Endogenous Opioid and Cannabinoid Mechanisms Are Involved in the Analgesic Effects of Celecoxib in the Central Nervous System
R.M. Rezende a P. Paiva-Lima a W.G.P. Dos Reis a V.M. Camêlo a A. Faraco b
Y.S. Bakhle c J.N. Francischi a
a Laboratory of Inflammation and Pain, Department of Pharmacology, Institute of Biological Sciences, and b Laboratory of Pharmaceutical Products, Pharmacy School, Federal University of Minas Gerais, Belo Horizonte , Brazil; c Leukocyte Biology, National Heart and Lung Institute, Faculty of Medicine, Imperial College, London , UK
Introduction
The selective cyclooxygenase (COX)-2 inhibitor cele-coxib has been extensively used in the treatment of osteo-arthritis and rheumatoid arthritis, with less gastrointes-tinal toxicity than the ‘classic’ nonsteroidal anti-inflam-matory drugs (NSAIDs) [1, 2] . This compound and other selective inhibitors of COX-2 (hereafter referred to as ‘coxibs’) exhibit the 3 characteristic biological activities observed for classic NSAIDs – antipyretic, anti-inflam-matory and analgesic [3] – attributed to their inhibition of prostaglandin biosynthesis [4] . However, in a well-es-tablished model of inflammatory pain induced by carra-geenan in rat paws [5, 6] , we found that celecoxib given systemically (s.c. injection) exerted antinociceptive ac-tions that differed from those of the classic NSAIDs, such as indomethacin or piroxicam [7] . In particular, the an-algesic actions of celecoxib were, unlike those of indo-methacin, sensitive to blockade by opioid receptor antag-onists [8] . This, along with other differences, has led us to propose that the analgesic actions of celecoxib we ob-served did not involve the inhibition of COX-2. Compa-rable analgesic effects in this model of inflammatory pain
Key Words
Celecoxib � Hypoalgesia � Opioids � Cannabinoid receptor � Fatty acid amide hydrolase � Central nervous system
Received: November 1, 2011 Accepted after revision: January 1, 2012 Published online: March 10, 2012
Prof. Janetti N. de Francischi Departamento de Farmacologia, ICB-UFMG Av. Antônio Carlos 6627 Pampulha, Belo Horizonte, MG 31901-270 (Brasil) Tel. +55 31 3409 2715, E-Mail janettif @ icb.ufmg.br
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Pharmacology 2012;89:127–136128
were also observed after giving celecoxib or its congener SC 236 to the central nervous system (CNS), by injection into the cerebral ventricles [9] .
We analyzed further this central analgesic effect of ce-lecoxib in terms of possible mechanisms, including the inhibition of COX-2. We used a structural analog of cele-coxib which does not inhibit COX-2 and assessed the con-tribution of two other endogenous systems, opioid and endocannabinoid, to the analgesic effects of celecoxib. Our results confirmed that COX-2 inhibition is not nec-essary for the characteristic analgesic effects, strength-ened the evidence for the participation of endogenous opioids and disclosed a new and crucial role played by cannabinoid (CB) CB 1 receptors in celecoxib-induced an-algesia.
Material and Methods
Animals All animal care and experimental procedures adhered to the
guidelines of the Committee for Research and Ethical Issues of IASP [10] and were approved by the local Ethics Committee for Animal Experimentation. Male Holtzman rats (total = 194), weighing 170–200 g and supplied by the Bioterism Center of the Federal University of Minas Gerais, were used in these experi-ments. After implantation of the cannula in the right lateral cere-bral ventricle (see below), animals were housed one per cage with food and water ad libitum, with light/dark cycles of 12/12 h, start-ing at 7.00 a.m. The animals used were killed humanely at the end of the 6-hour experimental period.
Reagents We used celecoxib (Celebra � ; Pfizer Pharmaceuticals LLC –
Caguas, Puerto Rico), � -funaltrexamine, naltrindole, nor-binal-torphimine and AM 251 (Tocris, UK), SR 144528 (a generous gift from Sanofi-Aventis, France), bestatin (Genaxxon Bioscience, Germany), URB 597 (Cayman Chemical, USA) and � -carrageen-an (Sigma, USA).
