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Peripheral μ-, κ- and δ-opioid receptors mediate the hypoalgesic effect of celecoxib in a rat model of thermal hyperalgesia
Life Sciences 86 (2010) 951–956
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Peripheral μ-, κ- and δ-opioid receptors mediate the hypoalgesic effect of celecoxib in a rat model of thermal hyperalgesia Jôice D. Correa a, Patrícia Paiva-Lima a, Rafael M. Rezende a, Webster G.P. Dos Reis a, Dalton L. Ferreira-Alves a, Y.S. Bakhle b, Janetti N. Francischi a,⁎ a b
Department of Pharmacology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil Leukocyte Biology, National Heart and Lung Institute, Faculty of Medicine, Imperial College, SW7 2AZ London, United Kingdom
a r t i c l e
i n f o
Article history: Received 6 January 2010 Accepted 26 April 2010 Keywords: Hypoalgesia Celecoxib Hargreave's method Opioid receptors Cyclooxygenase inhibition
a b s t r a c t Aims: The endogenous opioids mediate the analgesic effects of celecoxib in a model of mechanical hyperalgesia in rats. As responses to thermal stimuli may differ from those to mechanical stimuli, we have here assessed celecoxib in a rat model of thermal hyperalgesia and the possible involvement of endogenous opioids and their corresponding receptors in these effects. Main methods: Injection of carrageenan (CG) into one hind paw induced a dose-related hyperalgesia (decreased time for paw withdrawal) to thermal stimuli (infra-red light beam), over 6 h. Key findings: Celecoxib (sc) 30 min before CG (250 μg per paw) induced a dose-dependent reversal of hyperalgesia, with withdrawal times well above basal levels, characterizing development of hypoalgesia. Indomethacin (sc) reversed CG-induced hyperalgesia only to basal levels (an anti-hyperalgesic effect). Naltrexone (sc) prevented hypoalgesia after celecoxib but did not change the response to indomethacin. Local (intraplantar) injection of either a selective antagonist of μ-(beta-funaltrexamine), κ-(norbinaltorphimine) or of δ-(naltrindole) opioid receptors also reversed the hypoalgesic effects of celecoxib, without modifying the hyperalgesia due to CG or affecting the nociceptive thresholds in the non-injected paw. Significance: Our data show that celecoxib, unlike indomethacin, was hypoalgesic in this model of thermal hyperalgesia, and that this effect was mediated by peripheral μ-, κ- and δ-opioid receptors. © 2010 Elsevier Inc. All rights reserved.
Introduction Experimental models of nociception in small animals have played a fundamental role in our understanding of the mechanisms involved in pain (Sandkuhler 2009; Le Bars et al. 2001), as well as in the development of the new analgesic drugs (Penning et al. 1997; Chan et al. 1999). In such models, usually in rats or mice, mechanical or thermal nociceptive stimuli have been most frequently used over many decades (Le Bars et al. 2001). However, distinct neuroanatomical and/or neurochemical mechanisms may underlie the processing of thermal versus mechanical stimuli (Schepers et al. 2008; Hargreaves et al. 1988). We have already provided evidence for the participation of the endogenous opioid system in the mechanism of analgesia exerted by the selective inhibitor of COX-2, celecoxib (Francischi et al. 2002; França et al. 2006; Rezende et al. 2009). This evidence was obtained from a model of hyperalgesia induced by the intraplantar injection of
carrageenan in rats, using a mechanical stimulus. However, as summarized above, there are mechanistic differences in nociception of mechanical or thermal stimuli and we have therefore re-examined the effects of celecoxib and another selective inhibitor SC236, and the possible involvement of endogenous opioids, using thermal instead of mechanical stimulus. Indomethacin was used as a standard nonsteroidal anti-inflammatory analgesic drug. A further goal of the study was to characterize, pharmacologically, the involvement of opioids, using unselective (naltrexone) or selective antagonists of μ-(betafunaltrexamine), κ-(nor-binaltorphimine) and δ-(naltrindole) opioid receptors given locally into the rat hind paw. As classical opioid antagonists, these substances are devoid of intrinsic activity unless opioid agonists have been given previously or the endogenous system had been activated (Gutstein and Akil 2006). Methods Animals
⁎ Corresponding author. Department of Pharmacology, Institute of Biological Sciences, Federal University of Minas Gerais, Av. Antonio Carlos 6627, Belo Horizonte, Minas Gerais, Brazil. Tel.: +55 31 3409 2715. E-mail address: [email protected] (J.N. Francischi). 0024-3205/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2010.04.012
All animal care and experimental procedures complied with the guidelines of the International Association for the Study of Pain in conscious animals (Zimmermann 1983). No animals were re-used
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and after completion of the studies, experimental animals were humanely killed by specialized personal. Male Holtzman rats (N=170) from the UFMG Bioresources, weighing 180–200 g, were used throughout this study. The animals were left to adapt for 24 h under controlled experimental conditions (23–26 °C; light/dark cycles of 12/12 h with lights on at 7:00 h a.m.). Food and water were available ad libitum until 12 h before the experimental period. Carrageenan injections A range of concentrations of λ-carrageenan (CG 2.5–20 mg/ml) were prepared in sterile physiological saline (NaCl, 0.9 %) for injection into the rat hind paw in a volume of 0.1 ml at time zero. These concentrations are known to induce mechanical and thermal hyperalgesia in this tissue (Resende et al. 2001; Hargreaves et al. 1988; Vinegar et al. 1987). The contralateral paws received the same volume of saline at the same time.
Table 1 Summary of drug treatments (with the time lines) used in the study. Opioid antagonist (− 1 h)
COX inhibitors (− 30 min)
Nociceptive stimulus (zero time)
Dose of CG μg/paw
Refer to
– NAL – NAL – NAL – FNT/Veh Nor-BNI/Veh Naltrind/Veh
– VEH INDO INDO CX CX SC236 CX CX CX
CG CG CG CG CG CG CG CG CG CG
250, 1000, 2000 250 250 250 250 250 250 250 250 250
Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig.
1/Fig. 3 4 2 4 2 4 2 5 5 5
CG = carrageenan; NAL = naltrexone (3 mg/kg); INDO = indomethacin; CX = celecoxib; FNT = beta-funaltrexamine; Nor-BNI = nor-binaltorphimine; Naltrind = naltrindole; Veh = respective vehicle; – = physiological saline. All drugs injected subcutaneously were in a volume of 0.1 ml/100 g animal, and those intraplantarly injected were in a total volume of 0.1 ml/paw.
