Clinical and Experimental Pharmacology and Physiology (2015) 42, 246–255

doi: 10.1111/1440-1681.12347

ORIGINAL ARTICLE

Cannabinoid CB1 receptors mediate the effects of dipyrone Fernanda Crunfli,*† Fabiana C Vilela†‡ and Alexandre Giusti-Paiva*†§ ‡

*Department of Physiological Sciences, Institute of Biomedical Sciences, †Graduate Program in Health Biosciences, Faculty of Pharmaceutical Sciences, Federal University of Alfenas, Alfenas, MG, and §Multicenter Graduate Program in Physiological Sciences, Brazilian Society of Physiology, Sao Paulo, Brazil

SUMMARY Dipyrone is a non-steroidal anti-inflammatory drug used primarily as an analgesic and antipyretic. Some hypothesize that dipyrone activity can modulate other pathways, including endocannabinoid signalling. Thus, the aim of the present study was to evaluate the possible role of endocannabinoids in mediating dipyrone activity. This study is based on the tetrad effects of cannabinoids, namely an antinociceptive and cataleptic state, hypolocomotion and hypothermia. Dipyrone (500 mg/kg, i.p.) treatment decreased locomotor activity, increased the latency to a thermal analgesic response and induced a cataleptic and hypothermic state. These reactions are similar to the tetrad effects caused by the cannabinoid agonist WIN 55,212-2 (3 mg/kg, i.p.). The cannabinoid CB1 receptor antagonist AM251 (10 mg/kg, i.p.) reversed the effects of dipyrone on locomotor activity, the cataleptic response and thermal analgesia. Both AM251 (10 mg/kg, i.p.) and the transient receptor potential vanilloid 1 (TRPV1) antagonist capsazepine (10 mg/kg, i.p.) accentuated the reduction in body temperature caused by dipyrone. However, the CB2 receptor antagonist AM630 did not alter the hypothermic response to dipyrone. These results indicate involvement of the endocannabinoid system, especially CB1 receptors, in the analgesic and cataleptic effects of dipyrone, as well as hypolocomotion. However, cannabinoid receptors and TRPV1 were not involved in the hypothermic effects of dipyrone. We hypothesize that the mechanism of action of dipyrone may involve inhibition of cyclo-oxygenase and fatty acid amide hydrolase, which together provide additional arachidonic acid as substrate for endocannabinoid synthesis or other related molecules. This increase in endocannabinoid availability enhances CB1 receptor stimulation, contributing to the observed effects. Key words: dipyrone, endocannabinoid, tetrad effect.

Correspondence: Alexandre Giusti-Paiva (PhD), Institute of Biomedical Sciences, Federal University of Alfenas, UNIFAL, Rua Gabriel Monteiro da Silva, 700, Alfenas, 37130-000, MG, Brazil. Email: alexandre.paiva@ unifal-mg.edu.br Received 15 August 2014; revision 10 October 2014; accepted 14 October 2014. © 2014 Wiley Publishing Asia Pty Ltd

INTRODUCTION Dipyrone, or metamizole sodium, is a common antipyretic, analgesic and non-steroidal anti-inflammatory drug (NSAID) that has been used clinically for more than 80 years. In aqueous solution, dipyrone is immediately hydrolysed to 4-methylamino-antipyrine (MAA), which can be further metabolized to 4-amino-antipyrine (AA), 4-formylamino-antipyrine (FAA) or 4-acetyl-amino-antipyrine (AAA).1,2 The molecular mechanisms underlying the analgesic and antipyretic actions of dipyrone have long been under debate. It has been proposed that these effects depend, at least in part, on inhibition of cyclo-oxygenase (COX)-1 and COX-2 and decreased synthesis of prostaglandin (PG) E2.3 Although the inhibitory effect of dipyrone on COX has long been known, the exact mechanism by which MAA inhibits COX activity remains unclear. Other studies have shown that dipyrone may produce analgesia by acting on opioid receptors.3,4 Traditional NSAIDs inhibit COX activity by competing with arachidonic acid for its binding site.5,6 The search for additional mechanisms for NSAID-induced antinociception was stimulated by studies showing that the inhibition of prostaglandin synthesis in inflamed tissue is not the only pathway for this response. Antinociceptive mechanisms for NSAIDs may also involve the opioid system and the nitric oxide (NO)/cGMP/KATP signalling pathway.7,8 Other studies have linked NSAID activity with the endocannabinoid system.9 This suggestion of a novel mechanism for mild analgesics began with the discovery that paracetamol interacts with cannabinoid receptors in producing analgesia.9,10 Subsequent studies have shown that an arachidonic acid-conjugated metabolite of paracetamol is formed in the spinal cord and brain of mice through fatty acid amide hydrolase (FAAH), the primary catabolic enzyme regulating the endocannabinoid anandamide.11 In addition, NSAIDs inhibit COX and FAAH, and both these enzymes can also metabolise endocannabinoids. This suggests a plausible link between NSAIDs and endocannabinoids.12–14 By inhibiting COX and FAAH, NSAIDs may prevent the enzymatic cleavage of endocannabinoids, which induces analgesia through cannabinoid CB1 and CB2 receptors. Given the contentious relationship between dipyrone and NSAIDs and the popularity of dipyrone as an analgesic and antipyretic drug, the aim of the present study was to investigate the involvement of the endocannabinoid system in dipyrone-mediated

