J Neural Transm (2013) 120:1533–1538 DOI 10.1007/s00702-013-1052-7
TRANSLATIONAL NEUROSCIENCES - ORIGINAL ARTICLE
Involvement of cannabinoid CB1 receptors in the antinociceptive effect of dipyrone Pinar Elmas • Ahmet Ulugol
Received: 26 February 2013 / Accepted: 11 June 2013 / Published online: 20 June 2013 Ó Springer-Verlag Wien 2013
Abstract Cannabinoid CB1 receptors have been implicated in the antinociceptive effect of paracetamol. In the current study, we examined whether blockade of CB1 receptors prevent the analgesic activity of dipyrone, in a similar way to paracetamol. Hot-plate and tail-flick tests were used to assess the antinociceptive activity in mice. Dipyrone and WIN 55,212-2, a cannabinoid agonist, exerted significant antinociceptive effects in both hot-plate and tail flick tests. The CB1 receptor antagonist, AM-251 (3 mg/kg), at a dose which had no effect when used alone, did not alter the antinociceptive effect of dipyrone, whereas completely prevented the antinociceptive activity of WIN 55,212-2 in both thermal antinociceptive tests. Our findings suggest that, unlike paracetamol, cannabinoid CB1 receptors do not participate in the antinociceptive action of dipyrone when acute pain tests used. Keywords Dipyrone WIN 55,212-2 CB1 receptor Antinociception
Introduction Dipyrone (metamizol), a pirazolone derivative, is an antipyretic and analgesic drug widely used in most parts of the world. Although there are some concerns with
Submitted to ‘‘Pain in Europe VIII, 8th Congress of the European Federation of IASP Chapters’’, Florence, Italy, 9–12 October, 2013. P. Elmas A. Ulugol (&) Department of Medical Pharmacology, Faculty of Medicine, Trakya University, 22030 Edirne, Turkey e-mail:
[email protected]
respect to its safety, it is maintaining its popularity due to its low cost, high analgesic efficacy and reduced gastric side effects. Unlike classical non-steroidal anti-inflammatory drugs, it possesses little anti-inflammatory activity; and its antinociceptive mechanism of action is not totally elucidated. However, in addition to the traditional peripheral effect of NSAIDs, site of action of its antinociceptive effect is thought to be predominantly in central nervous system, including the periaqueductal gray matter (PAG), the rostral ventromedial medulla (RVM) and the spinal cord (Vanegas and Schaible 2001; Vanegas and Tortorici 2002). There are accumulating indications pointing to the role of cannabinoids in nociception, especially in chronic pain states (Gunduz et al. 2011a; Ulugol et al. 2004, 2006); unfortunately, serious adverse effects preclude their usage in the clinic. Cannabinoids produce antinociceptive action by activating descending inhibition via CB1 receptors, their site of action similar to dipyrone being at peripheral, spinal and supraspinal levels (Seyrek et al. 2010). Endogenous cannabinoids, such as anandamide and 2-arachidonylglycerol, are released tonically and act as the same way as exogenous cannabinoids. Drugs exerting their effects via augmentation of the endogenous cannabinoid tonus through inhibition of re-uptake or metabolism of endogenous cannabinoids are promising for pain therapy, due to their low central adverse effect potential compared to natural and synthetic cannabinoids (Schlosburg et al. 2009; Dogrul et al. 2012). Systemic and local antinociceptive effects of paracetamol, another non-opioid analgesic like dipyrone, have been shown to be mediated by cannabinoid CB1 receptors (Ottani et al. 2006; Dani et al. 2007). Moreover, fatty acid amide hydrolase (FAAH)-dependent metabolism into AM404 and reinforcement of descending serotonergic
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pathways by the endocannabinoid system through activation of CB1 receptors by this metabolite, are proposed to be involved in paracetamol-induced analgesia (Mallet et al. 2008). Very recently, two novel arachidonyl-conjugated metabolites of dipyrone were found and FAAH is suggested to be responsible for the formation of these metabolites (Rogosch et al. 2012). Unlike classical NSAIDs, both paracetamol and dipyrone are non-opioid analgesics possessing very little anti-inflammatory activity; moreover, opioid receptor activation and endogenous opioid tonus together with descending inhibitory pathways are also suggested to be involved both in paracetamol and dipyrone effects (Bujalska 2004; Hernandez-Delgadillo and Cruz 2006; Mallet et al. 2008; Sandrini et al. 2001; Vazquez et al. 2005, 2007). Considering the similarities between dipyrone and paracetamol, contribution of the endocannabinoid system in dipyrone-induced analgesia can be expected. We therefore aimed to examine the possible involvement of cannabinoid CB1 receptors in the antinociceptive effect of dipyrone.