Hyperalgesia assessment Assessment of hyperalgesia consisted of measurement of the threshold stimulus for nociceptive reaction (paw withdrawal) using a thermal stimulus (beam of infra-red light, 50 W) applied to the pads of the hind paws given by the Plantar Test apparatus (model 7371, Ugo Basile, Italy). This method is essentially described by Hargreaves et al. 1988. Animals were placed in a transparent acrylic square box with three compartments presenting the following dimensions: 17 cm width; 22 cm length; 10 cm depth. The square boxes were supported by a glass platform which allowed the stimulus from the radiant source, located below of the apparatus, to be applied. The light beam was turned off automatically by a photocell when the rat lifted the paw, allowing the measurement of time between start of light beam and the withdrawal of the paw (withdrawal latency). Right and left paws were measured within an interval of 2 min between stimulations and three measurements were averaged for each side. The nociceptive threshold was measured before (time zero) and 1, 2, 3, 4 and 6 h after carrageenan injection. Values were expressed as the difference (Δ in seconds, s) of right and left paw latencies. The experimenter was unaware of the treatments. Drug treatments Cyclooxygenase inhibitors (celecoxib, SC236 and indomethacin) and the non-selective opioid antagonist (naltrexone) were administered by subcutaneous (sc) route whereas the selective opioid antagonists (beta-funaltrexamine; nor-binaltorphimine and naltrindole) were administered intraplantarly (ipl). The doses used are indicated in the Results. Celecoxib and the opioid antagonists were diluted in physiological saline, whereas SC236 and indomethacin were dissolved in ethanol and then diluted in 5% Tween 80 (v/v) in saline (Francischi et al. 2002; França et al. 2006). Control animals were injected with the corresponding vehicles at the same time. Volumes for sc or ipl injections were, respectively, 0.1 ml/100 g or 0.1 ml per paw. Indications for used treatments were summarized in Table 1. Drug sources Celecoxib (Celebra, Searle & Co, Cáguas, Porto Rico); SC236 (Cayman Chemical Company; USA), beta-funaltrexamine, nor-binaltorphimine and naltrindole (Tocris; UK). Carrageenan, naltrexone and indomethacin were provided by Sigma-Aldrich (USA). Statistics Values are presented as mean (±standard error of the mean) from groups of 5 animals. In the inset to Fig. 1, a time course of the Δ
withdrawal latency (difference between latencies of right (with carrageenan), and left (with saline) paws), over 6 h is presented. In all other figures, we have integrated these responses by measuring the area under the time/Δ latency curve (AUC) to facilitate comparison between experimental conditions. Positive values (N0) of AUC indicate hypoalgesia development. AUC negative values were described as hyperalgesia when CG was administered and anti-hyperalgesia when combined with analgesics. One-way ANOVA followed by post hoc analysis (Bonferroni/ Dunnett's, as appropriate) was used to determine significance between means, accepting a difference when P b 0.05. Results Carrageenan (CG)-induced hyperalgesia to thermal stimuli To establish standard conditions for this study, we first assessed the dose–response relationship between the dose of CG injected (250 to 2000 μg per paw) and the nociceptive threshold to thermal stimuli. Nociceptive threshold decreased soon after the intraplantar injection of CG, reaching a minimum value within 2 h, and lasting for more 4 h, in the case of the highest dose of carrageenan used (2000 μg per paw), compared with the thresholds in paws injected with saline (controls; inset graph in Fig. 1). The corresponding AUC results (main graph in Fig. 1) clearly show a dose-related depth of hyperalgesia and match findings from the original description of this model of thermal hyperalgesia (Hargreaves et al. 1988). Effect of celecoxib in the model of thermal hyperalgesia To assess the analgesic effects of celecoxib in this model of thermal hyperalgesia, a range of doses were injected sc, 30 min before injection of CG into the paw. As shown in Fig. 2, celecoxib (0.3–30 mg/kg) induced a progressive and dose-dependent reversal of the lowered nociceptive threshold (hyperalgesia) induced by CG and this reversal produced thresholds well above the basal level at the higher doses of celecoxib (3–30 mg/kg), demonstrating the development of hypoalgesia. The hypoalgesic action was however lost at the highest dose of celecoxib (60 mg/kg), although a clear anti-hyperalgesic effect, i.e. reversal to basal thresholds, remained at about the same level seen with the lowest dose of the compound (0.3 mg/kg). Hypoalgesia was also observed in animals treated with another selective COX-2 inhibitor, SC236 (Fig. 2). Interestingly, indomethacin also given sc at a dose of 2 mg/kg, only exerted an anti-hyperalgesic effect (Fig. 2). Moreover, hypoalgesia to celecoxib was no longer detected in rat paws injected with a 4-fold bigger dose of carrageenan (1000 μg), as shown in Fig. 3.
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Fig. 1. Dose-related nociceptive thermal thresholds following administration of carrageenan in rat hind paws. Rats (n = 5/group) were injected with depicted doses of carrageenan (CG, in μg) or saline intraplantarly at time zero. The latency for thermal withdrawal of right and left paws given by a plantar test apparatus (Ugo Basile) was measured within an interval of 2 min between stimulations and three measurements were averaged for each side. The nociceptive thermal threshold was measured before (time zero) and 1, 2, 3, 4 and 6 h after carrageenan or saline (C) injection. Values were expressed as the difference (in seconds) of right and left paw latencies versus time shown in the inset and as area under the curves (AUC) in the main graph. *Significantly different from control (C) P b 0.05.
Involvement of endogenous opioid receptors in the hypoalgesia induced by celecoxib The involvement of endogenous opioids in celecoxib hypoalgesia was first tested by using a non-selective opioid receptor antagonist, naltrexone (3 mg/kg) given sc 30 min before a hypoalgesic dose of celecoxib (12 mg/kg). As shown in Fig. 4A, naltrexone reversed the hypoalgesic effect induced by celecoxib to the basal level, although the anti-hyperalgesic component of the response to celecoxib persisted. In contrast, even a 5-folder higher dose of indomethacin (10 mg/kg) than previously used (see Fig. 2) did not induce hypoalgesia nor its anti-hyperalgesic effect was affected by a standard naltrexone dose (3 mg/kg) given ½ h beforehand (Fig. 4B). To analyze further the type of opioid receptors that could be involved in this hypoalgesic response to celecoxib, an opioid antagonist, selective for μ-opioid receptors (beta-funaltrexamine, 50 μg/paw = 100 nmol), one selective for κ-opioid receptors (nor-binaltorphimine, 76 μg/
Fig. 2. Development of hypoalgesia to celecoxib in the thermal model of hyperalgesia. Celecoxib (CX; 0.3–60 mg/kg) or vehicle (saline; C) was subcutaneously administered ½ h before carrageenan (250 μg/paw at time zero) in rat hind paws. Nociceptive thermal thresholds, presented as AUC, were obtained as described in the legend of Fig. 1. For comparison, nociceptive thermal thresholds for another coxib (SC236) and indomethacin (Indo), a non-selective cyclooxygenase inhibitor, given by the same route, are shown at the left hand side of the Figure (dotted line). SC236 and INDO induced hypo and antihyperalgesia, respectively. # indicates hypoalgesia (valuesN zero) and * significantly different from C (P b 0.05).
paw = 100 nmol), or one selective for δ-opioid receptors (naltrindole, 48.7 μg/paw = 100 nmol) were injected locally into the paws. As shown in Fig. 5, beta-funaltrexamine given alone to the paw did not modify the hyperalgesia induced by carrageenan but when injected in animals treated with carrageenan and celecoxib, the raised threshold characteristic of hypoalgesia induced by celecoxib were returned to basal levels. Importantly, when the μ-opioid antagonist was given in the contralateral, non-inflamed paw, the hypoalgesia in the CG-treated paw was unaffected (Fig. 5A). Local injection of the κ-opioid receptor antagonist, nor-binaltorphimine also reversed the celecoxib-induced hypoalgesia to basal levels but allowed the anti-hyperalgesic component of the response to celecoxib to persist (Fig. 5B). Basically, the same profile was also seen, i.e. reversal of the hypoalgesic response due to celecoxib, when the delta-opioid antagonist naltrindole was used (Fig. 5C). Note that local treatment with either beta-funaltrexamine, nor-binaltorphimine or naltrindole, given alone to the inflamed paw, did not modify the hyperalgesia induced by CG, compared with data from Fig. 1, and were not different from those obtained with saline/ vehicle-treated animals (other figures).