CB1 receptors and effects of dipyrone tetrad effects of cannabinoids, namely analgesia, hypolocomotion, catalepsy and hypothermia.

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RESULTS Mice treated with saline or dipyrone (10–500 mg/kg, i.p.) were evaluated in four tests (hotplate, catalepsy, open field and temperature), with or without addition vehicle or WIN 55,212-2 (0.3, 1.0 or 3.0 mg/kg, i.p.) treatement. To evaluate the role of CB1 receptors in the effects of dipyrone, the mice were pretreated with vehicle or AM251 (3 mg/kg, i.p.) for 10 min before being injected with saline or dipyrone (500 mg/kg, i.p.) and evaluated in the hotplate, catalepsy and open field tests. For temperature evaluation only, mice were injected with AM251 (3 or 10 mg/kg, i.p.), AM630 (10 mg/kg, i.p.) or capsazepine (10 mg/kg, i.p.) 10 min before being injected with saline or dipyrone (200 mg/kg, i.p.).

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Evaluation of latency response in the hotplate test Two-way ANOVA revealed significant treatment (F4,130 = 12.83; P < 0.001), time (F5,130 = 8.864; P < 0.001) and time 9 treatment interaction (F20,130 = 2.745; P < 0.001) effects of dipyrone treatment (50–500 mg/kg, i.p.). Post hoc analysis revealed an increased analgesic response latency compared with the vehicletreated group 30 min (P < 0.05) and 60 min (P < 0.001) after the administration of 500 mg/kg dipyrone (Fig. 1a). Two-way ANOVA revealed significant treatment (F3,192 = 9.3; P < 0.001), but not time (F5,192 = 2.16; P = 0.06) or treatment 9 time interaction (F15,192 = 1,09; P = 0.37), effects of the cannabinoid agonist WIN 55,212-2 (Fig. 1b). Post hoc analysis revealed an increased latency 30 min and 1 h following treatment with WIN 55,212-2 (0.3 and 3 mg/kg; i.p.) compared with the vehicle-treated group (P < 0.05 for both; Fig. 1b). When animals were pretreated with the cannabinoid CB1 receptor antagonist AM251, the analgesic effects of dipyrone at 30 min were reversed (pretreatment factor: F1,39 = 3.813, P = 0.058; treatment factor: F1,39 = 14.44, P < 0.001; pretreatment 9 treatment interaction: F1,39 = 6.567; P < 0.05; Fig. 1c). Similarly, AM251 pretreatment reversed the effects of dipyrone at 60 min (pretreatment factor: F1,39 = 6.629, P < 0.05; treatment factor: F1,39 = 6.824, P < 0.05; pretreatment 9 treatment interaction: F1,39 = 4.91; P < 0.05) and 120 min (pretreatment factor: F1,39 = 2.899, P < 0.05; treatment factor: F1,39 = 7.952, P < 0.01; pretreatment 9 treatment interaction: F1,39 = 4.62; P < 0.05). Evaluation in the catalepsy test At 500 mg/kg, dipyrone induced catalepsy in rodents (two-way treatment factor: F4,385 = 19.95, P < 0.001; time factor: F5,385 = 7.58, P < 0.001; treatment 9 time interaction: F20,285 = 3.08, P < 0.001) at both 30 and 60 min after administration (Fig. 2a). Two-way ANOVA showed significant effects of treatment (F3,180 = 35.75; P < 0.001), time (F5,180 = 14.89, P < 0.001) and the treatment 9 time interaction (F15,180 = 2.072, P < 0.01) indicating that treatment with WIN 55,212-2 induced a cataleptic effect at 30, 60 and 240 min at higher doses (1.0 and 3.0 mg/kg) compared with the control group (Fig. 2b). ANOVA;