Cannulation of the Right Lateral Cerebral Ventricle Intracerebroventricular (i.c.v.) injection was performed as be-
fore [9] . Briefly, under anesthesia induced by ketamine and xyla-zine (10 and 2%, respectively; 1 ml/kg, i.p.-supplemented when necessary) unilateral stainless steel guide cannulas (22-gauge, 16-mm length) were placed within the right lateral cerebral ventricle of rats. Animals were positioned in a stereotaxic frame (Kopf In-struments, USA) with the tooth bar fixed 5.0 mm above the level of the interaural line. Guide cannulas were positioned using the bregma as reference point and according to coordinates adapted from the atlas of Paxinos and Watson [11] : posterior –1.5 mm, lat-eral –2.5 mm and ventral 3.0–3.4 mm below the skull surface. The guide cannulas were fixed with dental acrylic cement and an-chored by screws placed in the skull. After the surgical proce-dures, the animals were closely monitored for 4 h and used in experiments 1 week later.
Intracerebroventricular Injections Microinjections of drugs were performed with a 30-gauge in-
jection needle (17-mm length), connected to a polyethylene tub-ing (0.010 ID, Norton, USA) attached to a 10- � l Hamilton sy-ringe (Reno, USA). All i.c.v. injections were made in a volume of 5 � l. Celecoxib (5.5–44 � g [9] ) was diluted in sterile saline and cautiously injected into conscious animals 30 min before intra-plantar injection of carrageenan. � -Funaltrexamine, naltrin-dole, nor-binaltorphimine (5 � g each [12, 13] ), naltrexone (4 � g [9] ) and bestatin (40 � g [9] ) diluted in sterile saline, and AM 251 (10 � g [14] ), SR 144528 (10 � g [15] ) and URB 597 (0.01, 0.1 and 1 � g [16] ) diluted in DMSO sterile saline (30–70%) were cautious-ly injected i.c.v. 30 min before celecoxib. Control animals re-ceived the same volume of the corresponding drug vehicle. At the end of the experiments, rats were injected i.c.v. with 5% Evans Blue (5 � l) and then killed by cervical dislocation for confirma-tion of the appropriate location of the cannula. If a cannula was incorrectly positioned, the result from that particular animal was excluded.
Induction of Paw Inflammation Inflammation was induced in one hind paw (right) using � -
carrageenan (250 � g in 0.1 ml) diluted in sterile physiological sa-line (NaCl 0.9%) as the proinflammatory stimulus at time zero. The contralateral paw was injected with the same volume of phys-iological saline (0.1 ml). In earlier studies, this dose of carrageen-an was shown to induce paw hyperalgesia [7, 9, 17, 18] .
Measurement of Nociceptive Threshold in Paws The assessment of nociception consisted of measurement of
the threshold stimulus for nociceptive reaction (paw withdrawal) using a weight (with a maximum limit of 500 g) applied to the pads of hind paws by an experimenter using a Ugo Basile appara-tus, based on the Randall-Selitto method [19] . The threshold for eliciting a nociceptive response was measured before (time zero) and at 0.5, 1, 2, 3, 4 and 6 h after the intraplantar injection of car-rageenan. Results are presented as the absolute values of nocicep-tive threshold found for each hind paw at the indicated time-points or as the area under the curves (AUC) obtained by the trap-ezoidal rule from the time courses over the 2-hour interval (right/left paw values). Thus, a fall in nociceptive thresholds (hyperalge-sia) would generate a negative AUC and hypoalgesia would gener-ate positive AUC values.
Systemic AM 251 Treatment In one set of experiments, the CB 1 receptor antagonist, AM
251, was also administered subcutaneously (2 mg/kg, 0.1 ml/100 g of weight [20] ) 30 min before or after i.c.v. injection of cele-coxib.
Statistical Analysis Results are presented as the mean values ( 8 SEM) obtained
from groups of 4–5 animals for each condition. Differences be-tween mean values were considered statistically significant when comparisons between the control (vehicle + carrageenan) and treated (drug + carrageenan) animals – using 1-way ANOVA, fol-lowing Bonferroni’s post hoc test – gave p values ! 0.05.