Fig. 3. Celecoxib-induced hypoalgesia depends on carrageenan dose. Celecoxib (CX) at a selected dose (12 mg/kg) or its vehicle (SAL) was administered subcutaneously to rats ½ h before 250 or 1000 μg/paw carrageenan (CG) given intraplantarly. Nociceptive thermal thresholds (AUC) were obtained as described in Fig. 1. No change on the hyperalgesia induced by the high dose of carrageenan was observed. * significantly different from SAL; # significantly different from basal values.
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Fig. 4. Celecoxib, but not indomethacin, induced hypoalgesia was reversed by naltrexone. (A) Subcutaneous injection of celecoxib (CX; 12 mg/kg) induced hypoalgesia, raising nociceptive thresholds in the inflamed paws (CG) above the level in paws injected only with saline (SAL; #, P b 0.05). However, (B) indomethacin (INDO, sc, 10 mg/kg) was only anti-hyperalgesic in inflamed paws raising thresholds back to, but not above, the levels in saline-injected paws. Note also that naltrexone (NAL, 3 mg/kg; sc) given 30 min before CG reversed the celecoxib-induced hypoalgesia to basal levels (*, P b 0.05), but did not affect the anti-hyperalgesia shown by indomethacin. Nociceptive thermal thresholds (AUC) were obtained as described in Fig. 1.
Discussion Our experiments have shown that celecoxib, a selective inhibitor of COX-2 induced a state of hypoalgesia (nociceptive thresholds raised above normal levels) in a well-established model of inflammatory pain, using thermal stimuli (Le Bars et al. 2001; Hargreaves et al. 1988). We have also provided evidence that these actions of celecoxib were mediated by opioid receptors, most likely μ, κ and δ receptors located peripherally. We undertook these experiments to extend the results we had obtained in the same inflammatory pain model using mechanical stimuli, because there is considerable evidence that different painful stimuli (thermal vs. mechanical) can give rise to different effects in the same models (Schepers et al. 2008; Bennet et al. 2000; Mansikka et al. 2000). Overall, the results we report here are similar to those obtained with mechanical stimuli (Francischi et al. 2002; França et al. 2006) but with some important differences. As in previous work, we used here a relatively low dose of CG (250 μg per paw) as the standard inflammatory stimulus which produced a hyperalgesic state lasting about 6 h after the injection of CG. Higher doses of CG (up to 2000 μg per paw) induced greater intensity and duration of hyperalgesia and the analgesic effects of celecoxib were lost in these conditions of increased hyperalgesia. This loss of analgesic activity probably reflects a “competition” between the intensity of the inflammatory process
Fig. 5. Peripheral (local) reversal by opioid antagonists of celecoxib-induced hypoalgesia. A hundred nanomoles of each opioid antagonist (A: beta-funaltrexamine—FNT; B: norbinaltorphimine—Nor-BNI; C: naltrindole) was injected into the paw either ipsi (ipsi) or contralaterally (contra) 30 min before celecoxib (CX) or saline (SAL) subcutaneously and 1 h before 250 μg/paw carrageenan (CG). The nociceptive thermal values are presented as AUC from control (CG + opioid antagonist) and test animals. All three opioid antagonists reversed the hypoalgesia induced by celecoxib to basal level only following ipsi administration. * significant difference from vehicle-treatment (P b 0.05); # significant difference from basal values.
and the strength of the analgesic process. It is possible that a higher dose of celecoxib—we used 12 mg/kg and the IC50 provided by Penning et al. (1997) for celecoxib was about 30 mg/kg—would have been more effective against 2000 μg CG, but that was not the major aim of our work and was not further investigated. Using a thermal stimulus, although celecoxib still exhibited hypoalgesia with a bell-shaped dose–response curve, this hypoalgesia was observed over a wider dose range (3–30 mg/kg) than with mechanical stimuli (3–12 mg kg). Also the lowest dose of celecoxib used (0.3 mg/kg) was effective against thermal hyperalgesia, but showed no analgesic effect with mechanical stimuli. Thus the thermal stimulus provides a model more responsive to the analgesic effect of celecoxib, a finding compatible with those of Hargreaves et al. more than 20 years ago, who concluded that thermal stimuli yielded a more sensitive pain model. Since then, although progress has been made in understanding the neural basis of many commonly used nociceptive
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models (Le Bars et al. 2001), differences between thermal and mechanical stimuli are still being reported and analyzed. Recently Schepers et al. (2008) using these two stimuli in the same model (paws injected with complete Freunds' adjuvant) found that nor-BNI induced bilateral hypersensitivity to mechanical, but not to thermal stimuli. Similarly, mechanical and thermal stimuli were differently affected by NMDA receptor antagonists (Bennet et al. 2000) or by genetic deletion of NK-1 receptors (Mansikka et al. 2000). These differences have been analyzed in terms of different anatomical substrates (Schepers et al. 2008) or different mediators (Bennet et al. 2000; Mansikka et al. 2000) involved in mechanical or thermal hyperalgesia. However, in our model with either stimulus, there was the same hypoalgesia caused by celecoxib and the same susceptibility to opioid antagonists, either non-selective (Francischi et al. 2002; França et al. 2006) or selective (this study). Thus, the endogenous opioid mediation of coxib-induced hypoalgesia is not restricted to mechanical hyperalgesia and may operate in other modes of inflammatory pain. The bell-shaped curve may also be indirect evidence for opioid mediation of celecoxib-induced hypoalgesia, as such response curves have been noted for some time, with endogenous (Bujdosó et al. 2001; Hauser et al. 2005) or exogenous opiates (Hara et al. 1997; Silverman 2009; Sarton et al. 2008). Several explanations have been advanced including the pro-nociceptive effects of endogenous peptides (Hauser et al. 2005; Tan-No et al. 2009; Calo et al. 2000) and two forms of μ-opioid receptor, one mediating pro- and the other, anti-nociceptive effects (Sarton et al. 2008). However our experiments were not designed to decide between these possibilities. Another important aspect, common to both forms of hyperalgesia, was that in our model of inflammatory pain, analgesic effects in the contralateral, non-inflamed, paw were never seen with either celecoxib or indomethacin given systemically (sc) or centrally (icv), whereas morphine or paracetamol (sc) did modify the nociceptive threshold in contralateral paws (Ferreira et al. 1978; Rezende et al. 2008). This apparent inflammation-dependence of celecoxib's analgesia may reflect an increased number of opioid receptors (Zöllner et al. 2003; Stein et al. 2003) or, more speculatively, an increased release of endogenous opioid (s) in inflamed sites. If there were an inflammation-dependent, increased release of endogenous opioids, then this did not noticeably occur in the absence of celecoxib, as neither the selective antagonists (present study) nor naltrexone (França et al. 2006) modified the hyperalgesia induced by CG, a finding that contrasted with that of Schepers et al. (2008) who found the κ-receptor antagonist, nor-BNI, to potentiate hyperalgesia induced by CFA. Notwithstanding the overall similarity in responses between mechanical and thermal hyperalgesia, the present model of thermal hyperalgesia showed that naltrexone and selective antagonists of μ-, κ- and δ-opioid receptors reversed the analgesic effects of celecoxib in CG-treated paws, only to a normal, i.e., untreated, level, whereas for mechanical hyperalgesia, the effects of the celecoxib was totally reversed to thresholds equal to those seen with CG alone (França et al. 2006). As we have used the same inflammatory stimulus, the same strain of rats and the same dose of celecoxib given sc in both sets of experiments, this differential effect of opioid antagonists would imply a real difference in mechanisms between those mediating antihyperalgesia and those mediating hypoalgesia, with thermal stimuli. One simple explanation for our present results would be to postulate two separate mechanisms for celecoxib, one mediating the antihyperalgesia, i.e. return of the nociceptive thresholds to the basal level and the other responsible for the hypoalgesia, i.e, raising thresholds above basal levels. The first would involve PGs and COX, just like the effects of indomethacin (Vane 1971; Ferreira et al. 1978) and thus be resistant to naltrexone, whereas the second would involve opioid receptors and be sensitive to naltrexone and other opioid receptor antagonists. However such a scheme would only operate for thermal, and not mechanical, hyperalgesia, and would imply the involvement of
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two sets of neuronal pathways in the thermal hyperalgesia, in contrast to the one, opioid-mediated, pathway for mechanical hyperalgesia. Our findings that selective antagonists of μ-, κ- and δ-opioid receptors given to the paw were able to prevent celecoxib-induced thermal hypoalgesia imply that these receptors were involved at a local, peripheral site. Agonists for those receptors, morphine, bremazocine and SNC80, respectively, have been shown to induce anti-nociceptive effects (Ferreira et al. 1991; Amarante and Duarte 2002; Pacheco et al. 2005). Conclusion We have extended our analysis of coxib-induced hypoalgesia, demonstrating that the effects of celecoxib (and SC236) were not restricted to one type of nociceptive stimulus but were exhibited, albeit with some differences, in the model of thermal hyperalgesia. We were also able to confirm that with thermal stimuli, celecoxib still induced hypoalgesia, which was mediated by the endogenous opioid system acting via peripheral μ- and κ- and δ opioid receptors. Important questions still to be answered include the identity of the endogenous opioids and the cell types involved in this peripheral antinociceptive action of celecoxib. Conflict of interest The authors claim no conflict of interest.
