered significant. Statistical analysis was conducted using GraphPrism 5.0 for windows.
3. Results
3.1. Mechanical allodynia induced by paclitaxel
Mechanical allodynia was already observed two days after the first administration of paclitaxel and maintained throughout the experimental period of 14 days. Mechanical nociceptive threshold reduced from approximately 7.5 g to approximately 3.5 g on the 12th day after the first injection of paclitaxel. Sterile saline (control group) did not affect the nociceptive threshold (Fig. 1).
3.2. Effect induced by nicorandil, nicotinamide or nicotinic acid on the mechanical allodynia induced by paclitaxel Nicorandil (50, 100 and 150 mg/kg) was administered p.o. on the 14th day after the first paclitaxel injection. The animals received two doses of nicorandil at an interval of 2 h (0 h and 2 h). The three doses of nicorandil attenuated the mechanical allodynia induced by paclitaxel. The highest dose attenuated the mechanical allodynia at all evaluated times. The intermediate dose attenuated the mechanical allodynia at 1, 3 and 5 h, while the lowest dose induced an effect only at 1 and 3 h. CMC 0.5% did not affect the mechanical allodynia induced by paclitaxel (Fig. 2). In another experiment, the effect induced by nicorandil (150 mg/kg, p.o.) was compared to those induced by equimolar doses of nicotinamide (86.7 mg/kg, p.o.) or nicotinic acid (87.7 mg/kg, p.o.). While nicorandil, once again, attenuated the mechanical allodynia induced by paclitaxel at all evaluated times, nicotinamide and nicotinic acid were devoid of effect (Fig. 3).
3.3. Mechanical allodynia induced by CCI
Mechanical allodynia was already observed two days after the sciatic nerve injury and maintained throughout the experimental period of 14 days. Mechanical nociceptive threshold reduced from approximately 6 g to approximately 2 g after CCI. In shamoperated animals, the mechanical nociceptive threshold reduced from approximately 6 to approximately 5 g (Fig. 4).
3.4. Effect induced by nicorandil on the mechanical allodynia induced by CCI Nicorandil (150 mg/kg) was administered p.o. on the 14th day after the sciatic nerve injury. The animals received two doses of nicorandil at an interval of 2 h (0 h and 2 h). Nicorandil attenuated the mechanical allodynia at 1 and 5 h. CMC 0.5% did not affect the mechanical allodynia induced by CCI (Fig. 5).
3.5. Effect induced by nicorandil on the motor activity
To investigate whether the inhibition of the nociceptive behavior induced by nicorandil is not the result of a motor impairment, we evaluated the effect induced by the drug on the motor activity of the animals on the rota-rod apparatus. Nicorandil (50, 100 and 150 mg/kg, p.o.) was administered p.o. twice at an interval of 2 h. The motor activity was not changed 1, 3, 5 and 7 h after the first dose of nicorandil. However, a single dose of phenobarbital (50 mg/kg, p.o.) markedly reduced the time mice spent in the apparatus (Table 1).
3.6. Effects induced by naltrexone, cyproheptadine or glibenclamide on the antiallodynic activity of nicorandil
To investigate putative mechanisms mediating the antiallodynic activity of nicorandil, opioidergic (naltrexone) and serotonergic (cyproheptadine) antagonists and an ATP-dependent potassium channel blocker (glibenclamide) were used. Mechanical allodynia was induced by paclitaxel, as previously described, and evaluated 14 days after the first injection. Nicorandil (150 mg/kg, p.o., two doses 2 h apart) reduced the mechanical allodynia induced by paclitaxel. The antinociceptive activity of nicorandil was partially attenuated by previous (15 min) administration of naltrexone (5 and 10
mg/kg, i.p.; Fig. 6) or cyproheptadine (5 and 10 mg/kg, i.p.; Fig. 7), but not by previous (30 min) administration of glibenclamide (20 and 40 mg/kg, p.o.; Fig. 8) A single dose of naltrexone (10 mg/kg, p.o.; Fig. 6), cyproheptadine (10 mg/kg, p.o.; Fig. 7) or glibenclamide (40 mg/kg, p.o.; Fig. 8), per se, failed to affect the mechanical allodynia induced by paclitaxel.
4. Discussion
Neuropathic pain is highly refractory to many of the available analgesic drugs and its proper pharmacological management still represents a challenge (Gilron et al., 2015). Thus, preclinical and clinical investigations of new drugs that may provide adequate and safe relief of this debilitating category of pain are warranted. In the present study, we provide preliminary results indicating that nicorandil may be a promising drug to be investigated aiming to identify a new alternative in the pharmacological management of neuropathic pain. To investigate new treatments that may provide relief of neuropathic pain, experimental models that mimic the marked features of this condition are essential. Repeated injections of paclitaxel have been shown to induce a painful peripheral neuropathy in rodents characterized by mechanical allodynia and thermal hyperalgesia (Polomano et al., 2001), thus mimicking the neuropathic pain that may occur in cancer patients treated with this chemotherapeutic drug (Scripture et al., 2006). We reproduced such results by demonstrating that repeated injections of paclitaxel induced a long lasting mechanical allodynia in mice that was already established two days after the first injection. It has been shown that a variety of clinical approved drugs, including pregabalin (Ito et al., 2012; Salat et al., 2014), gabapentin (Ledeboer et al., 2006), duloxetine, mexiletine (Ito et al., 2012), morphine, methadone (Pascual et al., 2010), etodolac (Ito et al., 2012) and leflunomide (Brito et al., 2017), among others, may inhibit the sensitization induced by paclitaxel in rodents. In the present study, we demonstrated that the nicorandil, a NO donor that has been approved for the treatment of patients with myocardial ischemia, also inhibits the mechanical allodynia induced by paclitaxel or nerve injury in mice. As there is evidence that nicorandil (Gantenbein et al., 1995), as well as other potassium channel openers (Kobayashi et al., 2008), may exhibit anticonvulsant activity in experimental models, suggesting a central depressant effect, we investigated the