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Fig. 1 Response latency in a hotplate test recorded 30, 60, 120, 240 and 360 min after treatment with (a) dipyrone (n = 5–6 mice per group), (b) the cannabinoid CB1 receptor agonist WIN 55,212-2 (n = 9 mice per group) and (c) pretreatment with the cannabinoid receptor antagonist AM251 followed by dipyrone (n = 9–11 mice per group). Data are the mean  SEM. *P < 0.05, **P < 0.01, ***P < 0.001 compared with control groups; †P < 0.05, ††P < 0.01 compared with vehicle + dipyrone.

The cataleptic effect of dipyrone at 30 min was reversed by pretreatment with the cannabinoid CB1 receptor antagonist AM251 (pretreatment factor: F1,30 = 5.693, P < 0.05; treatment factor: F1,30 = 10.91, P < 0.01; pretreatment 9 treatment interaction: F1,30 = 5.853; P < 0.05; Fig. 2c). Similarly, AM251 pretreatment reversed the effects of dipyrone at 60 min (pretreatment factor: F1,30 = 14.81, P < 0.001; treatment factor: F1,30 = 38.8, P < 0.001; pretreatment 9 treatment interaction: F1,30 = 15.04; P < 0.001). Evaluation in the open field test In the open field test, dipyrone treatment decreased the total number of line crossings (F4,51 = 6.087; P < 0.001) as well as crossings in the central (F4,51 = 3.673; P < 0.01) and peripheral (F4,51 = 6.163; P < 0.001) areas and the number of rearings (F4,51 = 17.06; P < 0.001) compared with the control group. This

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Fig. 2 Response to a catalepsy test recorded 30, 60, 120, 240 and 360 min after the administration of (a) dipyrone (n = 13–16 mice per group), (b) the cannabinoid CB1 receptor agonist WIN 55,212-2 (n = 8–9 mice per group) and (c) pretreatment with the cannabinoid antagonist AM251 followed by dipyrone (n = 9–10 mice per group). Data are the mean  SEM. *P < 0.05, **P < 0.01, ***P < 0.001 compared with control groups; ††P < 0.01, †††P < 0.001 compared with vehicle + dipyrone.

indicates that 500 mg/kg dipyrone decreases locomotor activity (Fig. 3a). Treatment with the CB1 receptor agonist WIN 55,212-2 (3 mg/ kg) decreased the total number of line crossings (F3,20 = 5.69; P < 0.01) as well as the crossings in peripheral (F3,20 = 4478; P < 0.05) and central (F3,20 = 11.56, P < 0.001) areas and the number of rearings (F3,20 = 3.751; P < 0.05) compared with the control group (Fig. 3b). Pretreatment with AM251 (3 mg/kg) reversed the effects of dipyrone on locomotion, as evidenced by a reduction in the total number of line crossings (two-way ANOVA; treatment factor: F1,32 = 11.74, P < 0.01; pretreatment factor: F1,32 = 1.21, P > 0.05; pretreatment 9 treatment interaction: F1,32 = 6.778, P < 0.05; Fig. 3c). Similarly, AM251 reversed the reduction in line crossings in peripheral areas (treatment factor: F1,32 = 6.225, P < 0.05; pretreatment factor: F1,32 = 2.865, P > 0.05; pretreatment 9 treatment interaction: F1,32 = 8.372, P < 0.01) and central areas (treatment factor: F1,32 = 13.04, P < 0.01; pretreatment factor: F1,32 = 0.183, P > 0.05; pretreatment 9 treatment

Fig. 3 Effects of treatment with (a) dipyrone (n = 10 mice per group), (b) the cannabinoid CB1 receptor agonist WIN 55,212-2 (n = 6 mice per group) and (c) pretreatment with the cannabinoid antagonist AM251 followed by dipyrone (n = 9 mice per group) on total, peripheral and central line crossing and the number of rearings in the open field test. Data are the mean  SEM. *P < 0.05, **P < 0.01, ***P < 0.001 compared with control groups; †P < 0.05 compared with vehicle + dipyrone.