Acknowledgment Financial support: Conselho Nacional de Pesquisa (CNPq), Fundação de Amparo à Pesquisa de Minas Gerais (FAPEMIG) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. References Amarante LH, Duarte IDG. The κ-opioid agonist (±)-bremazocine elicits peripheral antinociception by activation of the L-arginine/nitric oxide/cyclic GMP pathway. European Journal of Pharmacology 454, 19–23, 2002. Bennet AD, Everhart AW, Hulsebosch CE. Intrathecal administration of an NMDA or a non-NMDA receptor antagonist reduces mechanical but not thermal allodynia in a rodent model of chronic central pain after spinal cord injury. Brain Research 859, 72–82, 2000. Bujdosó E, Jászberényi M, Tömböly C, Tóth G, Telegdy G. Behavioral and neuroendocrine actions of endomorphin-2. Peptides 22, 1459–1463, 2001. Calo G, Guerrini R, Rizzi A, Salvadori S, Regoli D. Pharmacology ofnociceptin and its receptor: a novel therapeutic target. British Journal of Pharmacology 129, 1261–1283, 2000. Chan CC, Boyce S, Brideau C, Charleson S, Cromlish W, Ethier D, Evans J, Ford-Hutchinson AW, Forrest M, Gauthier JY, Gordon R, Gresser M, Guay J, Kargman S, Kennedy B, Leblanc Y, Leger S, Mancini J, O'Neill GP, Quellet M, Patrick D, Percival MD, Perrier H, Prasit P, Rodger I, Tagari P, Therien M, Vickers P, Visco D, Wang Z, Webb J, Wong E, Xu L-J, Young RN, Zamboni R, Riendeau D. Rofecoxib (Vioxx, MK-0966; 4-(4´methylsulfonylphenyl)-3-phenyl-2-(5H)-furanone): a potent and orally active cyclooxygenase-2 inhibitor. Pharmacological and biochemical profiles. The Journal of Pharmacology and Experimental Therapeutics 290, 551–560, 1999. Ferreira SH, Duarte ID, Lorenzetti BB. The molecular mechanism of action of peripheral morphine analgesia: stimulation of the cGMP system via nitric oxide release. European Journal of Pharmacology 201 (1), 121–122, 1991. Ferreira SH, Nakamura M, Castro MAS. The hyperalgesic effects of prostacyclin and prostaglandin E2. Prostaglandins 16, 31–37, 1978. França DS, Ferreira-Alves DL, Duarte ID, Ribeiro MC, Rezende RM, Bakhle YS, Francischi JN. Endogenous opioids mediate the hypoalgesia induced by inhibitors of cyclooxygenase-2 in rat paws treated with carrageenan. Neuropharmacology 51, 37–43, 2006. Francischi JN, Chaves CT, Moura ACL, Lima AS, Rocha OA, Ferreira-Alves DL, Bakhle YS. Selective inhibitors of cyclo-oxygenase-2 (COX-2) induce hypoalgesia in a rat paw model of inflammation. British Journal of Pharmacology 37, 837–844, 2002. Gutstein HB, Akil H. Opioid analgesics (Chapter 23). In: Brunton, LL, Lazo, JS, Parker, KL (Eds.), The Pharmacological Basis of Therapeutics, 11e. The McGraw-Hill companies, United States of America, pp. 569–620, 2006. Hara N, Minami T, Okuda-Ashitaka E, Sugimoto T, Sakai M, Onaka M, Mori H, Imanishi T, Shingu K, Ito S. Characterization of nociceptin hyperalgesia and allodynia in conscious mice. British Journal of Pharmacology 121, 401–408, 1997. Hargreaves K, Dubner R, Brown F, Flores C, Joris J. A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain 32 (1), 77–88, 1988.
956
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Hauser KF, Aldrich JV, Anderson KJ, Bakalkin G, Christie MJ, Hall ED, Knapp PE, Scheff SW, Singh IN, Vissel B, Woods AS, Yakovleva T, Shippenberg TS. Pathobiology of dynorphins in trauma and disease. Frontiers in Bioscience 10, 216–235, 2005. Le Bars D, Gozariu M, Cadden SW. Animal models of nociception. Pharmacological Reviews 53 (4), 597–652, 2001. Mansikka H, Sheth RN, DeVries C, Lee H, Winchurch R, Raja SN. Nerve injury-induced mechanical but not thermal hyperalgesia is attenuated in neurokinin-1 receptor knockout mice. Experimental Neurology 162, 343–349, 2000. Pacheco DF, Reis GML, Castro MSA, Francischi JN, Duarte IDG. Delta-opioid receptor agonist SNC80 elicits peripheral antinociception via d1 and d2 receptors and activation of the L-arginine/nitric oxide/cGMP pathway. Life Sciences 78, 54–60, 2005. Penning TB, Talley JJ, Bertenshaw SR, Carter JS, Collins PW, Docter S, Graneto MJ, Lee LF, Malecha JW, Miyashiro JM, Rogers RS, Rogier DJ, Yu SS, Anderson GD, Burton EG, Cogburn JN, Gregory SA, Koboldt CM, Perkins WE, Seibert K, Veenhuizen AW, Zhang YY, Isakson PC. Synthesis and biological evaluation of the 1, 5-diarylpyrazole class of cyclooxygenase-2 inhibitors: identification of 4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-y1] benzenesulfonamide (SC-58635, Celecoxib). Journal of Medicinal Chemistry 40, 1347–1365, 1997. Resende MA, Reis WGP, Pereira LSM, Ferreira W, De Lima ME, Santoro MM, Francischi JN. Hyperalgesia and edema responses induced by rat peripheral blood mononuclear cells incubated with carrageenan. Inflammation 25, 277–285, 2001. Rezende RM, Dos Reis WGP, Duarte ID, Lima PP, Bakhle YS, Francischi JN. The analgesic actions of centrally administered celecoxib are mediated by endogenous opioids. Pain 142 (1–2), 94–100, 2009. Rezende RM, França DS, Menezes GB, Dos Reis WGP, Bakhle YS, Francischi JN. Different mechanisms underlie the analgesic actions of paracetamol and dipyrone in a rat model of inflammatory pain. British Journal of Pharmacology 153, 760–768, 2008.