Materials and methods Animals and ethics Male Balb-c albino mice (Center of the Laboratory Animals, Trakya University), weighing 20–30 g, were used. Animals were housed in groups of ten and maintained under 12–12 h light–dark cycles at the temperature of 21 ± 2 °C; water and food were provided ad libitum. This study was performed according to the guidelines of the Ethical Committee of the International Association for the Study of Pain (Zimmermann 1983), and the experimental protocols were approved by the local ‘‘Animal Care Ethics Committee’’.
P. Elmas, A. Ulugol
latency - baseline latency)/cut-off latency] 9 100 (Isguzar et al. 2012).
time - baseline
Tail-flick test A standardized tail-flick test (Commat, Ankara, Turkey) was also used to assess the antinociceptive activity. Briefly, radiant heat was focused on the dorsal surface of the mouse tail and the time until the tail flicked was measured by means of a photocell and an automatic timer. At the beginning of the experiments, the stimulus intensity was adjusted to a baseline tail-withdrawal latency of 3–5 s in all rats. A cut-off time of 15 s was set to avoid tissue damage. Tail-flick test times before and after drugs were similar to those of hot-plate test. Test latencies were converted to the percentage of the MPE according to the formula: %MPE = [(post-drug latency - baseline latency)/cut-off time - baseline latency] 9 100. Experimental design After testing different doses of dipyrone (150, 300, 600 mg/kg, i.p.) and WIN 55,212-2 (1, 3, 10 mg/kg, i.p.), the effect of the CB1 receptor antagonist, AM-251 (3 mg/ kg, i.p.), on the antinociceptive effects of dipyrone and WIN 55,212-2 was studied. Drug doses were chosen from previous studies (Gunduz et al. 2011b, c; Yilmaz and Ulugol 2009). Prior to drug treatments, mice were tested on the hot-plate and tail flick tests for a few days in order to achieve a stable control response. Both hot-plate and tailflick test were performed in all animals and each animal was used only once (n = 10 in each group); the tests were always performed consecutively and in the same order for the standardization, tail-flick being the first. Drugs
Hot-plate test To perform hot-plate test, a conventional hot/cold-plate apparatus (Ugo Basile, Comerio, Italy) was used. On an electrically heated metal plate that was maintained at a temperature of 55 ± 0.5 °C, the animals were placed one at a time. By means of an electronic timer, response latencies either to licking of one hind-paw or to jump were recorded. A cut-off time of 25 s was set to avoid tissue damage. Each mouse was tested before drug treatment and the values were averaged to obtain a baseline, post-drug latencies were evaluated 30 min after the analgesics and 45 min after AM-251. Test latencies were converted to the percentage of the maximal possible effect (%MPE) according to the formula: %MPE = [(post-drug
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Dipyrone was purchased from Santa Cruz, WIN 55,212-2 from Sigma, and AM-251 from Tocris. Dipyrone was dissolved in saline, while WIN 55,212-2 and AM-251 were administered in 20 % DMSO, 1 % Tween 80, 1 % ethanol, 78 % saline. All of the drugs were given i.p. in a volume of 0.05 ml/10 g body weight. Control animals were injected i.p. with 0.9 % saline according to the same time schedule. Statistical analysis To compare the data of the groups analysis of variance (ANOVA), followed by Bonferroni t test, was carried out. Values of P \ 0.05 were considered to be significant. All data are expressed as mean ± SEM.
Involvement of cannabinoid CB1 receptors
Results Antinociceptive effects of dipyrone and WIN 55,212-2 With its highest dose, 600 mg/kg, dipyrone elicited a significant antinociceptive effect in both hot-plate and tailflick tests; 300 mg/kg of dipyrone also increased hot-plate latencies during the first hour (Fig. 1). WIN 55,212-2 also exerted antinociceptive effects with its doses of 3 and 10 mg/kg in both tests (Fig. 2). Effect of CB1 receptor antagonism on the antinociceptive effects of dipyrone and WIN 55,212-2 The CB1 receptor antagonist, AM-251 (3 mg/kg), at a dose which had no effect when given by itself, did not alter the antinociceptive effect of dipyrone, whereas completely prevented the antinociceptive effect of WIN 55,212-2 in both hot-plate and tail-flick tests (Figs. 3, 4).