interaction: F1,32 = 9.169, P < 0.01), but did not alter the reduction in rearing (treatment factor: F1,32 = 202.6, P < 0.001; pretreatment factor: F1,32 = 0.784, P > 0.05; pretreatment 9 treatment interaction: F1,32 = 0.014, P > 0.05). Effects of dipyrone on body temperature Administration of dipyrone (50, 200 and 500 mg/kg) decreased body temperature, as evidenced by a reduction in the thermal index (F4,31 = 11.07; P < 0.001; Fig. 4a,b). The time points included in the thermal index were based on the duration of 200 mg/kg dipyrone-induced hypothermia. This dose was also used to evaluate a possible mechanism for dipyroneinduced hypothermia. Body temperature was also reduced by administration of WIN 55,212-2 (F2,26 = 7.828; P < 0.001; Fig. 4c,d). Pretreatment with AM251 (3 mg/kg) accentuated dipyroneinduced hypothermia (treatment factor: F1,30 = 50,79, P < 0.001; pretreatment factor: F1,30 = 5.71, P < 0.05; pretreatment 9 treatment interaction: F1,30 = 5.035, P < 0.05), similar to the effect of 10 mg/kg AM251 (treatment factor: F1,29 = 62,87, P < 0.001; pretreatment factor: F1,29 = 4.023, P = 0.054; pretreatment 9 treatment interaction: F1,29 = 7.179, P < 0.05; Fig. 5a). However, the hypothermia induced by dipyrone was not affected

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CB1 receptors and effects of dipyrone

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Fig. 4 Time course of the effect of (a,b) dipyrone (n = 6–8 mice per group) and (c,d) the cannabinoid CB1 receptor agonist WIN 55,212-2 (n = 7–8 mice per group) on body temperature (a,c) and thermal index (b,d). Data are the mean  SEM. *P < 0.05, **P < 0.01, ***P < 0.001 compared with saline or vehicle control groups.

by pretreatment with the CB2 receptor antagonist AM630 (treatment factor: F1,16 = 31.88, P < 0.001; pretreatment factor: F1,16 = 4.151, P = 0.059; pretreatment 9 treatment interaction: F1,16 = 0,002, P > 0.05; Fig. 5b). In addition, the hypothermic effect of dipyrone was accentuated by pretreatment with the transient receptor potential vanilloid 1 (TRPV1) antagonist capsazepine (treatment factor: F1,30 = 110.8, P < 0.001; pretreatment factor: F1,30 = 28.56, P < 0.001; pretreatment 9 treatment interaction: F1,30 = 26.75, P < 0.001; Fig. 5c).

DISCUSSION The results of the present study revealed that dipyrone treatment produced an analgesic effect in response to a thermal stimulus, a decrease in locomotor activity accompanied by a cataleptic state

and hypothermia. These observations are similar to those following treatment with the cannabinoid CB1 receptor agonist WIN 55,212-2. In addition, the CB1 receptor antagonist AM251 was able to reverse the dipyrone-induced effects, with the exception of hypothermia. Indeed, AM251 and the TRPV1 receptor antagonist capsazepine accentuated the hypothermia evoked by dipyrone, and the CB2 cannabinoid receptor antagonist AM630 did not alter this response. These findings indicate that the mechanism of action of dipyrone may involve inhibition of COX and FAAH, because these enzymes are also related to the endocannabinoid system. The molecular mechanisms of the analgesic and antipyretic actions of dipyrone have long been under debate.15 These actions seem to depend on COX inhibition and decreased PGE2 synthesis,3 but the exact mechanisms remain unclear. Based on these suggested interactions between COX and the endocannabinoid

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Fig. 5 Time course of changes in body temperature and thermal index following drug treatment. (a) Vehicle or AM251 pretreatment followed by saline or dipyrone (n = 6–8 mice per group). (b) Vehicle or AM630 pretreatment followed by saline or dipyrone (n = 6 mice per group). (c) Vehicle or capsazepine pretreatment followed by saline or dipyrone (n = 7–8 mice per group). Data are the mean  SEM. **P < 0.01, ***P < 0.001 compared with the vehicle + saline group; ††P < 0.01, †††P < 0.001 compared with vehicle + dipyrone.