Sandkuhler J. Models and mechanisms of hyperalgesia and allodynia. Physiological Reviews 89, 707–758, 2009. Sarton E, Teppema L, Dahan A. Naloxone reversal of opioid-induced respiratory depression with special emphasis on the partial agonist/antagonist buprenorphine. Advances in Experimental Medicine and Biology 605, 486–491, 2008. Schepers RJ, Mahoney JL, Gehrke BJ, Shippenberg TS. Endogenous kappa-opioid receptor systems inhibit hyperalgesia associated with localized peripheral inflammation. Pain 138, 423–439, 2008. Silverman SM. Opioid induced hyperalgesia: clinical implications for the pain practitioner. Pain Physician 12, 679–684, 2009. Stein C, Schäfer M, Machelska H. Attacking pain at its source: new perspectives on opioids. Natural Medicines 9, 1003–1008, 2003. Tan-No K, Takahashi H, Nakagawasai O, Niijima F, Sakurada S, Bakalkin G, Terenius L, Tadano T. Nociceptive behavior induced by the endogenous opioid peptides dynorphins in uninjured mice: evidence with intrathecal N-ethylmaleimide inhibiting dynorphin degradation. International Review of Neurobiology 85, 191–205, 2009. Vane JR. Inhibition of prostaglandin synthesis as a mechanism of action for aspirin like drugs. Nature: New Biology 231, 232–235, 1971. Vinegar R, Truax JF, Selph JL, Johnston PR, Venable AL, McKenzie KK. Pathway to carrageenan-induced inflammation in the hind limb of the rat. Federation Proceedings 46, 118–126, 1987. Zimmermann M. Ethical guidelines for investigations of experimental pain in conscious animals. Pain 6, 109–110, 1983. Zöllner C, Shaquira MA, Bopaiah CP, Mousa S, Stein C, Schäfer M. Painful inflammationinduced increase in μ-opioid receptor binding and G-protein coupling in primary afferent neurons. Molecular Pharmacology 64, 202–210, 2003.
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Life Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i f e s c i e
Peripheral μ-, κ- and δ-opioid receptors mediate the hypoalgesic effect of celecoxib in a rat model of thermal hyperalgesia Jôice D. Correa a, Patrícia Paiva-Lima a, Rafael M. Rezende a, Webster G.P. Dos Reis a, Dalton L. Ferreira-Alves a, Y.S. Bakhle b, Janetti N. Francischi a,⁎ a b
Department of Pharmacology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil Leukocyte Biology, National Heart and Lung Institute, Faculty of Medicine, Imperial College, SW7 2AZ London, United Kingdom
a r t i c l e
i n f o
Article history: Received 6 January 2010 Accepted 26 April 2010 Keywords: Hypoalgesia Celecoxib Hargreave's method Opioid receptors Cyclooxygenase inhibition
a b s t r a c t Aims: The endogenous opioids mediate the analgesic effects of celecoxib in a model of mechanical hyperalgesia in rats. As responses to thermal stimuli may differ from those to mechanical stimuli, we have here assessed celecoxib in a rat model of thermal hyperalgesia and the possible involvement of endogenous opioids and their corresponding receptors in these effects. Main methods: Injection of carrageenan (CG) into one hind paw induced a dose-related hyperalgesia (decreased time for paw withdrawal) to thermal stimuli (infra-red light beam), over 6 h. Key findings: Celecoxib (sc) 30 min before CG (250 μg per paw) induced a dose-dependent reversal of hyperalgesia, with withdrawal times well above basal levels, characterizing development of hypoalgesia. Indomethacin (sc) reversed CG-induced hyperalgesia only to basal levels (an anti-hyperalgesic effect). Naltrexone (sc) prevented hypoalgesia after celecoxib but did not change the response to indomethacin. Local (intraplantar) injection of either a selective antagonist of μ-(beta-funaltrexamine), κ-(norbinaltorphimine) or of δ-(naltrindole) opioid receptors also reversed the hypoalgesic effects of celecoxib, without modifying the hyperalgesia due to CG or affecting the nociceptive thresholds in the non-injected paw. Significance: Our data show that celecoxib, unlike indomethacin, was hypoalgesic in this model of thermal hyperalgesia, and that this effect was mediated by peripheral μ-, κ- and δ-opioid receptors. © 2010 Elsevier Inc. All rights reserved.
Introduction Experimental models of nociception in small animals have played a fundamental role in our understanding of the mechanisms involved in pain (Sandkuhler 2009; Le Bars et al. 2001), as well as in the development of the new analgesic drugs (Penning et al. 1997; Chan et al. 1999). In such models, usually in rats or mice, mechanical or thermal nociceptive stimuli have been most frequently used over many decades (Le Bars et al. 2001). However, distinct neuroanatomical and/or neurochemical mechanisms may underlie the processing of thermal versus mechanical stimuli (Schepers et al. 2008; Hargreaves et al. 1988). We have already provided evidence for the participation of the endogenous opioid system in the mechanism of analgesia exerted by the selective inhibitor of COX-2, celecoxib (Francischi et al. 2002; França et al. 2006; Rezende et al. 2009). This evidence was obtained from a model of hyperalgesia induced by the intraplantar injection of
carrageenan in rats, using a mechanical stimulus. However, as summarized above, there are mechanistic differences in nociception of mechanical or thermal stimuli and we have therefore re-examined the effects of celecoxib and another selective inhibitor SC236, and the possible involvement of endogenous opioids, using thermal instead of mechanical stimulus. Indomethacin was used as a standard nonsteroidal anti-inflammatory analgesic drug. A further goal of the study was to characterize, pharmacologically, the involvement of opioids, using unselective (naltrexone) or selective antagonists of μ-(betafunaltrexamine), κ-(nor-binaltorphimine) and δ-(naltrindole) opioid receptors given locally into the rat hind paw. As classical opioid antagonists, these substances are devoid of intrinsic activity unless opioid agonists have been given previously or the endogenous system had been activated (Gutstein and Akil 2006). Methods Animals
⁎ Corresponding author. Department of Pharmacology, Institute of Biological Sciences, Federal University of Minas Gerais, Av. Antonio Carlos 6627, Belo Horizonte, Minas Gerais, Brazil. Tel.: +55 31 3409 2715. E-mail address: [email protected] (J.N. Francischi). 0024-3205/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2010.04.012
All animal care and experimental procedures complied with the guidelines of the International Association for the Study of Pain in conscious animals (Zimmermann 1983). No animals were re-used
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and after completion of the studies, experimental animals were humanely killed by specialized personal. Male Holtzman rats (N=170) from the UFMG Bioresources, weighing 180–200 g, were used throughout this study. The animals were left to adapt for 24 h under controlled experimental conditions (23–26 °C; light/dark cycles of 12/12 h with lights on at 7:00 h a.m.). Food and water were available ad libitum until 12 h before the experimental period. Carrageenan injections A range of concentrations of λ-carrageenan (CG 2.5–20 mg/ml) were prepared in sterile physiological saline (NaCl, 0.9 %) for injection into the rat hind paw in a volume of 0.1 ml at time zero. These concentrations are known to induce mechanical and thermal hyperalgesia in this tissue (Resende et al. 2001; Hargreaves et al. 1988; Vinegar et al. 1987). The contralateral paws received the same volume of saline at the same time.