effect induced by nicorandil on the performance of the animals on the rotating rod. This is important to evaluate whether the activity of nicorandil is not the result of a confounding effect on motor coordination or muscle tone. As the three doses of nicorandil used in the present study did not change the motor coordination of the animals, it is unlikely that inhibition of the nociceptive behavior is due to motor incoordination or muscle relaxing effect. Although we have recently demonstrated that nicorandil exhibits activity in models of nociceptive and inflammatory pain (Dutra et al., 2013; Cesar et al., 2014; Dutra et al, 2015), this is the first demonstration that this nicotinamide nitrate derivative also exhibits activity in a well known model of neuropathic pain induced by a chemotherapeutic drug. The antiallodynic effect induced by nicorandil was not restricted to the model of neuropathic pain induced by paclitaxel, but was also observed in the model of neuropathic pain induced by a different stimulus, the chronic constriction of the sciatic nerve. Remarkably, in both models, the antiallodynic effect induced by nicorandil occurred when the sensitization was fully established and after acute administration. The neuropathic pain induced by paclitaxel has been associated with a variety of changes including increased expression of inflammatory mediators that can induce cascading effects on glial cells and neurons, in addition to altered activity of ion channels, neurotransmission and intracellular signaling and disrupted cell structures (Scripture et al., 2006; Boyette-Davis et al., 2015). A myriad of changes also seems to underlie the increased sensitization induced by nerve injury, such as abnormalities in injured and uninjured afferents supplying the lesion site, central sensitization, altered glial cells activity, reduced central inhibition and altered autonomic nervous system (Campbell and Meyer, 2006; Nickel et al., 2012). Thus, the antiallodynic effect induced by nicorandil may result from the action at different targets and may occur directly or indirectly. Nicorandil is vasodilator drug that results from the coupling of a NO donor to nicotinamide, a molecule whose activity in experimental models of pain has been demonstrated (Stevens et al., 2007; Godin et al., 2011). Nicorandil goes through to extensive hepatic metabolism and the main biotransformation pathway leads to formation of nicotinamide and nicotinic acid (Frydman et al., 1989). It has been recently suggested that these two molecules may mediate a known side effect induced by nicorandil, the skin and oral lesions observed in patients under prolonged treatment
(Trechot et al., 2015). Thus, a hypothesis that may be put forward is that nicorandil induces its effects indirectly by releasing nicotinamide or nicotinic acid in vivo. However, this hypothesis is unlikely as doses of nicotinamide (86.7 mg/kg) and nicotinic acid (87.7 mg/kg) equimolar to the highest dose of nicorandil (150 mg/kg) used in the present study were devoid of effect. We have previously shown that both nicotinamide (Godin et al., 2011; Dutra et al., 2013) and nicorandil (Dutra et al., 2013) induce antinociceptive effect in models of nociceptive and inflammatory pain. However, in these models nicotinamide induced antinociceptive effect when administered at doses much higher than those of nicorandil. Similarly, higher doses of nicotinic acid are needed to induce effects in these models (Godin et al., 2012). Whether nicotinamide and nicotinic acid in higher doses also induce antinociceptive effects in models of neuropathic pain is a question that has to be investigated, but the results of the present and previous (Godin et al., 2011; Dutra et al., 2013) studies clearly indicate that the coupling of a NO donor to nicotinamide greatly increases the activity of the resulting molecule, nicorandil. Although nicorandil is a NO donor, results from our previous study (Dutra et al., 2013) provided mixed cues about the role of this gaseous transmitter in the antinociceptive activity this drug in models of inflammatory and nociceptive pain. It seems that NO does not appear to be an essential mediator of the antinociceptive activity of nicorandil in the model of nociceptive response induced by formaldehyde. Many studies have shown that nicorandil exhibits different activities when compared to other NO donors (Taira, 1987; Edwards and Weston, 1990; Greenberg et al., 1991). In experimental models of neuropathic pain, NO donors have also provided mixed results. Some studies have shown that NO donors may increase (Naik et al., 2006) or reduce (Chen et al., 2000) mechanical allodynia induced by nerve injury in rodents, while others have shown that the effects induced by these drugs may be dose related (Souza and Prado, 2001). Thus, the role of NO in the antinociceptive activity of nicorandil and related compounds in models of neuropathic pain is still not clear. We also investigated other mechanisms that could contribute to the antiallodynic activity of nicorandil in the neuropathic pain model induced by paclitaxel. Previous administration of naltrexone partially attenuated the antiallodynic activity of nicorandil, indicating that it depends in part on the activation of opioidergic pathways. It is not clear whether nicorandil directly interacts with opioid receptors or stimulates the release of endogenous opioid peptides, actions that could contribute to its antinociceptive
activity. We have also shown that the activity of nicorandil in the model of nociceptive response induced by formaldehyde, a mixed model of nociceptive and inflammatory pain, is attenuated by an opioid antagonist (Dutra et al., 2015). However, as naltrexone is a non-selective opioid receptor antagonist (Crabtree, 1984), no conclusion about the role of a specific opioid receptor in the antinociceptive activity of nicorandil in both models of pain may be put forward. We also demonstrated that cyproheptadine, a nonselective serotonergic antagonist, attenuates the antinociceptive activity of nicorandil. Inhibition of nicorandil activity in animals previously treated with cyproheptadine indicates that this NO donor could facilitate neurotransmission mediated by serotonin, a neurotransmitter that plays a relevant role in the central nociceptive processing (Sommer, 2006; Marks et al., 2009; Viguier et al., 2013). Finally, although it is well known that ATP-dependent potassium channel blockers, such as glibenclamide, attenuate the antinociceptive activity of various drugs (Alves and Duarte, 2002; Sachs et al., 2004), we failed to demonstrate that this drug affects the antiallodynic activity of nicorandil. In our previous study (Dutra et al., 2013), we also failed to demonstrate that activation of ATP-dependent potassium channels mediate the activity of nicorandil in the model of nociceptive response induced by formaldehyde. In conclusion, we demonstrated that nicorandil exhibits activity in two experimental models of neuropathic pain. This activity was observed when the mechanical allodynia induced by paclitaxel or CCI was fully established and did not result from motor incoordination or muscle relaxing effect. Activation of opioidergic and serotonergic pathways partially mediates the antinociceptive activity of nicorandil. It is unlikely that this activity requires biotransformation to nicotinamide or nicotinic acid. The results show that nicorandil should be further evaluated aiming to identify a new alternative in the pharmacological management of neuropathic pain.