Mechanisms of Celecoxib Analgesia Pharmacology 2012;89:127–136 129
Results
Characterization of Basal Conditions: Celecoxib-Induced Analgesia in Carrageenan-Injected Rat Paws
Carrageenan injected into the right hind paw induced a fall in the nociceptive threshold of that paw, demon-strating the hyperalgesia characteristic of this model of inflammatory pain (see fig. 1 and 6 for AUC and other figs. for the time course). Pretreatment of animals with i.c.v. celecoxib (5.5, 11, 22 and 44 � g) dose-dependently raised the threshold in the carrageenan-injected (in-flamed) paw to values well above that found under basal conditions (time = 0) for up to 1 h after carrageenan. An antihyperalgesic effect was maintained until the third hour after carrageenan with the higher doses of celecox-ib (22 and 44 � g). These data are summarized as AUC in figure 1 . Nociceptive thresholds were affected by celecox-ib only in the inflamed paw and those in the contralat-eral, uninflamed paw were not changed either by the in-jection of carrageenan or by i.c.v. celecoxib (data not shown), confirming our previous data [9] . As celecoxib
induced unilateral hypoalgesia, i.e. in the inflamed paw only, subsequent results will show only data from the right, inflamed paw.
Mechanisms of Analgesia Induced by i.c.v. Administration of Celecoxib Because we proposed that the analgesic actions of cele-
coxib in our model are not causally related to its inhibition of COX-2, we tested a structural analog of celecoxib, OSU 03012, lacking COX-2 inhibitory activity [21] . We injected this compound i.c.v., at a dose of 22 � g, 30 min before car-rageenan injection to the paw, i.e. exactly the same proto-col as used for celecoxib, and measured nociceptive thresholds over the next 6 h. As shown in figure 2 , OSU 03012 prevented the carrageenan-induced hyperalgesia and raised the nociceptive threshold to above normal lev-els, a response very similar to that seen after the same dose of celecoxib (22 � g) in a different set of animals. Impor-tantly, treatment with OSU 03012 did not affect the noci-ceptive thresholds of the contralateral, noninflamed paws, as noted above for celecoxib (data not shown).
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Fig. 1. Effect of celecoxib (given i.c.v.) on the hyperalgesia induced by carrageenan in rat paws. Results are presented as AUC of no-ciceptive threshold versus time, over 2 h, calculated using the trapezoidal rule (means 8 SEM). Negative values of AUC repre-sent hyperalgesia (a fall in nociceptive threshold) and positive val-ues represent hypoalgesia (a threshold raised above basal). All rats were injected with carrageenan (250 � g in 100 � l) in the right paw and saline (100 � l) in the left paw. Animals were pretreated (i.c.v. injection; 5 � l) with either vehicle (C) or celecoxib (CX; 5.5, 11, 22 or 44 � g) 30 min before carrageenan. The data show a dose-relat-ed reversal of hyperalgesia and induction of hypoalgesia by cele-coxib. * Significantly different from vehicle (C) values. # Signifi-cantly raised above basal threshold: 1-way ANOVA, p ! 0.05.
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Fig. 2. Time course of the effect of the celecoxib analog, OSU 03012, injected i.c.v. on the hyperalgesia induced by carrageenan in rat paws. All rats were injected with carrageenan (250 � g in 100 � l) in the right paw and saline (100 � l) in the left paw. Animals were pretreated (i.c.v. injection; 5 � l) with either vehicle (C) or OSU 03012 (OSU; 22 � g) 30 min before carrageenan. Nociceptive thresholds (mean 8 SEM) were measured at the times indicated, for 6 h after carrageenan injection. Centrally injected OSU 03012 induced hypoalgesia for at least 1 h, as did celecoxib (22 � g, i.c.v.), shown here for comparison. * Significantly different from vehicle (C) values. # Significantly raised above basal threshold: 1-way ANOVA, p ! 0.05.
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Involvement of � - and � -Opioid Receptors in Analgesia Induced by Celecoxib We showed previously [8, 9] that celecoxib-induced
analgesia in this model was prevented by the nonselective opioid receptor antagonist, naltrexone. We used selective antagonists to define more clearly the opioid receptors involved. Thus, � -funaltrexamine, naltrindole or nor-binaltorphimine (5 � g each) selective for � -, � - and � -opioid receptors, respectively, were given i.c.v. 30 min be-fore i.c.v. injection of celecoxib. As shown in figure 3 a and b, � -funaltrexamine or naltrindole completely prevented the analgesia induced by celecoxib, without affecting the contralateral paw (data not shown). However, nor-binal-torphimine ( fig. 3 c) did not modify celecoxib’s effects. Note that none of the antagonists used affected the car-rageenan-induced hyperalgesia.