Table 1 Summary of drug treatments (with the time lines) used in the study. Opioid antagonist (− 1 h)
COX inhibitors (− 30 min)
Nociceptive stimulus (zero time)
Dose of CG μg/paw
Refer to
– NAL – NAL – NAL – FNT/Veh Nor-BNI/Veh Naltrind/Veh
– VEH INDO INDO CX CX SC236 CX CX CX
CG CG CG CG CG CG CG CG CG CG
250, 1000, 2000 250 250 250 250 250 250 250 250 250
Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig.
1/Fig. 3 4 2 4 2 4 2 5 5 5
CG = carrageenan; NAL = naltrexone (3 mg/kg); INDO = indomethacin; CX = celecoxib; FNT = beta-funaltrexamine; Nor-BNI = nor-binaltorphimine; Naltrind = naltrindole; Veh = respective vehicle; – = physiological saline. All drugs injected subcutaneously were in a volume of 0.1 ml/100 g animal, and those intraplantarly injected were in a total volume of 0.1 ml/paw.
Hyperalgesia assessment Assessment of hyperalgesia consisted of measurement of the threshold stimulus for nociceptive reaction (paw withdrawal) using a thermal stimulus (beam of infra-red light, 50 W) applied to the pads of the hind paws given by the Plantar Test apparatus (model 7371, Ugo Basile, Italy). This method is essentially described by Hargreaves et al. 1988. Animals were placed in a transparent acrylic square box with three compartments presenting the following dimensions: 17 cm width; 22 cm length; 10 cm depth. The square boxes were supported by a glass platform which allowed the stimulus from the radiant source, located below of the apparatus, to be applied. The light beam was turned off automatically by a photocell when the rat lifted the paw, allowing the measurement of time between start of light beam and the withdrawal of the paw (withdrawal latency). Right and left paws were measured within an interval of 2 min between stimulations and three measurements were averaged for each side. The nociceptive threshold was measured before (time zero) and 1, 2, 3, 4 and 6 h after carrageenan injection. Values were expressed as the difference (Δ in seconds, s) of right and left paw latencies. The experimenter was unaware of the treatments. Drug treatments Cyclooxygenase inhibitors (celecoxib, SC236 and indomethacin) and the non-selective opioid antagonist (naltrexone) were administered by subcutaneous (sc) route whereas the selective opioid antagonists (beta-funaltrexamine; nor-binaltorphimine and naltrindole) were administered intraplantarly (ipl). The doses used are indicated in the Results. Celecoxib and the opioid antagonists were diluted in physiological saline, whereas SC236 and indomethacin were dissolved in ethanol and then diluted in 5% Tween 80 (v/v) in saline (Francischi et al. 2002; França et al. 2006). Control animals were injected with the corresponding vehicles at the same time. Volumes for sc or ipl injections were, respectively, 0.1 ml/100 g or 0.1 ml per paw. Indications for used treatments were summarized in Table 1. Drug sources Celecoxib (Celebra, Searle & Co, Cáguas, Porto Rico); SC236 (Cayman Chemical Company; USA), beta-funaltrexamine, nor-binaltorphimine and naltrindole (Tocris; UK). Carrageenan, naltrexone and indomethacin were provided by Sigma-Aldrich (USA). Statistics Values are presented as mean (±standard error of the mean) from groups of 5 animals. In the inset to Fig. 1, a time course of the Δ
withdrawal latency (difference between latencies of right (with carrageenan), and left (with saline) paws), over 6 h is presented. In all other figures, we have integrated these responses by measuring the area under the time/Δ latency curve (AUC) to facilitate comparison between experimental conditions. Positive values (N0) of AUC indicate hypoalgesia development. AUC negative values were described as hyperalgesia when CG was administered and anti-hyperalgesia when combined with analgesics. One-way ANOVA followed by post hoc analysis (Bonferroni/ Dunnett's, as appropriate) was used to determine significance between means, accepting a difference when P b 0.05. Results Carrageenan (CG)-induced hyperalgesia to thermal stimuli To establish standard conditions for this study, we first assessed the dose–response relationship between the dose of CG injected (250 to 2000 μg per paw) and the nociceptive threshold to thermal stimuli. Nociceptive threshold decreased soon after the intraplantar injection of CG, reaching a minimum value within 2 h, and lasting for more 4 h, in the case of the highest dose of carrageenan used (2000 μg per paw), compared with the thresholds in paws injected with saline (controls; inset graph in Fig. 1). The corresponding AUC results (main graph in Fig. 1) clearly show a dose-related depth of hyperalgesia and match findings from the original description of this model of thermal hyperalgesia (Hargreaves et al. 1988). Effect of celecoxib in the model of thermal hyperalgesia To assess the analgesic effects of celecoxib in this model of thermal hyperalgesia, a range of doses were injected sc, 30 min before injection of CG into the paw. As shown in Fig. 2, celecoxib (0.3–30 mg/kg) induced a progressive and dose-dependent reversal of the lowered nociceptive threshold (hyperalgesia) induced by CG and this reversal produced thresholds well above the basal level at the higher doses of celecoxib (3–30 mg/kg), demonstrating the development of hypoalgesia. The hypoalgesic action was however lost at the highest dose of celecoxib (60 mg/kg), although a clear anti-hyperalgesic effect, i.e. reversal to basal thresholds, remained at about the same level seen with the lowest dose of the compound (0.3 mg/kg). Hypoalgesia was also observed in animals treated with another selective COX-2 inhibitor, SC236 (Fig. 2). Interestingly, indomethacin also given sc at a dose of 2 mg/kg, only exerted an anti-hyperalgesic effect (Fig. 2). Moreover, hypoalgesia to celecoxib was no longer detected in rat paws injected with a 4-fold bigger dose of carrageenan (1000 μg), as shown in Fig. 3.
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Fig. 1. Dose-related nociceptive thermal thresholds following administration of carrageenan in rat hind paws. Rats (n = 5/group) were injected with depicted doses of carrageenan (CG, in μg) or saline intraplantarly at time zero. The latency for thermal withdrawal of right and left paws given by a plantar test apparatus (Ugo Basile) was measured within an interval of 2 min between stimulations and three measurements were averaged for each side. The nociceptive thermal threshold was measured before (time zero) and 1, 2, 3, 4 and 6 h after carrageenan or saline (C) injection. Values were expressed as the difference (in seconds) of right and left paw latencies versus time shown in the inset and as area under the curves (AUC) in the main graph. *Significantly different from control (C) P b 0.05.