Acknowledgments
We thank Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Pró-Reitoria de Pesquisa/Universidade Federal de Minas Gerais for financial support.
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Fig. 1. Mechanical allodynia induced by repeated administrations of paclitaxel. Paclitaxel (2 mg/kg, cumulative dose 8 mg/kg, i.p.) or saline (2 ml/kg, i.p.) were administered on four alternate days. Mechanical nociceptive thresholds were evaluated on alternate days until 14 days after the first injection of paclitaxel or saline. *** indicates significant difference from saline group (P < 0.001). n = 6.
Fig. 2. Effects induced by two administrations of nicorandil (50, 100 or 150 mg/kg, p.o.) on the mechanical allodynia induced by paclitaxel. Mechanical nociceptive thresholds were evaluated 1, 3, 5 and 7 h after the first administration of nicorandil or CMC 0.5% (8 ml/kg) on the 14th day after the first injection of paclitaxel. Baseline represents the mechanical nociceptive threshold before injection of paclitaxel. A represents the temporal course and B represents the area under the curve. *, ** and *** indicate significant differences from CMC group (P < 0.05, P < 0.01 and P < 0.001, respectively). n = 6.
Fig. 3. Effects induced by two administrations of nicorandil (150 mg/kg, p.o.) or two administrations of equimolar doses of nicotinamide (86.7 mg/kg, p.o.) or nicotinic acid (87.7 mg/kg, p.o.) on the mechanical allodynia induced by injection of paclitaxel. Mechanical nociceptive thresholds were evaluated 1, 3, 5 and 7 h after the first administration of nicorandil, nicotinamide, nicotinic acid or CMC 0.5% (8 ml/kg) on the 14th day after the first injection of paclitaxel. Baseline represents the mechanical nociceptive threshold before injection of paclitaxel. A represents the temporal course and B represents the area under the curve. *, ** and *** indicate significant differences from CMC group (P < 0.05, P < 0.01 and P < 0.001, respectively). n = 6.
Fig. 4. Mechanical allodynia induced by chronic constriction injury (CCI) of the sciatic nerve. Mechanical thresholds were evaluated on alternate days until 14 days after CCI or sham surgery. *** indicates significant difference from sham-surgery group (P < 0.001). n = 6. Fig. 5. Effects induced by two administrations of nicorandil (150 mg/kg, p.o.) on the mechanical allodynia induced by CCI. Mechanical nociceptive thresholds were evaluated 1, 3, 5 and 7 h after the first administration of nicorandil or CMC 0.5% (8 ml/kg) on the 14th day after CCI. Baseline represents the mechanical nociceptive
threshold before CCI. * and ** indicate significant differences from CMC group (P < 0.05 and P < 0.01, respectively). n = 6.
Fig. 6. Effects induced by two administrations of nicorandil (NIC; 150 mg/kg, p.o.) on the mechanical allodynia induced by paclitaxel in animals previously (15 min) treated with naltrexone (Nal; 5 or 10 mg/kg, i.p.) or saline (2 ml/kg, i.p.). Mechanical allodynia was evaluated 1, 3, 5 and 7 h after the first administration of nicorandil on the 14th day after the first injection of paclitaxel. Baseline represents the mechanical nociceptive threshold before injection of paclitaxel. A represents the temporal course and B represents the area under the curve. *** indicates significant difference from saline/CMC group (p <0.001). #, ## and ### indicate significant differences from saline/NIC 150 group (P < 0.05, P < 0.01 and P < 0.001, respectively). n = 6. Fig. 7. Effects induced by two administrations of nicorandil (NIC; 150 mg/kg, p.o.) on the mechanical allodynia induced by paclitaxel in animals previously (15 min) treated with cyproheptadine (Cypro; 5 or 10 mg/kg, i.p.) or saline (2 ml/kg, i.p.). Mechanical allodynia was evaluated 1, 3, 5 and 7 h after the first administration of nicorandil on the 14th day after the first injection of paclitaxel. Baseline represents the mechanical nociceptive threshold before injection of paclitaxel. A represents the temporal course and B represents the area under the curve. * and ** indicate significant differences from saline/CMC group (P < 0.05 and P < 0.01, respectivelly). # indicates significant difference from saline/NIC 150 group (P < 0.05). n = 6.
Fig. 8. Effects induced by two administrations of nicorandil (NIC; 150 mg/kg, p.o.) on the mechanical allodynia induced by paclitaxel in animals previously (30 min) treated with glibenclamide (Glib; 20 or 40 mg/kg, p.o.) or saline (8 ml/kg, p.o.). Mechanical allodynia was evaluated 1, 3, 5 and 7 h after the first administration of nicorandil on the 14th day after the first injection of paclitaxel. Baseline represents the mechanical nociceptive threshold before injection of paclitaxel. A represents the temporal course and B represents the area under the curve. *, ** and *** indicate significant differences from saline/CMC group (P < 0.05, P < 0.01 and P < 0.001, respectively). n = 6.
Table 1 Effects induced by two administrations of nicorandil or a single dose of phenobarbital or CMC on the time spent on the rotating rod. Treatment
Time spent on the rotating rod (s) Baseline
1h
3h
5h
7h
CMC 0.5% 8 ml/kg
120±0
120±0
120±0
120±0
120±0
Nicorandil 50 mg/kg
120±0
120±0
120±0
120±0
120±0
Nicorandil 100 mg/kg
120±0
120±0
120±0
120±0
120±0
Nicorandil 150 mg/kg
120±0
120±0
120±0
120±0
120±0
120±0
b
a
120±0
120±0
Phenobarbital 50 mg/kg
45±24
78±24
Motor activity was evaluated 1, 3, 5 and 7 h after the first administration of nicorandil, phenobarbital or CMC. a
and b indicate significant differences from CMC group (P < 0.05 and P < 0.001, respectively). n = 5-6.