system, the aim of the present study was to evaluate the involvement of endocannabinoids in the actions of dipyrone using the tetrad test as an experimental paradigm. The tetrad test is based on cannabinoid receptor-mediated effects on behaviour and is widely used for screening drugs. These effects, namely antinociception, hypolocomotion, catalepsy (impaired initiation of movement) and hypothermia, are together called the ‘tetrad model’ of cannabimimetic activity.16,17 Despite the fact that the tetrad effects do not exhaustively represent the myriad behavioural and autonomic actions of cannabinoids, it is one of the best available measures of the cannabimimetic activity of drugs and has been

used extensively to identify and classify cannabinoid compounds.18,19 In the present study we observed that dipyrone treatment resulted in very similar effects to those generated by cannabinoids, suggesting a possible role of this system in the mechanism of action of dipyrone. The therapeutic potential of cannabinoids has been the topic of extensive investigation following the discovery of cannabinoid receptors and their endogenous ligands.20 Multiple lines of evidence indicate that endocannabinoids serve as natural pain suppressors. Although it is now clear that cannabinoids suppress nociceptive neuro-

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CB1 receptors and effects of dipyrone transmission, more work is needed to establish the clinical usefulness of these compounds.21 In the present study, dipyrone and CB1 receptor agonist treatment increased the latency response in a hotplate test, indicating an analgesic effect of dipyrone.22 The analgesic properties of the endocannabinoid system have also been demonstrated for other drugs used extensively for the treatment of pain, such as paracetamol, ibuprofen and indomethacin.9,23–25 The CB1 receptor seems to be involved in analgesic effects and other responses, possibly by inhibiting the activity of adenylate cyclase.16,26 In our studies, we observed that pretreatment with the CB1 receptor antagonist AM251 reversed the analgesic effect of dipyrone. Thus, two possible mechanisms can be suggested. First, dipyrone inhibits COX-1/2, which provides more arachidonic acid as a substrate for the synthesis of prostaglandins and other prostanoids, endocannabinoids, leukotrienes and lipoxins,27 which, in turn, act on cannabinoid receptors. Although it is not clear which arachidonic acid pathways contribute to the synthesis of endocannabinoids (anandamide or other), Pestonjamasp and Burstein28 showed the anandamide levels increased only when RAW264.7 cells were pretreated with indomethacin, a COX-2 inhibitor. In addition, other NSAIDs seem to be able to interact with the COX-2 pathway, such as indomethacin and flurbiprofen.29–32 Second, this mobilization and availability of arachidonic acid to produce cannabinoids could act on other related receptors, including the vanilloid receptor (TRPV1), which is considered an important therapeutic target in pain treatment; both agonists and antagonists of TRPV1 are being evaluated as potential analgesics for clinical use.33 In fact, the effects of dipyrone on the endogenous cannabinoid system are controversial. Previous studies demonstrated that AM251 and AM630 (CB1 and CB2 receptor antagonists, respectively) were not able to decrease the peripheral antinociceptive effect when dipyrone was injected into the hind paw of rats and animals were tested using the von Frey34 or hotplate and tail-flick tests.35 The divergence of results may be related to the lower temperature used by us on hotplate tests. Plone et al.36 suggested that lower hotplate temperatures reduce the intensity of the noxious stimulus. In addition, a lower hotplate temperature test shows greater sensitivity and offers more statistical power. Furthermore, Suh et al.37 demonstrated greater sensitivity at a 50°C than 55°C hotplate. Nevertheless, dos Santos et al.38 showed that, similar to dipyrone, the metabolites 4-MAA and 4-AA reduce PGE2-induced hyperalgesia in a nociceptive paw electronic pressure meter test. The antihyperalgesic effect of 4-MAA is mediated by cGMP activation and KATP channel opening. In contrast, the antihyperalgesic effect of 4-AA is associated with cannabinoid CB1, but not CB2, receptor activation.38 The association between the bioactive metabolite of dipyrone and the cannabinoid system, particularly CB1 receptors, supports our hypothesis that cannabinoid CB1 receptors mediate the effects of dipyrone. In addition, all metabolites of dipyrone show a low binding to plasma proteins and are distributed homogeneously and quickly throughout the body due to their ability to easily cross the blood–brain barrier, allowing a central action of dipyrone. Recently, Escobar et al.39 showed that antagonizing CB1 cannabinoid receptors in the periaqueductal grey (PAG) or the rostral ventrolateral medulla (RVLM) reduced the antinociceptive effect