Involvement of endogenous opioid receptors in the hypoalgesia induced by celecoxib The involvement of endogenous opioids in celecoxib hypoalgesia was first tested by using a non-selective opioid receptor antagonist, naltrexone (3 mg/kg) given sc 30 min before a hypoalgesic dose of celecoxib (12 mg/kg). As shown in Fig. 4A, naltrexone reversed the hypoalgesic effect induced by celecoxib to the basal level, although the anti-hyperalgesic component of the response to celecoxib persisted. In contrast, even a 5-folder higher dose of indomethacin (10 mg/kg) than previously used (see Fig. 2) did not induce hypoalgesia nor its anti-hyperalgesic effect was affected by a standard naltrexone dose (3 mg/kg) given ½ h beforehand (Fig. 4B). To analyze further the type of opioid receptors that could be involved in this hypoalgesic response to celecoxib, an opioid antagonist, selective for μ-opioid receptors (beta-funaltrexamine, 50 μg/paw = 100 nmol), one selective for κ-opioid receptors (nor-binaltorphimine, 76 μg/
Fig. 2. Development of hypoalgesia to celecoxib in the thermal model of hyperalgesia. Celecoxib (CX; 0.3–60 mg/kg) or vehicle (saline; C) was subcutaneously administered ½ h before carrageenan (250 μg/paw at time zero) in rat hind paws. Nociceptive thermal thresholds, presented as AUC, were obtained as described in the legend of Fig. 1. For comparison, nociceptive thermal thresholds for another coxib (SC236) and indomethacin (Indo), a non-selective cyclooxygenase inhibitor, given by the same route, are shown at the left hand side of the Figure (dotted line). SC236 and INDO induced hypo and antihyperalgesia, respectively. # indicates hypoalgesia (valuesN zero) and * significantly different from C (P b 0.05).
paw = 100 nmol), or one selective for δ-opioid receptors (naltrindole, 48.7 μg/paw = 100 nmol) were injected locally into the paws. As shown in Fig. 5, beta-funaltrexamine given alone to the paw did not modify the hyperalgesia induced by carrageenan but when injected in animals treated with carrageenan and celecoxib, the raised threshold characteristic of hypoalgesia induced by celecoxib were returned to basal levels. Importantly, when the μ-opioid antagonist was given in the contralateral, non-inflamed paw, the hypoalgesia in the CG-treated paw was unaffected (Fig. 5A). Local injection of the κ-opioid receptor antagonist, nor-binaltorphimine also reversed the celecoxib-induced hypoalgesia to basal levels but allowed the anti-hyperalgesic component of the response to celecoxib to persist (Fig. 5B). Basically, the same profile was also seen, i.e. reversal of the hypoalgesic response due to celecoxib, when the delta-opioid antagonist naltrindole was used (Fig. 5C). Note that local treatment with either beta-funaltrexamine, nor-binaltorphimine or naltrindole, given alone to the inflamed paw, did not modify the hyperalgesia induced by CG, compared with data from Fig. 1, and were not different from those obtained with saline/ vehicle-treated animals (other figures).
Fig. 3. Celecoxib-induced hypoalgesia depends on carrageenan dose. Celecoxib (CX) at a selected dose (12 mg/kg) or its vehicle (SAL) was administered subcutaneously to rats ½ h before 250 or 1000 μg/paw carrageenan (CG) given intraplantarly. Nociceptive thermal thresholds (AUC) were obtained as described in Fig. 1. No change on the hyperalgesia induced by the high dose of carrageenan was observed. * significantly different from SAL; # significantly different from basal values.
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Fig. 4. Celecoxib, but not indomethacin, induced hypoalgesia was reversed by naltrexone. (A) Subcutaneous injection of celecoxib (CX; 12 mg/kg) induced hypoalgesia, raising nociceptive thresholds in the inflamed paws (CG) above the level in paws injected only with saline (SAL; #, P b 0.05). However, (B) indomethacin (INDO, sc, 10 mg/kg) was only anti-hyperalgesic in inflamed paws raising thresholds back to, but not above, the levels in saline-injected paws. Note also that naltrexone (NAL, 3 mg/kg; sc) given 30 min before CG reversed the celecoxib-induced hypoalgesia to basal levels (*, P b 0.05), but did not affect the anti-hyperalgesia shown by indomethacin. Nociceptive thermal thresholds (AUC) were obtained as described in Fig. 1.
Discussion Our experiments have shown that celecoxib, a selective inhibitor of COX-2 induced a state of hypoalgesia (nociceptive thresholds raised above normal levels) in a well-established model of inflammatory pain, using thermal stimuli (Le Bars et al. 2001; Hargreaves et al. 1988). We have also provided evidence that these actions of celecoxib were mediated by opioid receptors, most likely μ, κ and δ receptors located peripherally. We undertook these experiments to extend the results we had obtained in the same inflammatory pain model using mechanical stimuli, because there is considerable evidence that different painful stimuli (thermal vs. mechanical) can give rise to different effects in the same models (Schepers et al. 2008; Bennet et al. 2000; Mansikka et al. 2000). Overall, the results we report here are similar to those obtained with mechanical stimuli (Francischi et al. 2002; França et al. 2006) but with some important differences. As in previous work, we used here a relatively low dose of CG (250 μg per paw) as the standard inflammatory stimulus which produced a hyperalgesic state lasting about 6 h after the injection of CG. Higher doses of CG (up to 2000 μg per paw) induced greater intensity and duration of hyperalgesia and the analgesic effects of celecoxib were lost in these conditions of increased hyperalgesia. This loss of analgesic activity probably reflects a “competition” between the intensity of the inflammatory process
Fig. 5. Peripheral (local) reversal by opioid antagonists of celecoxib-induced hypoalgesia. A hundred nanomoles of each opioid antagonist (A: beta-funaltrexamine—FNT; B: norbinaltorphimine—Nor-BNI; C: naltrindole) was injected into the paw either ipsi (ipsi) or contralaterally (contra) 30 min before celecoxib (CX) or saline (SAL) subcutaneously and 1 h before 250 μg/paw carrageenan (CG). The nociceptive thermal values are presented as AUC from control (CG + opioid antagonist) and test animals. All three opioid antagonists reversed the hypoalgesia induced by celecoxib to basal level only following ipsi administration. * significant difference from vehicle-treatment (P b 0.05); # significant difference from basal values.