PII: DOI: Reference:
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S0014-2999(18)30089-X https://doi.org/10.1016/j.ejphar.2018.02.014 EJP71667
To appear in: European Journal of Pharmacology Received date: 19 December 2017 Revised date: 7 February 2018 Accepted date: 9 February 2018 Cite this article as: Marcela I. Morais, Felipe F. Rodrigues, Sarah O.A.M. Costa, Franciele A. Goulart, Fábio C. Costa, Ivo S.F. Melo, Paulo S.A. Augusto, Marcela M.G.B. Dutra, Ângelo de Fátima, Márcio M. Coelho and Renes R. Machado, Nicorandil inhibits mechanical allodynia induced by paclitaxel by activating opioidergic and serotonergic mechanisms, European Journal of Pharmacology, https://doi.org/10.1016/j.ejphar.2018.02.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Nicorandil inhibits mechanical allodynia induced by paclitaxel by activating opioidergic and serotonergic mechanisms
Marcela I. Morais1, Felipe F. Rodrigues1, Sarah O. A. M. Costa1, Franciele A. Goulart1, Fábio C. Costa1, Ivo S. F. Melo1, Paulo S. A. Augusto1, Marcela M. G. B. Dutra1,3, Ângelo de Fátima2, Márcio M. Coelho1, Renes R. Machado1,*
1
Departamento de Produtos Farmacêuticos, Faculdade de Farmácia, Universidade
Federal de Minas Gerais, Avenida Antônio Carlos, 6627, CEP 31270-901, Belo Horizonte, MG, Brazil 2
Departamento de Química, Instituto de Ciências Exatas, Universidade Federal de
Minas Gerais, Avenida Antônio Carlos, 6627, CEP 31270-901, Belo Horizonte, MG, Brazil 3
Centro Universitário Newton Paiva, Avenida Silva Lobo, 1730, CEP 30460-000, Belo
Horizonte, MG, Brazil.
*
Corresponding author: Departamento de Produtos Farmacêuticos, Faculdade de
Farmácia, Universidade Federal de Minas Gerais, Avenida Antônio Carlos, 6627, 31270-901, Belo Horizonte, MG, Brazil. Tel.: + 55 31 3409 6965, fax: + 55 31 3409 6935; [email protected] ABSTRACT
Recently, we demonstrated that nicorandil exhibits activities in models of inflammatory and nociceptive pain. In the present study, we extended this investigation by evaluating the effects of nicorandil in models of neuropathic pain induced by paclitaxel or nerve injury in mice. Four intraperitoneal (i.p.) injections of paclitaxel (2 mg/kg.day, cumulative dose 8 mg/kg) or chronic constriction injury (CCI) of the sciatic nerve induced a long lasting mechanical allodynia. Per os (p.o.) administration of two doses of
nicorandil (50, 100 and 150 mg/kg) on the 14th day after the first paclitaxel injection attenuated the mechanical allodynia. Equimolar doses of nicotinamide (86.7 mg/kg, p.o.) or nicotinic acid (87.7 mg/kg, p.o.) were devoid of effect. Mechanical allodynia induced by CCI was also attenuated by p.o. administration of two doses of nicorandil (150 mg/kg) on the 14th day after nerve injury. Nicorandil (50, 100 and 150 mg/kg, p.o.) did not affect motor activity. The antinociceptive activity of nicorandil in the model of mechanical allodynia induced by paclitaxel was partially attenuated by naltrexone (5 and 10 mg/kg, i.p.) or cyproheptadine (5 and 10 mg/kg, i.p.), but not by glibenclamide (20 and 40 mg/kg, p.o.). Concluding, nicorandil exhibits activity in experimental models of neuropathic pain when mechanical allodynia is fully established. Activation of opioidergic and serotonergic pathways mediates the antinociceptive activity of nicorandil. It is unlikely that this activity requires biotransformation to nicotinamide or nicotinic acid. Nicorandil should be further evaluated aiming to identify a new alternative in the pharmacological management of neuropathic pain.
Keywords: Pain; Neuropathic pain; Nicorandil; Paclitaxel; Chronic constriction injury.
1. Introduction
Neuropathic pain is triggered by injuries or diseases affecting the central and peripheral nervous systems. This modality of chronic pain represents a manifestation of maladaptive plasticity in the nervous system and the affected individuals may report spontaneous lancinating, shooting and/or burning pain, tingling sensation and amplified responses to noxious (hyperalgesia) and innocuous (allodynia) stimuli (Woolf, 2004; Costigan et al., 2009; Jensen et al., 2011).
Although the commonly used pharmacotherapy of neuropathic pain includes many drugs such as antiepileptics, antidepressants (tricyclic antidepressants, selective serotonin reuptake inhibitors, serotonin-norepinephrine reuptake inhibitors), opioid analgesics and local anesthetics, shortcomings are well recognized as many patients do not experience sufficient relief (Baron et al., 2010). The reduced efficacy of the available drugs may be associated with the heterogeneity of causes, symptoms and underlying mechanisms of neuropathic pain (Beniczky et al., 2005). Acknowledging the shortcomings of the current available pharmacotherapy of neuropathic pain prompts investigation of other alternatives. Among the alternatives that have been evaluated aiming to identify new therapies that promote the relief of neuropathic pain is drug repositioning. This involves the identification of new functions of clinical approved drugs thus allowing the development of new and safe pharmacotherapies in shorter period of time and with lower costs (Ashburn and Thor, 2004). Nicorandil, a nicotinamide nitrate derivative (2-nicotinamide ethyl nitrate), is a vasodilator drug that has been approved is some countries for the treatment of patients with angina pectoris (Frampton et al., 1992; El-Moselby et al., 2009). It has been suggested that release of nitric oxide (NO), activation of guanylyl cyclase and opening of ATP-dependent potassium channels may contribute to the vasodilator activity of nicorandil (Kukovetz et al., 1992; Yasuda et al., 2001; Barbato, 2005). Although the vasodilation may represent an important activity contributing to the pain relief in patients with myocardial ischemia (Abrams, 1985; Fung, 1993), it has been acknowledged that NO donors may also exhibit an intrinsic analgesic activity. Central or peripheral administration of NO donors exhibit antinociceptive and anti-hyperalgesic effects in different experimental models of pain that are not associated with ischemia (Duarte et al., 1990; Ferreira et al., 1992; Soares et al., 2000; Sousa and Prado, 2001). Recently, we demonstrated that nicorandil, as other NO donors, also exhibits activities in models of inflammatory and nociceptive pain (Dutra et al., 2013; Cesar et al., 2014; Dutra et al., 2015). Activation of guanylyl cyclase (Dutra et al., 2013) and opioid receptors (Dutra et al., 2015) are suggested as mechanisms that could mediate the antinociceptive activity of nicorandil in these models. In the present study, we extended the investigation on the antinociceptive activity of nicorandil by evaluating its effects in the models of neuropathic pain induced by paclitaxel or nerve injury in mice and also evaluated the possible mechanisms involved in the antinociceptive activity of this drug.