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of an intralateral–ventrolateral PAG microinjection of dipyrone. Our results corroborate with the hypothesis that cannabinoid CB1 receptors participate in the antinociceptive action in an acute pain test.39 Both dipyrone and CB1 receptor agonist treatment increased the immobility time in animals, leading to a cataleptic state. Volunteer studies have consistently shown impairment of a variety of motor tasks when treated with CB1 agonists.40,41 In rats, dosedependent ataxia and catalepsy were observed after systemic administration of cannabinoids.42 In addition, D9-tetrahydrocannabinol (THC) induced catalepsy-like immobilization43,44 and this behaviour was antagonized by SR141716A (rimonabant; a CB1 receptor antagonist). In addition, microinjection of D9-THC into regions that express high levels of CB1 receptors, such as the nucleus accumbens, amygdala or hypothalamus, stimulated catalepsy-like immobilization.45 Furthermore, the cataleptic effect produced by dipyrone was reversed by pretreatment with a CB1 receptor antagonist, supporting the hypothesis that the endocannabinoid system is involved in the mechanism of action of dipyrone. However, we cannot exclude that the increase in endogenous levels of cannabinoids, such as anandamide, can have effects that are not specific to CB1 receptors.46 There are currently no reports regarding the involvement of TRPV1 on the induction of catalepsy. Pamplona et al.27 have shown that inhibition of COX with aspirin enhances the effects of anandamide in vivo in a catalepsy test, confirming the proposed role for COX in the metabolism of endocannabinoids.31 The psychoactive effects of cannabinoids are mediated by CB1 receptors; the tetrad effects of THC are abolished in mutant mice lacking expression of CB1 receptors,47,48 or after CB1 receptor blockade.49–51 Cannabinoids affect motor behaviour through CB1 receptors that are abundant in different parts of the basal ganglia, regulating glutamatergic and GABAergic systems within the same neuronal network. Thus, cannabinoid receptors may modulate excitatory and inhibitory neuronal transmission in the basal ganglia and thus provide dual control of movement.52–54 Consistently, pharmacological treatments with CB1 receptor agonists exert biphasic effects, especially on motor activity; low doses induce motor activity and high doses suppress motor activity or induce catalepsy.53,55–58 In the open field test, we showed that animals treated with high doses of dipyrone and WIN 55,212-2 were underactive. This hypolocomotive phenotype is one of the characteristics included in the tetrad effect of cannabinoids. The antagonist AM251 reversed dipyrone-induced hypolocomotion, suggesting involvement of CB1 receptors in the actions of dipyrone and supporting our hypothesis that dipyrone activity is integrated with the endocannabanoid system. Finally, hypothermia was the last parameter of the tetrad test used to determine the effects of dipyrone on the endocannabinoid system. The presence of the dipyrone metabolites 4-MAA and 4-AA in cerebrospinal fluid and the hypothalamus was associated with inhibition of PGE2 synthesis and a reduction in lipopolysaccharide-induced fever;59,60 however, 4-MAA has also been shown to be antipyretic in PGE2-independent fever induced by Tityus serrulatus venom.59 This suggests that 4-MAA is responsible for the PGE2-independent antipyretic activity of dipyrone59 because it was not mechanistically linked to the inhibition of hypothalamic PGE2 synthesis.60 Both dipyrone and cannabinoid agonist treatment evoked hypothermia and a CB1