Involvement of CBs in Celecoxib-Induced Analgesia Although the results obtained thus far supported the
involvement of the opioid system in celecoxib-induced analgesia, celecoxib itself is not an agonist at opioid recep-tors [22] and so celecoxib may be acting indirectly, by re-leasing endogenous opioids. In one example of endoge-nous opioid release, Ibrahim et al. [23] showed that activa-tion of CB receptors induced � -endorphin release. We therefore tested two CB receptor antagonists, AM 251 se-lective for CB 1 and SR 144528 selective for CB 2 receptors, in our model. AM 251 ( fig. 4 a), but not SR 144528 ( fig. 4 b), given i.c.v. 30 min before celecoxib, completely prevented the analgesic effects induced by celecoxib. Moreover, nei-
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Fig. 3. Effects of selective antagonists of � -, � - or � -opioid recep-tors on the analgesic effects of celecoxib. All rats were injected with carrageenan (250 � g in 100 � l) in the right paw and saline (100 � l) in the left paw. Animals were pretreated (i.c.v. injection; 5 � l) with the antagonist (5 � g or saline) 30 min before celecoxib, and with celecoxib (CX; 22 � g) or saline (SAL) 30 min before car-rageenan. Nociceptive thresholds (mean 8 SEM) were measured at the times indicated, for 6 h after carrageenan injection. a Pre-treatment with the � -opioid antagonist, funaltrexamine (FNT) abolished the analgesic effects of celecoxib (22 � g). Funaltrex-amine given without celecoxib (FNT + SAL) did not affect the hyperalgesia induced by carrageenan. b Pretreatment with the � -opioid antagonist naltrindole (NTD) also totally blocked the an-algesic effects of celecoxib without affecting carrageenan-induced hyperalgesia (NTD + SAL). c By contrast, the � -opioid antagonist, norbinaltorphimine (BNI), did not modify celecoxib-induced hy-poalgesia or the carrageenan-induced hyperalgesia. * Significant-ly different from corresponding values without celecoxib treat-ments. * * Significantly different from SAL + CX values. # Signifi-cantly raised above basal threshold: 1-way ANOVA, p ! 0.05.
Mechanisms of Celecoxib Analgesia Pharmacology 2012;89:127–136 131
ther antagonist given alone affected carrageenan-induced hyperalgesia ( figs. 4 a, b) or basal nociceptive thresholds of contralateral, noninflamed paws (data not shown).
If, as suggested by these effects of AM 251, celecoxib were acting through the CB 1 receptor to release endoge-nous opioids, then the effects of bestatin, demonstrated in our previous work [9] , which depend on endogenous opioid peptides, should be affected by AM 251 as well. In the next set of experiments to test this proposition, we gave AM 251 by s.c. injection (2 mg/kg, 0.1 ml/100 g of weight) at the same time as i.c.v. bestatin (pretreatment) or 30 min after i.c.v. celecoxib injection and therefore at the same time as the intraplantar carrageenan injection (after treatment, relative to celecoxib). The combination of bestatin and low-dose celecoxib induced analgesia as expected, but this analgesia was prevented by pretreat-ment with AM 251 ( fig. 5 a). Interestingly, when the CB 1 receptor antagonist was given, 30 min after celecoxib, it did not affect the analgesia induced by the combination of bestatin and low-dose celecoxib ( fig. 5 b).
Effect of an Irreversible Inhibitor of Fatty Acid Amide Hydrolase on Hypoalgesia Induced by Celecoxib
If celecoxib was acting through the CB 1 receptor, this action could represent a direct or indirect (by release of
endocannabinoids) activation of the receptor. A major endocannabinoid is anandamide, which is inactivated by the fatty acid amide hydrolase (FAAH) and inhibition of FAAH should potentiate the actions of such endogenous CBs [24–26] . We used URB 597, an irreversible inhibitor of FAAH, given i.c.v. at doses of 0.01, 0.1 or 1 � g (0.025–2.5 nmol), 30 min before i.c.v. injection of celecoxib and its effects are shown in figure 6 . URB 597 dose-depend-ently prevented celecoxib-induced analgesia, with com-plete inhibition at 1 � g. However, URB 597, at the highest dose (1 � g), did not affect carrageenan-induced hyperal-gesia ( fig. 6 ) or the basal nociceptive thresholds of contra-lateral, noninflamed paws (data not shown).