2. Materials and methods
2.1. Animals
Male Swiss mice (25–30 g) were kept at home cage with food and water ad libitum except when the protocol required per os (p.o.) administration. In this case, the animals were subjected to food restriction of at least 10 h before the experiment. Three days before the experiments, 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. The Ethics Committee on Animal Experimentation of the Federal University of Minas Gerais (Protocol 339/2015) approved the study and ethical guidelines for investigation of experimental pain in conscious animals were followed (Zimmermann, 1983).
2.2. Drugs
Nicorandil (purity >99.0%) was synthesized at the Department of Chemistry, Federal University of Minas Gerais. Nicotinamide, nicotinic acid, naltrexone, glibenclamide, cyproheptadine, carboxymethylcellulose (CMC), paclitaxel (SigmaAldrich, USA), phenobarbital (Sanofi-Aventis, Brazil), ketamine and xylazine (Ceva Sante Animale, Brazil) were obtained commercially. Solutions of paclitaxel, phenobarbital, naltrexone, glibenclamide and cyproheptadine were prepared in sterile saline. Suspensions of nicorandil, nicotinamide and nicotinic acid were prepared in CMC (0.5% w/v in sterile saline). All solutions or suspensions were prepared immediately before each experiment.
2.3. Mechanical allodynia induced by paclitaxel
Paclitaxel was used to induce a sensitization state thus mimicking a model of neuropathic pain induced by an antineoplastic (Smith et al., 2004). Paclitaxel (2 mg/kg.day, cumulative dose 8 mg/kg) or saline (2 ml/kg; control group) were administered intraperitoneally (i.p.) on four alternate days (days 1, 3, 5 and 7).
2.4. Mechanical allodynia induced by chronic constriction injury (CCI) of the sciatic nerve
The method described by Bennet and Xie (1988), with adaptations to mice, was used. Briefly, in mice anesthetized with ketamine (90 mg/kg, i.p.) plus xylazine (9 mg/kg, i.p.), three ligatures of 5.0 chromic gut (Brasuture, Brazil) were tightly tied around the sciatic nerve at the level of the midthigh. In sham-operated mice, the same surgical procedure was followed, except the ligatures of the nerve.
2.5. Evaluation of the mechanical allodynia
An electronic von Frey apparatus (Model EFF 301, Insight, Brazil) was used to evaluate mechanical allodynia. After acclimatization of the animals to the experimental apparatus (1 h/day during 2 days), the basal paw withdrawal threshold of each animal was determined (mean of three measurements). The animals were divided into the experimental groups in such a way that the mean withdrawal thresholds of the different groups were similar. The paw withdrawal threshold of each animal was measured every 2 days during 14 days after the first injection of paclitaxel or CCI.
2.6. Evaluation of the effects induced by nicorandil, nicotinamide or nicotinic acid on the mechanical allodynia induced by paclitaxel
Initially, nicorandil (50, 100 and 150 mg/kg) or vehicle (CMC 0.5%, 8 ml/kg) were administered p.o. on the 14th day after the first paclitaxel injection. The animals received two doses of nicorandil or CMC at an interval of 2 h (0 h and 2 h). Two doses of nicorandil were used as its effect is not long lasting (Dutra et al., 2015). Mechanical allodynia was evaluated 1, 3, 5 and 7 h after the first dose of nicorandil. In another protocol, the effects induced by nicorandil were compared to those induced by nicotinamide and nicotinic acid. The animals received two doses of nicorandil (150 mg/kg) or two equimolar doses of nicotinamide (86.7 mg/kg) or nicotinic acid (87.7 mg/kg), at an interval of 2 h, on the 14th day after the first paclitaxel injection. Mechanical allodynia was evaluated 1, 3, 5 and 7 h after the first dose of nicorandil, nicotinamide or nicotinic acid.
2.7. Evaluation of the effects induced by nicorandil on the mechanical allodynia induced by CCI
Nicorandil (150 mg/kg) or vehicle (CMC 0.5%, 8 ml/kg) were administered p.o. on the 14th day after CCI. The animals received two doses of nicorandil at an interval of 2 h (0 h and 2 h). Mechanical allodynia was evaluated 1, 3, 5 and 7 h after the first dose of nicorandil.
2.8. Evaluation of the effects induced by naltrexone, cyproheptadine or glibenclamide on the antiallodynic activity of nicorandil
Nicorandil (150 mg/kg) or vehicle (CMC 0.5%, 8 ml/kg) were administered p.o. on the 14th day after the first paclitaxel injection. The animals received two doses of nicorandil at an interval of 2 h (0 h and 2 h). To evaluate the mechanisms mediating the antiallodynic activity of nicorandil, naltrexone (5 or 10 mg/kg, i.p., -15 min), cyproheptadine (5 or 10 mg/kg, i.p., -15 min), glibenclamide (20 or 40 mg/kg, p.o., -30 min) or vehicle (saline 2 ml/kg, i.p., -15 min or saline 8 ml/kg, p.o., -30 min) were administered before the first dose of nicorandil (150 mg/kg, p.o.). Mechanical allodynia was evaluated 1, 3, 5 and 7 h after the first dose of nicorandil.
2.9. Evaluation of the effects induced by nicorandil on the motor activity
The motor activity of the animals was evaluated on a rota-rod apparatus. Two days before the evaluation, the animals were trained on the apparatus. To evaluate the motor activity, the animals were placed on the rota-rod (14 rpm) and the time they spent on it was determined. The cut-off time was 120 s. After obtaining baseline values, the animals received two doses of nicorandil (50, 100 or 150 mg/kg, p.o) or vehicle (CMC 0.5%; 8 ml/kg) at an interval of 2 h. Another group of animals was treated with a single dose of phenobarbital (50 mg/kg, p.o.; positive control). Motor activity was evaluated 1, 3, 5 and 7 h after the first dose of nicorandil or phenobarbital.