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receptor antagonist blocked the analgesic, hypolocomotion and catalepsy responses to dipyrone. This could indicate the involvement of cannabinoid receptors in the dipyrone-stimulated hypothermic response. However, pretreatment with the cannabinoid antagonist AM251 accentuated the hypothermic response evoked by dipyrone. In addition, pretreatment with AM251 and treatment with the vanilloid receptor antagonist capsazepine further increased dipyrone-triggered hypothermia, suggesting no involvement of these receptors in dipyrone-regulated antipyretic activity. Finally, the cannabinoid CB2 receptor was blocked with AM630; this treatment did not alter hypothermia triggered by dipyrone. Rogosch et al.15 suggested a new hypothesis for the analgesic effect of dipyrone: that an increase in arachidonic acid levels by COX inhibition could act in a combinatory manner with the acylation of the primary metabolite of dipyrone (MAA) to stimulate CB1 receptors. Hence, Rogosch et al.15 postulated that dipyrone acts as a prodrug for two substances that potentially elicit analgesic effects through the endocannabinoid system. A similar analgesic mechanism has been shown for paracetamol-induced antinociception, which involves the cannabinoid system,9,11 and suggests a strategy for developing central nervous system analgesics based on coexpression of FAAH and TRPV1 in the brain.9 Thus, both paracetamol and dipyrone are converted to arachidonoyl amides, which, in turn, activate CB1 receptors.9–11,24,25 It is likely that other drugs may be converted to fatty acid derivatives that are responsible for at least part of their pharmacological activity.24 Moreover, knowledge of this dual mechanism of analgesia by arachidonoyl amides would allow deliberate development of pain relievers with higher activity than dipyrone and paracetamol and allow for lower dosages.9–11,24,25 Inhibition of COX-1 and COX-2 by dipyrone can be accomplished by activation of cannabinoid receptors (CB1 and CB2), suggesting that these receptors are candidate targets for the development of analgesic drugs.61 G€uhring et al.29 proposed a possible interaction between the analgesic activity of COX and endocannabinoids in a behavioural study; they observed further evidence for indomethacin activity at the spinal level by at least three mechanisms. First, indomethacin blocks COX-1/2, resulting in greater amounts of arachidonic acid for endocannabinoid synthesis. Second, it lowers NO production, reducing activation of endocannabinoid transporters and hence endocannabinoid degradation. Third, indomethacin inhibits FAAH, contributing to the preservation of endocannabinoids.29 In other words, the findings of that study suggest that indomethacin is able to enhance endocannabinoid tone by interacting with the endocannabinoid system at several levels and contributing to its analgesic effects.29,61 In conclusion, the endocannabinoid system is involved in the actions of dipyrone in eliciting analgesia, catalepsy and hypolocomotion, but its involvement in hypothermic responses is uncertain. These effects, with the exception of the hypothermic response, occur via activation of CB1 receptors. Indeed, hypothermic effects are accentuated by antagonists of CB1 receptors and TRPV1. Based on these observations and previous reports, our hypothesis is that the mechanism of action of dipyrone may involve increased availability of arachidonic acid as a substrate for the synthesis of endocannabinoids or other related molecules. This increase in endocannabinoid availability, in turn,

increases CB1 receptor stimulation, contributing to the observed effects.

METHODS Animals Adult Swiss male mice (40  5 g; Central Animal Facility, Federal University of Alfenas) were used in the present study. Mice were given commercial feed and water ad libitum throughout the experiment and were adapted for 7 days in a 23  2°C room in polypropylene boxes. For the experiments, mice were divided into groups and maintained separately. All procedures were conducted in accordance with Animals in Research: Reporting In vivo Experiments (ARRIVE) guidelines62 and with the approval of the Ethics Committee of the Federal University of Alfenas (no. 360/2011). Drug administration All drugs were administered by intraperitoneal injection. Dipyrone (Sigma, St Louis, MO, USA) was dissolved in sterile isotonic saline (0.9% NaCl). The CB1 receptor antagonist AM251 (1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide) and agonist (R)-(+)-WIN 55,212-2 mesylate salt were purchased from Sigma-Aldrich (St Louis, MO, USA) and were dissolved in a solution containing 0.9% NaCl, Tween and dimethyl sulphoxide (DMSO) at a ratio of 8 : 1 : 1. The CB2 cannabinoid receptor antagonist AM630 (6-iodo-2-methyl-1-[2(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)methanon) was purchased from Tocris (Ellisville, MO, USA) and the TRPV1 receptor antagonist capsazepine (N-[2-(4-chlorophenyl)ethyl]1,3,4,5-tetrahydro-7,8-dihydroxy-2H-2-benzazepine-2-carbothioamide) was purchased from Sigma. Both were dissolved in a solution containing 0.9% NaCl, Tween and DMSO at a ratio of 8 : 1 : 1. The experimental protocol was as follows. First, mice were treated with saline (control group) or dipyrone (10, 50, 200 or 500 mg/kg, i.p.) and were evaluated in four tests: hotplate (analgesia), catalepsy, open field (locomotion) and temperature. Second, mice were treated with vehicle (control group) or WIN 55,212-2 (0.3, 1.0 or 3.0 mg/kg, i.p.) and were then evaluated in the same four tests. Third, mice were pretreated with vehicle or AM251 (3 mg/kg, i.p.) and then, 10 min later, were injected with saline or dipyrone (500 mg/kg, i.p.). The mice were then evaluated in the hotplate, catalepsy and open field tests. Fourth, for temperature evaluation only, mice were pretreated with AM251 (3 or 10 mg/kg, i.p.), AM630 (10 mg/kg, i.p.) or capsazepine (10 mg/kg, i.p.) for 10 min before being injected with saline or dipyrone (200 mg/kg, i.p.). Hotplate test This test is used to measure the latency of responses to a thermal stimulus and is suitable for evaluating analgesic actions. Mice were placed individually on a hotplate (50  1°C) in a clear acrylic case. The time required for the first response (licking paws, jumping or quickly removing paws) was recorded. The maximum time allowed was 30 s to prevent tissue damage.