Discussion
Our results show that, in a model of inflammatory pain in rats, celecoxib given i.c.v. induced analgesia medi-ated by the activation of � - and � -opioid receptors and by involving the CB 1 but not the CB 2 receptor. The simplest explanation of these results is that the analgesic actions of celecoxib in this model were exerted indirectly by the release of endogenous opioid peptides following the acti-vation of CB 1 receptors. The results also support the con-
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Fig. 4. Time-course of the effects of selective antagonists of CB 1 or CB 2 receptors on the analgesia induced by celecoxib after car-rageenan. All rats were injected with carrageenan (250 � g in 100 � l) in the right paw and saline (100 � l) in the left paw. Animals were pretreated (i.c.v. injection; 5 � l) with AM 251 (10 � g) or SR 144528 (SR; 10 � g) 1 h before carrageenan, and with celecoxib (CX; 22 � g) 30 min before carrageenan. Nociceptive thresholds (mean 8 SEM) were measured at the times indicated, for 6 h after carrageenan injection. a Pretreatment with the CB 1 receptor an-
tagonist AM 251 totally blocked the analgesic effect of celecoxib, without affecting the hyperalgesia induced by carrageenan. b However, the CB 2 antagonist SR 144528 (SR) did not affect either celecoxib-induced hypoalgesia or carrageenan-induced hyperal-gesia, when given alone (SR + SAL). * Significantly different from values without celecoxib treatment. * * Significantly different from VEH + CX values. # Significantly different above basal threshold: 1-way ANOVA, p ! 0.05. VEH = Vehicle.
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cept that inhibition of COX-2 by celecoxib was not rele-vant to this analgesic action.
We proposed a lack of causality between COX-2 inhi-bition and celecoxib-induced analgesia from our first ex-periments disclosing the characteristic hypoalgesic ef-fects of celecoxib compared with the antihyperalgesic ef-fects of other NSAIDs, such as indomethacin and piroxicam [7] . The divergence between COX-2 inhibition and celecoxib-induced analgesia was recently strength-ened by the results with lumiracoxib, a more potent and more selective COX-2 inhibitor than celecoxib [27] , which, like the classical NSAIDs, did not induce hypoal-gesia but only provided antihyperalgesia [28] . Moreover, given that indomethacin and piroxicam inhibit COX-1 more potently than COX-2 [29] and that the selective COX-1 inhibitor SC 560 [30] also showed only antihyper-algesia [8] , it would appear that the inhibition of prosta-glandin biosynthesis in general was not a causal event in celecoxib-induced hypoalgesia. Finally, in this series of experiments, we found that OSU 03012, a structural ana-log of celecoxib but lacking COX inhibitory activity [21] , induced analgesia identical to that induced by celecoxib. The simplest explanation of all these results is that, in our
model, celecoxib induced analgesia by a mechanism oth-er than the inhibition of COX-2.
The initial indication of opioid involvement in cele-coxib-induced hypoalgesia was that it was reversed bynaltrexone, whereas analgesia after indomethacin was re-sistant [8] . Also, chronic treatment of rats with celecoxib, but not with indomethacin, induced a tolerance to its an-algesic effect [8, 31] . In addition, rats made tolerant to morphine in our model were cross-tolerant to SC 236, another structural analog of celecoxib and selective in-hibitor of COX-2, and rats tolerant to celecoxib were also cross-tolerant to the analgesic effects of morphine [8, 31] . On the basis of these indications of opioid involvement in celecoxib-induced analgesia, we tested the involvement of specific opioid receptors in this effect. Either � -funal-trexamine or naltrindole (but not nor-binaltorphimine) was effective in preventing celecoxib-induced analgesia, suggesting that the activation of � - and/or � -opioid re-ceptors was a critical component of celecoxib-induced analgesia. However, this activation of opioid receptors is unlikely to be a direct agonist effect on these receptors as celecoxib is not a ligand for opioid receptors [22] . This would therefore imply an indirect activation, i.e. the re-
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Fig. 5. Reversal by AM 251 of celecoxib-induced analgesia is time-dependent. All rats were injected with carrageenan (250 � g in 100 � l) in the right paw and saline (100 � l) in the left paw. Animals were pretreated (i.c.v. injection) with bestatin (BE; 40 � g) 1 h be-fore carrageenan, and celecoxib (CX; 5.5 � g) 30 min before car-rageenan. Nociceptive thresholds (mean 8 SEM) were measured at the times indicated, for 6 h after carrageenan injection. This lower dose of celecoxib (SAL + VEH + CX 5.5) did not affect car-rageenan-induced hyperalgesia, but, combined with bestatin (BE 40 + VEH + CX 5.5), did induce hypoalgesia. AM 251 given s.c. before celecoxib abolished the analgesic effects of the combina-
tion of bestatin + celecoxib. Note that bestatin and AM 251, in the absence of celecoxib, did not modify carrageenan-induced hyper-algesia. b The same combination of bestatin and celecoxib (BE + CX 5.5 + VEH) induced hypoalgesia for at least 1 h, but when AM 251 was given (s.c.) 30 min after celecoxib, i.e 30 min later than in a , no reversal of celecoxib-induced hypoalgesia was observed. * Significantly different from values without celecoxib treatment. * * Significantly different from BE + VEH + CX values. # Signifi-cantly different above basal threshold: 1-way ANOVA, p ! 0.05. VEH = Vehicle.