2.10. Statistical analysis
Results were expressed as mean ± standard error of the mean. Results were analyzed by using two-way ANOVA followed by Bonferroni post test (temporal course of the mechanical allodynia induced by paclitaxel or CCI) or one-way ANOVA followed by Dunnett's test (area under the curve of the mechanical allodynia induced by paclitaxel and motor activity). A P < 0.05 was considered significant. Statistical analysis was conducted using GraphPrism 5.0 for windows.
3. Results
3.1. Mechanical allodynia induced by paclitaxel
Mechanical allodynia was already observed two days after the first administration of paclitaxel and maintained throughout the experimental period of 14 days. Mechanical nociceptive threshold reduced from approximately 7.5 g to approximately 3.5 g on the 12th day after the first injection of paclitaxel. Sterile saline (control group) did not affect the nociceptive threshold (Fig. 1).
3.2. Effect induced by nicorandil, nicotinamide or nicotinic acid on the mechanical allodynia induced by paclitaxel Nicorandil (50, 100 and 150 mg/kg) was administered p.o. on the 14th day after the first paclitaxel injection. The animals received two doses of nicorandil at an interval of 2 h (0 h and 2 h). The three doses of nicorandil attenuated the mechanical allodynia induced by paclitaxel. The highest dose attenuated the mechanical allodynia at all evaluated times. The intermediate dose attenuated the mechanical allodynia at 1, 3 and 5 h, while the lowest dose induced an effect only at 1 and 3 h. CMC 0.5% did not affect the mechanical allodynia induced by paclitaxel (Fig. 2). In another experiment, the effect induced by nicorandil (150 mg/kg, p.o.) was compared to those induced by equimolar doses of nicotinamide (86.7 mg/kg, p.o.) or nicotinic acid (87.7 mg/kg, p.o.). While nicorandil, once again, attenuated the mechanical allodynia induced by paclitaxel at all evaluated times, nicotinamide and nicotinic acid were devoid of effect (Fig. 3).
3.3. Mechanical allodynia induced by CCI
Mechanical allodynia was already observed two days after the sciatic nerve injury and maintained throughout the experimental period of 14 days. Mechanical nociceptive threshold reduced from approximately 6 g to approximately 2 g after CCI. In shamoperated animals, the mechanical nociceptive threshold reduced from approximately 6 to approximately 5 g (Fig. 4).
3.4. Effect induced by nicorandil on the mechanical allodynia induced by CCI Nicorandil (150 mg/kg) was administered p.o. on the 14th day after the sciatic nerve injury. The animals received two doses of nicorandil at an interval of 2 h (0 h and 2 h). Nicorandil attenuated the mechanical allodynia at 1 and 5 h. CMC 0.5% did not affect the mechanical allodynia induced by CCI (Fig. 5).
3.5. Effect induced by nicorandil on the motor activity
To investigate whether the inhibition of the nociceptive behavior induced by nicorandil is not the result of a motor impairment, we evaluated the effect induced by the drug on the motor activity of the animals on the rota-rod apparatus. Nicorandil (50, 100 and 150 mg/kg, p.o.) was administered p.o. twice at an interval of 2 h. The motor activity was not changed 1, 3, 5 and 7 h after the first dose of nicorandil. However, a single dose of phenobarbital (50 mg/kg, p.o.) markedly reduced the time mice spent in the apparatus (Table 1).
3.6. Effects induced by naltrexone, cyproheptadine or glibenclamide on the antiallodynic activity of nicorandil
To investigate putative mechanisms mediating the antiallodynic activity of nicorandil, opioidergic (naltrexone) and serotonergic (cyproheptadine) antagonists and an ATP-dependent potassium channel blocker (glibenclamide) were used. Mechanical allodynia was induced by paclitaxel, as previously described, and evaluated 14 days after the first injection. Nicorandil (150 mg/kg, p.o., two doses 2 h apart) reduced the mechanical allodynia induced by paclitaxel. The antinociceptive activity of nicorandil was partially attenuated by previous (15 min) administration of naltrexone (5 and 10
mg/kg, i.p.; Fig. 6) or cyproheptadine (5 and 10 mg/kg, i.p.; Fig. 7), but not by previous (30 min) administration of glibenclamide (20 and 40 mg/kg, p.o.; Fig. 8) A single dose of naltrexone (10 mg/kg, p.o.; Fig. 6), cyproheptadine (10 mg/kg, p.o.; Fig. 7) or glibenclamide (40 mg/kg, p.o.; Fig. 8), per se, failed to affect the mechanical allodynia induced by paclitaxel.