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CB1 receptors and effects of dipyrone Responses were evaluated 0, 30, 60, 120, 240 and 360 min after drug injection. Catalepsy Catalepsy was evaluated by placing the mouse with both forelegs over a horizontal glass bar (diameter 0.5 cm) 4.5 cm above the floor. The length of time (s) the mouse maintained this position was recorded for up to 300 s.62 The test was considered finished when at least one forepaw touched the floor or the mouse climbed up onto the bar. Latencies were measured immediately prior to and up to 6 h after drug or vehicle administration. Open field behavioural test Locomotor activity was quantified for 5 min in an open field apparatus consisting of a circular arena (30 cm diameter, 45 cm walls and a floor divided into 12 areas). A 15 cm diameter circle in the centre of the arena divided into four regions was defined as the central area. The eight areas along the walls were considered as peripheral areas. The number of peripheral (adjacent to the wall) and central (away from the wall) areas that the rat entered, with all four paws and rearing, was recorded for 5 min.63 Body temperature measurement Body temperature was measured continuously by biotelemetry, as described previously.63 Mice were anaesthetized with 2,2,2tribromoethanol (Aldrich, Milwaukee, WI, USA; 250 mg/kg, i.p.). A paramedian laparotomy was performed for the insertion of a biotelemetry probe capsule (model ER-3000 temperature; Mini-Mitter, Sunriver, OR, USA) into the abdominal cavity. The wound was then sutured and the implanted capsule was used for the measurement of core body temperature. After surgery, mice were treated with benzylpenicillin (100 000 U, i.m.) and housed individually. One week after surgery, cages were placed on a telemetry receiver (model ER-3000; Mini-Mitter) connected to a computer and body temperature was recorded at 10 min intervals during the experiments. Data were collected using VITAL VIEW software (Mini-Mitter). The thermal index (°C 9 min) were used to improve data analysis and was calculated as an area under the curve of body temperature measurements, as described previously.64 Statistical analysis Data are expressed as the mean  SEM. Comparisons of different groups were performed with one- or two-way ANOVA with post hoc comparisons for differences (Newman–Keuls’ test). A log transformation was performed on catalepsy data prior to statistical analysis.65 Differences were considered significant at twotailed P < 0.05. All statistical tests were performed using Prism 6.0 (GraphPad Software, San Diego, CA, USA).

ACKNOWLEDGEMENTS The authors are grateful for the excellent technical support of Marina F Ven^ancio and Jose dos Reis Pereira. This work was

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supported by Fundacß~ao de Amparo a Pesquisa do estado de Minas Gerais (FAPEMIG), Conselho Nacional de desenvolvimento Cientıfico e Tecnol ogico (CNPq) and Coordenacß~ao de Aperfeicßoamento de pessoal de nıvel superior (CAPES).

DISCLOSURE None of the authors have a conflict of interest to declare.

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Cannabinoid CB1 receptors mediate the effects of dipyrone.

Dipyrone is a non-steroidal anti-inflammatory drug used primarily as an analgesic and antipyretic. Some hypothesize that dipyrone activity can modulat...
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