Discussion The results presented in this study demonstrate that dipyrone administered intraperitoneally exerted a significant antinociceptive effect as expected, when assessed in two popular thermal nociceptive tests, hot-plate and tail-flick. However, unlike paracetamol, blockade of cannabinoid CB1 receptors did not prevent the analgesic activity of dipyrone. In addition to its action on peripheral sites, the antinociceptive effect of dipyrone is suspected to be centrally mediated for a long time. Now, it is known that it exerts its effect partly due to its action on central nervous system structures, such as PAG, RVM and spinal cord, the most important pain-related areas (Hernandez and Vanegas 2001; Vasquez and Vanegas 2000). Then, descending
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inhibitory pathways are triggered, in relation with the contribution of endogenous opioids at these pain-related structures (Vanegas and Tortorici 2002). Most importantly, as well as its analgesic efficacy through descending pathways when applied spinally or supraspinally, systemic dipyrone also appears to act by activating descending inhibition, which points to the clinically relevance of this effect (Neugebauer et al. 1994; Vazquez et al. 2005). Although there are a few reports indicating that the participation of endogenous opioid peptides is minimal in the analgesic effect of dipyrone (Beirith et al. 1998; Taylor et al. 1998), a large body of evidence now suggests that dipyrone releases endogenous opioids and activates descending inhibitory pathways (Akman et al. 1996; Hernandez and Vanegas 2001; Hernandez-Delgadillo and Cruz 2006; Neugebauer et al. 1994; Vasquez and Vanegas 2000; Vazquez et al. 2005, 2007). Mechanisms other than endogenous opioid release are also proposed for the antinociceptive effect of dipyrone. Among these, cyclooxygenase inhibition is surely well characterized elsewhere (Campos et al. 1999). Interactions with the l-arginine-nitric oxide pathway and/or the glutamatergic system are also suggested (Beirith et al. 1998; Hernandez-Delgadillo and Cruz 2006; Lorenzetti and Ferreira 1996; Siebel et al. 2004), although there are a few adverse opinions (Yilmaz and Ulugol 2009). Recently, two unknown arachidonyl-conjugated metabolites of dipyrone are found and were positively tested for cannabinoid receptor binding, although this does not directly demonstrate that these molecules have cannabinoid activity (Rogosch et al. 2012). FAAH is suggested to be responsible for the conversion of the known metabolites of dipyrone to these arachidonyl amides, and activation of cannabinoid receptors is proposed as a novel mechanism of action for the antinociceptive effect of dipyrone (Rogosch et al. 2012). Accordingly, Escobar et al. (2012) has shown that the antinociceptive effect of dipyrone is reduced by a
Fig. 1 Time course of the antinociceptive effect of different doses (150, 300, 600 mg/kg, n = 10 for each group) of dipyrone in the hotplate and tail-flick tests. (*P \ 0.05, **P \ 0.01, compared to corresponding values in the control group; MPE = maximal possible effect)
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Fig. 2 Time course of the antinociceptive effect of different doses (1, 3, 10 mg/kg, n = 10 for each group) of WIN 55,212-2 in the hot-plate and tail-flick tests. (*P \ 0.05, **P \ 0.01, compared to corresponding values in the control group; MPE = maximal possible effect)
Fig. 3 Effect of the cannabinoid CB1 receptor antagonist, AM-251 (3 mg/kg, n = 10 for each group), on the antinociceptive effect of dipyrone in the hot-plate and tail-flick tests. AM-251 was injected 15 min before, and tests were performed 30 min after the administration of dipyrone. (**P \ 0.01, compared to corresponding values in the control group; MPE = maximal possible effect)
Fig. 4 Effect of the cannabinoid CB1 receptor antagonist, AM-251 (3 mg/kg, n = 10 for each group), on the antinociceptive effect of WIN 55,212-2 in the hot-plate and tail-flick tests. AM-251 was injected 15 min before, and tests were performed 30 min after the
administration of WIN 55,212-2. (**P \ 0.01, compared to corresponding values in the control group; #P \ 0.05, ##P \ 0.01, compared to corresponding values in the WIN 55,212-2 group; MPE = maximal possible effect)
microinjection of a cannabinoid receptor antagonist into the PAG or RVM, and pointed to the participation of cannabinoidergic system in this effect. On the other hand, a short time ago, Schlosburg et al. (2012) reported that some pharmacological effects of dipyrone, including its thermal antinociceptive action, are not mediated by endogenous
cannabinoid system. This latter observation, suggesting a non-cannabinoid receptor mechanism of action for dipyrone effects, is performed under non-inflammatory conditions similar to our work and in line with our findings. The contribution of cannabinoids to the analgesic action of dipyrone in the study of Escobar et al. (2012), on the
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Involvement of cannabinoid CB1 receptors
contrary, was under inflammatory conditions and subsequent to the supraspinal injection. Taken together, these do not exclude the possible involvement of cannabinoid system to the antinociceptive effects of dipyrone when other pain models (neuropathic, inflammation, visceral, etc.) or different routes of administration are used. As a result, our findings in the present experiments indicate that cannabinoid CB1 receptors do not mediate the antinociceptive effect of dipyrone. CB1 receptors may still have role in this effect of dipyrone in inflammatory and/or neuropathic states, nevertheless our results make an additive contribution to the unknown effects of a worldwide used analgesic. Acknowledgments This work was supported by a grant from Trakya University Research Council (TUBAP-2012/58). The authors have no conflicts of interests to report.
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