4. Discussion
Neuropathic pain is highly refractory to many of the available analgesic drugs and its proper pharmacological management still represents a challenge (Gilron et al., 2015). Thus, preclinical and clinical investigations of new drugs that may provide adequate and safe relief of this debilitating category of pain are warranted. In the present study, we provide preliminary results indicating that nicorandil may be a promising drug to be investigated aiming to identify a new alternative in the pharmacological management of neuropathic pain. To investigate new treatments that may provide relief of neuropathic pain, experimental models that mimic the marked features of this condition are essential. Repeated injections of paclitaxel have been shown to induce a painful peripheral neuropathy in rodents characterized by mechanical allodynia and thermal hyperalgesia (Polomano et al., 2001), thus mimicking the neuropathic pain that may occur in cancer patients treated with this chemotherapeutic drug (Scripture et al., 2006). We reproduced such results by demonstrating that repeated injections of paclitaxel induced a long lasting mechanical allodynia in mice that was already established two days after the first injection. It has been shown that a variety of clinical approved drugs, including pregabalin (Ito et al., 2012; Salat et al., 2014), gabapentin (Ledeboer et al., 2006), duloxetine, mexiletine (Ito et al., 2012), morphine, methadone (Pascual et al., 2010), etodolac (Ito et al., 2012) and leflunomide (Brito et al., 2017), among others, may inhibit the sensitization induced by paclitaxel in rodents. In the present study, we demonstrated that the nicorandil, a NO donor that has been approved for the treatment of patients with myocardial ischemia, also inhibits the mechanical allodynia induced by paclitaxel or nerve injury in mice. As there is evidence that nicorandil (Gantenbein et al., 1995), as well as other potassium channel openers (Kobayashi et al., 2008), may exhibit anticonvulsant activity in experimental models, suggesting a central depressant effect, we investigated the
effect induced by nicorandil on the performance of the animals on the rotating rod. This is important to evaluate whether the activity of nicorandil is not the result of a confounding effect on motor coordination or muscle tone. As the three doses of nicorandil used in the present study did not change the motor coordination of the animals, it is unlikely that inhibition of the nociceptive behavior is due to motor incoordination or muscle relaxing effect. Although we have recently demonstrated that nicorandil exhibits activity in models of nociceptive and inflammatory pain (Dutra et al., 2013; Cesar et al., 2014; Dutra et al, 2015), this is the first demonstration that this nicotinamide nitrate derivative also exhibits activity in a well known model of neuropathic pain induced by a chemotherapeutic drug. The antiallodynic effect induced by nicorandil was not restricted to the model of neuropathic pain induced by paclitaxel, but was also observed in the model of neuropathic pain induced by a different stimulus, the chronic constriction of the sciatic nerve. Remarkably, in both models, the antiallodynic effect induced by nicorandil occurred when the sensitization was fully established and after acute administration. The neuropathic pain induced by paclitaxel has been associated with a variety of changes including increased expression of inflammatory mediators that can induce cascading effects on glial cells and neurons, in addition to altered activity of ion channels, neurotransmission and intracellular signaling and disrupted cell structures (Scripture et al., 2006; Boyette-Davis et al., 2015). A myriad of changes also seems to underlie the increased sensitization induced by nerve injury, such as abnormalities in injured and uninjured afferents supplying the lesion site, central sensitization, altered glial cells activity, reduced central inhibition and altered autonomic nervous system (Campbell and Meyer, 2006; Nickel et al., 2012). Thus, the antiallodynic effect induced by nicorandil may result from the action at different targets and may occur directly or indirectly. Nicorandil is vasodilator drug that results from the coupling of a NO donor to nicotinamide, a molecule whose activity in experimental models of pain has been demonstrated (Stevens et al., 2007; Godin et al., 2011). Nicorandil goes through to extensive hepatic metabolism and the main biotransformation pathway leads to formation of nicotinamide and nicotinic acid (Frydman et al., 1989). It has been recently suggested that these two molecules may mediate a known side effect induced by nicorandil, the skin and oral lesions observed in patients under prolonged treatment
(Trechot et al., 2015). Thus, a hypothesis that may be put forward is that nicorandil induces its effects indirectly by releasing nicotinamide or nicotinic acid in vivo. However, this hypothesis is unlikely as doses of nicotinamide (86.7 mg/kg) and nicotinic acid (87.7 mg/kg) equimolar to the highest dose of nicorandil (150 mg/kg) used in the present study were devoid of effect. We have previously shown that both nicotinamide (Godin et al., 2011; Dutra et al., 2013) and nicorandil (Dutra et al., 2013) induce antinociceptive effect in models of nociceptive and inflammatory pain. However, in these models nicotinamide induced antinociceptive effect when administered at doses much higher than those of nicorandil. Similarly, higher doses of nicotinic acid are needed to induce effects in these models (Godin et al., 2012). Whether nicotinamide and nicotinic acid in higher doses also induce antinociceptive effects in models of neuropathic pain is a question that has to be investigated, but the results of the present and previous (Godin et al., 2011; Dutra et al., 2013) studies clearly indicate that the coupling of a NO donor to nicotinamide greatly increases the activity of the resulting molecule, nicorandil. Although nicorandil is a NO donor, results from our previous study (Dutra et al., 2013) provided mixed cues about the role of this gaseous transmitter in the antinociceptive activity this drug in models of inflammatory and nociceptive pain. It seems that NO does not appear to be an essential mediator of the antinociceptive activity of nicorandil in the model of nociceptive response induced by formaldehyde. Many studies have shown that nicorandil exhibits different activities when compared to other NO donors (Taira, 1987; Edwards and Weston, 1990; Greenberg et al., 1991). In experimental models of neuropathic pain, NO donors have also provided mixed results. Some studies have shown that NO donors may increase (Naik et al., 2006) or reduce (Chen et al., 2000) mechanical allodynia induced by nerve injury in rodents, while others have shown that the effects induced by these drugs may be dose related (Souza and Prado, 2001). Thus, the role of NO in the antinociceptive activity of nicorandil and related compounds in models of neuropathic pain is still not clear. We also investigated other mechanisms that could contribute to the antiallodynic activity of nicorandil in the neuropathic pain model induced by paclitaxel. Previous administration of naltrexone partially attenuated the antiallodynic activity of nicorandil, indicating that it depends in part on the activation of opioidergic pathways. It is not clear whether nicorandil directly interacts with opioid receptors or stimulates the release of endogenous opioid peptides, actions that could contribute to its antinociceptive
activity. We have also shown that the activity of nicorandil in the model of nociceptive response induced by formaldehyde, a mixed model of nociceptive and inflammatory pain, is attenuated by an opioid antagonist (Dutra et al., 2015). However, as naltrexone is a non-selective opioid receptor antagonist (Crabtree, 1984), no conclusion about the role of a specific opioid receptor in the antinociceptive activity of nicorandil in both models of pain may be put forward. We also demonstrated that cyproheptadine, a nonselective serotonergic antagonist, attenuates the antinociceptive activity of nicorandil. Inhibition of nicorandil activity in animals previously treated with cyproheptadine indicates that this NO donor could facilitate neurotransmission mediated by serotonin, a neurotransmitter that plays a relevant role in the central nociceptive processing (Sommer, 2006; Marks et al., 2009; Viguier et al., 2013). Finally, although it is well known that ATP-dependent potassium channel blockers, such as glibenclamide, attenuate the antinociceptive activity of various drugs (Alves and Duarte, 2002; Sachs et al., 2004), we failed to demonstrate that this drug affects the antiallodynic activity of nicorandil. In our previous study (Dutra et al., 2013), we also failed to demonstrate that activation of ATP-dependent potassium channels mediate the activity of nicorandil in the model of nociceptive response induced by formaldehyde. In conclusion, we demonstrated that nicorandil exhibits activity in two experimental models of neuropathic pain. This activity was observed when the mechanical allodynia induced by paclitaxel or CCI was fully established and did not result from motor incoordination or muscle relaxing effect. Activation of opioidergic and serotonergic pathways partially mediates the antinociceptive activity of nicorandil. It is unlikely that this activity requires biotransformation to nicotinamide or nicotinic acid. The results show that nicorandil should be further evaluated aiming to identify a new alternative in the pharmacological management of neuropathic pain.