Mechanisms of Celecoxib Analgesia Pharmacology 2012;89:127–136 133
lease of endogenous opioids which would be the agonists for the � - or � -opioid receptors. The most likely opioids to fit this receptor profile are the enkephalins and � -en-dorphin and these opioid peptides are found in the pe-riphery and in the CNS [32, 33] . The metabolism and in-activation of either is blocked by bestatin, and would thus be compatible with the potentiating effects we have ob-served with this peptidase inhibitor in celecoxib-induced analgesia [9] .
Some years ago, the release of � -endorphin in rat paws by a CB agonist (AM1241) was reported by Ibrahim et al. [23] . This finding prompted us to test the involvement of the CBs in the possible release of endogenous opioids af-ter i.c.v. celecoxib, using two selective CB receptor an-tagonists. Only AM 251, the CB 1 receptor antagonist, pre-vented celecoxib-induced analgesia, clearly implying that these receptors were involved. Causal connections be-tween the CB and opioid systems have been frequently reported [34] and anatomical studies have shown a simi-lar distribution of opioids and CB receptors in several structures within the CNS [35–38] . Other evidence, par-ticularly relevant in our context, includes the ability of opioid or CB antagonists to reverse opioid or CB-induced analgesia [39] and cross-tolerance between both systems [39, 40] . CB agonists, such as � 9 -tetrahydrocannabinol and CP 55,940, a nonselective CB agonist and AM 1241, a selective CB 2 agonist, all released endogenous opioids after binding at their receptors, an effect contributing to the subsequent analgesia [23, 41–45] .
If the release of endogenous opioids was subsequent to the activation of CB 1 receptors, was celecoxib the CB 1 ag-onist or was there an additional step, the release of an endocannabinoid agonist at CB 1 receptors? There are at least two pieces of evidence against celecoxib being the CB 1 receptor agonist. First, in all the extensive work on the interactions between COX and the endocannabinoid system [46, 47] , direct CB receptor agonist activity was not shown or postulated for COX inhibitors. Second, our finding, that inhibition of FAAH affected the interaction between celecoxib and the endocannabinoid system, im-plies strongly that the endocannabinoids themselves were the CB 1 receptor agonist(s) involved.
If then, celecoxib was acting indirectly to activate CB 1 receptors, how was that achieved? Most of the COX-en-docannabinoid interactions have been attributed to the inhibition of COX because COX (and particularly COX-2) will metabolize both anandamide and 2-AG to prod-ucts lacking CB receptor activity [46, 47] . Thus, inhibi-tion of COX would have effects comparable to the inhibi-tion of other endocannabinoid clearance mechanisms.
However, in our model, the celecoxib-induced hypoalge-sia was unconnected to COX inhibition, as already dis-cussed above, so this mechanism would not explain our findings. We have therefore been forced to postulate the release of a CB 1 receptor agonist, which in turn releases endogenous opioid to achieve the final analgesic effect.