Acknowledgments
We thank Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Pró-Reitoria de Pesquisa/Universidade Federal de Minas Gerais for financial support.
References
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Fig. 1. Mechanical allodynia induced by repeated administrations of paclitaxel. Paclitaxel (2 mg/kg, cumulative dose 8 mg/kg, i.p.) or saline (2 ml/kg, i.p.) were administered on four alternate days. Mechanical nociceptive thresholds were evaluated on alternate days until 14 days after the first injection of paclitaxel or saline. *** indicates significant difference from saline group (P < 0.001). n = 6.
Fig. 2. Effects induced by two administrations of nicorandil (50, 100 or 150 mg/kg, p.o.) on the mechanical allodynia induced by paclitaxel. Mechanical nociceptive thresholds were evaluated 1, 3, 5 and 7 h after the first administration of nicorandil or CMC 0.5% (8 ml/kg) on the 14th day after the first injection of paclitaxel. Baseline represents the mechanical nociceptive threshold before injection of paclitaxel. A represents the temporal course and B represents the area under the curve. *, ** and *** indicate significant differences from CMC group (P < 0.05, P < 0.01 and P < 0.001, respectively). n = 6.
Fig. 3. Effects induced by two administrations of nicorandil (150 mg/kg, p.o.) or two administrations of equimolar doses of nicotinamide (86.7 mg/kg, p.o.) or nicotinic acid (87.7 mg/kg, p.o.) on the mechanical allodynia induced by injection of paclitaxel. Mechanical nociceptive thresholds were evaluated 1, 3, 5 and 7 h after the first administration of nicorandil, nicotinamide, nicotinic acid or CMC 0.5% (8 ml/kg) on the 14th day after the first injection of paclitaxel. Baseline represents the mechanical nociceptive threshold before injection of paclitaxel. A represents the temporal course and B represents the area under the curve. *, ** and *** indicate significant differences from CMC group (P < 0.05, P < 0.01 and P < 0.001, respectively). n = 6.
Fig. 4. Mechanical allodynia induced by chronic constriction injury (CCI) of the sciatic nerve. Mechanical thresholds were evaluated on alternate days until 14 days after CCI or sham surgery. *** indicates significant difference from sham-surgery group (P < 0.001). n = 6. Fig. 5. Effects induced by two administrations of nicorandil (150 mg/kg, p.o.) on the mechanical allodynia induced by CCI. Mechanical nociceptive thresholds were evaluated 1, 3, 5 and 7 h after the first administration of nicorandil or CMC 0.5% (8 ml/kg) on the 14th day after CCI. Baseline represents the mechanical nociceptive
threshold before CCI. * and ** indicate significant differences from CMC group (P < 0.05 and P < 0.01, respectively). n = 6.
Fig. 6. Effects induced by two administrations of nicorandil (NIC; 150 mg/kg, p.o.) on the mechanical allodynia induced by paclitaxel in animals previously (15 min) treated with naltrexone (Nal; 5 or 10 mg/kg, i.p.) or saline (2 ml/kg, i.p.). Mechanical allodynia was evaluated 1, 3, 5 and 7 h after the first administration of nicorandil on the 14th day after the first injection of paclitaxel. Baseline represents the mechanical nociceptive threshold before injection of paclitaxel. A represents the temporal course and B represents the area under the curve. *** indicates significant difference from saline/CMC group (p <0.001). #, ## and ### indicate significant differences from saline/NIC 150 group (P < 0.05, P < 0.01 and P < 0.001, respectively). n = 6. Fig. 7. Effects induced by two administrations of nicorandil (NIC; 150 mg/kg, p.o.) on the mechanical allodynia induced by paclitaxel in animals previously (15 min) treated with cyproheptadine (Cypro; 5 or 10 mg/kg, i.p.) or saline (2 ml/kg, i.p.). Mechanical allodynia was evaluated 1, 3, 5 and 7 h after the first administration of nicorandil on the 14th day after the first injection of paclitaxel. Baseline represents the mechanical nociceptive threshold before injection of paclitaxel. A represents the temporal course and B represents the area under the curve. * and ** indicate significant differences from saline/CMC group (P < 0.05 and P < 0.01, respectivelly). # indicates significant difference from saline/NIC 150 group (P < 0.05). n = 6.
Fig. 8. Effects induced by two administrations of nicorandil (NIC; 150 mg/kg, p.o.) on the mechanical allodynia induced by paclitaxel in animals previously (30 min) treated with glibenclamide (Glib; 20 or 40 mg/kg, p.o.) or saline (8 ml/kg, p.o.). Mechanical allodynia was evaluated 1, 3, 5 and 7 h after the first administration of nicorandil on the 14th day after the first injection of paclitaxel. Baseline represents the mechanical nociceptive threshold before injection of paclitaxel. A represents the temporal course and B represents the area under the curve. *, ** and *** indicate significant differences from saline/CMC group (P < 0.05, P < 0.01 and P < 0.001, respectively). n = 6.
Table 1 Effects induced by two administrations of nicorandil or a single dose of phenobarbital or CMC on the time spent on the rotating rod. Treatment
Time spent on the rotating rod (s) Baseline
1h
3h
5h
7h
CMC 0.5% 8 ml/kg
120±0
120±0
120±0
120±0
120±0
Nicorandil 50 mg/kg
120±0
120±0
120±0
120±0
120±0
Nicorandil 100 mg/kg
120±0
120±0
120±0
120±0
120±0
Nicorandil 150 mg/kg
120±0
120±0
120±0
120±0
120±0
120±0
b
a
120±0
120±0
Phenobarbital 50 mg/kg
45±24
78±24
Motor activity was evaluated 1, 3, 5 and 7 h after the first administration of nicorandil, phenobarbital or CMC. a
and b indicate significant differences from CMC group (P < 0.05 and P < 0.001, respectively). n = 5-6.