Such a proposition creates further problems. The most prevalent endocannabinoid, anandamide, is not able to stimulate synthesis or release of any type of endogenous opioid [43, 44, 48] . However, the marked effect of FAAH inhibition by URB 597 on celecoxib-induced hypoalgesia suggested that anandamide, a known substrate for FAAH, was involved. Inhibition of FAAH, the major inactivation pathway for anandamide, leads to potentiation of the ef-fects of anandamide in the CNS [49] or in the periphery [50] , but we have found URB 597 to inhibit celecoxib-induced analgesia. This particular paradox may be re-solved by noting that hyperalgesia has been reported fol-
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URB 0.1 + CX 22
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Fig. 6. Dose-dependent effects of the FAAH inhibitor URB 597 on celecoxib-induced analgesia after carrageenan. Results are pre-sented as AUC (means 8 SEM) of nociceptive threshold versus time, over 2 h, calculated using the trapezoidal rule. All rats were injected with carrageenan (250 � g in 100 � l) in the right paw and saline (100 � l in the left paw). Animals were pretreated with URB 597 (URB; i.c.v. 0.01, 0.1 or 1.0 � g) 1 h before carrageenan, and celecoxib (CX; i.c.v. 22 � g) 30 min before carrageenan. The first results (left-hand bar; URB + Sal) show that the highest dose of URB 597 (1 � g, i.c.v.), in the absence of celecoxib, did not affect carrageenan-induced hyperalgesia. The hypoalgesia induced by celecoxib (Sal + CX 22) was not changed by the lowest dose of URB 597 (URB 0.01 + CX 22), but higher doses of the FAAH inhibitor progressively reversed celecoxib-induced analgesia (URB 0.1 + CX 22; URB 1 + CX 22). * Significantly different from values with-out celecoxib treatment. * * Significantly different from SAL + CX values. # Significantly different above basal threshold: 1-way ANOVA, p ! 0.05.
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lowing synthetic CB receptor agonists [51] or capsaicin [52] , delivered to the periaqueductal grey and that [53] found stimulation of TRPV1 channels after inhibition of FAAH in rat periaqueductal grey slices. Anandamide is an agonist at TRPV1 channels [54] and our results may thus represent an increase in the hyperalgesic pathways (mediated by TRPV1 channels), relative to the analgesic pathways, following FAAH inhibition.
We cannot discount the other major endocannabi-noid, 2-AG, as the CB 1 receptor agonist stimulating opi-oid release in our experiments. Although inhibition of FAAH usually only affects anandamide and inhibition of monoacylglycerol lipase [55] usually only affects 2-AG levels [56] , there are relevant reports of URB 597 increas-ing 2-AG levels [51, 57] . However, unlike anandamide, 2-AG is not an agonist at TRPV1 channels [54] and so the reversal of celecoxib-induced hypoalgesia by URB 597 would not be explained by increased 2-AG levels. Never-theless, both anandamide and 2-AG can be released to-gether [58, 59] and that might be the case in our experi-ments.
One clear limitation of our work is that only one noci-ceptive assay system was used and there are many other models of inflammatory and of noninflammatory pain [60] with different mechanisms and hence the possibility of different outcomes. Our preference for our model was based on its value in predicting clinical outcomes for the anti-inflammatory aspects of NSAIDs [61] , but clearly now that the mechanisms underlying NSAID action seem not to apply to celecoxib-induced analgesia, our findings need to be extended to other pain models.
We have analyzed celecoxib’s action after central ad-ministration by i.c.v. injection in these experiments but it is arguable that celecoxib has most of its actions in the periphery, especially after oral administration. Further-more, celecoxib crosses the blood-brain barrier less ex-tensively than other coxibs [62] making a central site of action less likely. Therefore, our present series of experi-ments will need to be repeated with the peripheral ad-ministration of celecoxib to confirm the role of CB recep-tors in celecoxib-induced analgesia.
In summary, our results have strengthened the propo-sition that celecoxib acts, in our model, by releasing en-dogenous opioid peptides and subsequent activation of � - and/or � -opioid receptors, rather than by inhibiting COX-2. The results also disclosed the activation of CB 1 receptors as another causal link in celecoxib-induced central analgesia. Although the direct activation of CB 1 receptors by celecoxib and mechanisms involving COX inhibition are unlikely, how celecoxib induces the activa-tion of CB 1 receptors remains to be established. Never-theless, the possibility raised by our work of a non-COX-based, new analgesic mechanism – to explain the relief of inflammatory pain by celecoxib – is clearly worth pursu-ing.
Acknowledgements
This work was supported by the Conselho Nacional de Pes-quisa (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação de Amparo à Pesquisa de Minas Gerais (FAPEMIG MG).
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