and the strength of the analgesic process. It is possible that a higher dose of celecoxib—we used 12 mg/kg and the IC50 provided by Penning et al. (1997) for celecoxib was about 30 mg/kg—would have been more effective against 2000 μg CG, but that was not the major aim of our work and was not further investigated. Using a thermal stimulus, although celecoxib still exhibited hypoalgesia with a bell-shaped dose–response curve, this hypoalgesia was observed over a wider dose range (3–30 mg/kg) than with mechanical stimuli (3–12 mg kg). Also the lowest dose of celecoxib used (0.3 mg/kg) was effective against thermal hyperalgesia, but showed no analgesic effect with mechanical stimuli. Thus the thermal stimulus provides a model more responsive to the analgesic effect of celecoxib, a finding compatible with those of Hargreaves et al. more than 20 years ago, who concluded that thermal stimuli yielded a more sensitive pain model. Since then, although progress has been made in understanding the neural basis of many commonly used nociceptive
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models (Le Bars et al. 2001), differences between thermal and mechanical stimuli are still being reported and analyzed. Recently Schepers et al. (2008) using these two stimuli in the same model (paws injected with complete Freunds' adjuvant) found that nor-BNI induced bilateral hypersensitivity to mechanical, but not to thermal stimuli. Similarly, mechanical and thermal stimuli were differently affected by NMDA receptor antagonists (Bennet et al. 2000) or by genetic deletion of NK-1 receptors (Mansikka et al. 2000). These differences have been analyzed in terms of different anatomical substrates (Schepers et al. 2008) or different mediators (Bennet et al. 2000; Mansikka et al. 2000) involved in mechanical or thermal hyperalgesia. However, in our model with either stimulus, there was the same hypoalgesia caused by celecoxib and the same susceptibility to opioid antagonists, either non-selective (Francischi et al. 2002; França et al. 2006) or selective (this study). Thus, the endogenous opioid mediation of coxib-induced hypoalgesia is not restricted to mechanical hyperalgesia and may operate in other modes of inflammatory pain. The bell-shaped curve may also be indirect evidence for opioid mediation of celecoxib-induced hypoalgesia, as such response curves have been noted for some time, with endogenous (Bujdosó et al. 2001; Hauser et al. 2005) or exogenous opiates (Hara et al. 1997; Silverman 2009; Sarton et al. 2008). Several explanations have been advanced including the pro-nociceptive effects of endogenous peptides (Hauser et al. 2005; Tan-No et al. 2009; Calo et al. 2000) and two forms of μ-opioid receptor, one mediating pro- and the other, anti-nociceptive effects (Sarton et al. 2008). However our experiments were not designed to decide between these possibilities. Another important aspect, common to both forms of hyperalgesia, was that in our model of inflammatory pain, analgesic effects in the contralateral, non-inflamed, paw were never seen with either celecoxib or indomethacin given systemically (sc) or centrally (icv), whereas morphine or paracetamol (sc) did modify the nociceptive threshold in contralateral paws (Ferreira et al. 1978; Rezende et al. 2008). This apparent inflammation-dependence of celecoxib's analgesia may reflect an increased number of opioid receptors (Zöllner et al. 2003; Stein et al. 2003) or, more speculatively, an increased release of endogenous opioid (s) in inflamed sites. If there were an inflammation-dependent, increased release of endogenous opioids, then this did not noticeably occur in the absence of celecoxib, as neither the selective antagonists (present study) nor naltrexone (França et al. 2006) modified the hyperalgesia induced by CG, a finding that contrasted with that of Schepers et al. (2008) who found the κ-receptor antagonist, nor-BNI, to potentiate hyperalgesia induced by CFA. Notwithstanding the overall similarity in responses between mechanical and thermal hyperalgesia, the present model of thermal hyperalgesia showed that naltrexone and selective antagonists of μ-, κ- and δ-opioid receptors reversed the analgesic effects of celecoxib in CG-treated paws, only to a normal, i.e., untreated, level, whereas for mechanical hyperalgesia, the effects of the celecoxib was totally reversed to thresholds equal to those seen with CG alone (França et al. 2006). As we have used the same inflammatory stimulus, the same strain of rats and the same dose of celecoxib given sc in both sets of experiments, this differential effect of opioid antagonists would imply a real difference in mechanisms between those mediating antihyperalgesia and those mediating hypoalgesia, with thermal stimuli. One simple explanation for our present results would be to postulate two separate mechanisms for celecoxib, one mediating the antihyperalgesia, i.e. return of the nociceptive thresholds to the basal level and the other responsible for the hypoalgesia, i.e, raising thresholds above basal levels. The first would involve PGs and COX, just like the effects of indomethacin (Vane 1971; Ferreira et al. 1978) and thus be resistant to naltrexone, whereas the second would involve opioid receptors and be sensitive to naltrexone and other opioid receptor antagonists. However such a scheme would only operate for thermal, and not mechanical, hyperalgesia, and would imply the involvement of
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two sets of neuronal pathways in the thermal hyperalgesia, in contrast to the one, opioid-mediated, pathway for mechanical hyperalgesia. Our findings that selective antagonists of μ-, κ- and δ-opioid receptors given to the paw were able to prevent celecoxib-induced thermal hypoalgesia imply that these receptors were involved at a local, peripheral site. Agonists for those receptors, morphine, bremazocine and SNC80, respectively, have been shown to induce anti-nociceptive effects (Ferreira et al. 1991; Amarante and Duarte 2002; Pacheco et al. 2005). Conclusion We have extended our analysis of coxib-induced hypoalgesia, demonstrating that the effects of celecoxib (and SC236) were not restricted to one type of nociceptive stimulus but were exhibited, albeit with some differences, in the model of thermal hyperalgesia. We were also able to confirm that with thermal stimuli, celecoxib still induced hypoalgesia, which was mediated by the endogenous opioid system acting via peripheral μ- and κ- and δ opioid receptors. Important questions still to be answered include the identity of the endogenous opioids and the cell types involved in this peripheral antinociceptive action of celecoxib. Conflict of interest The authors claim no conflict of interest.
Acknowledgment Financial support: Conselho Nacional de Pesquisa (CNPq), Fundação de Amparo à Pesquisa de Minas Gerais (FAPEMIG) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. References Amarante LH, Duarte IDG. The κ-opioid agonist (±)-bremazocine elicits peripheral antinociception by activation of the L-arginine/nitric oxide/cyclic GMP pathway. European Journal of Pharmacology 454, 19–23, 2002. Bennet AD, Everhart AW, Hulsebosch CE. Intrathecal administration of an NMDA or a non-NMDA receptor antagonist reduces mechanical but not thermal allodynia in a rodent model of chronic central pain after spinal cord injury. Brain Research 859, 72–82, 2000. Bujdosó E, Jászberényi M, Tömböly C, Tóth G, Telegdy G. Behavioral and neuroendocrine actions of endomorphin-2. Peptides 22, 1459–1463, 2001. Calo G, Guerrini R, Rizzi A, Salvadori S, Regoli D. Pharmacology ofnociceptin and its receptor: a novel therapeutic target. British Journal of Pharmacology 129, 1261–1283, 2000. Chan CC, Boyce S, Brideau C, Charleson S, Cromlish W, Ethier D, Evans J, Ford-Hutchinson AW, Forrest M, Gauthier JY, Gordon R, Gresser M, Guay J, Kargman S, Kennedy B, Leblanc Y, Leger S, Mancini J, O'Neill GP, Quellet M, Patrick D, Percival MD, Perrier H, Prasit P, Rodger I, Tagari P, Therien M, Vickers P, Visco D, Wang Z, Webb J, Wong E, Xu L-J, Young RN, Zamboni R, Riendeau D. Rofecoxib (Vioxx, MK-0966; 4-(4´methylsulfonylphenyl)-3-phenyl-2-(5H)-furanone): a potent and orally active cyclooxygenase-2 inhibitor. Pharmacological and biochemical profiles. The Journal of Pharmacology and Experimental Therapeutics 290, 551–560, 1999. Ferreira SH, Duarte ID, Lorenzetti BB. The molecular mechanism of action of peripheral morphine analgesia: stimulation of the cGMP system via nitric oxide release. European Journal of Pharmacology 201 (1), 121–122, 1991. Ferreira SH, Nakamura M, Castro MAS. The hyperalgesic effects of prostacyclin and prostaglandin E2. Prostaglandins 16, 31–37, 1978. França DS, Ferreira-Alves DL, Duarte ID, Ribeiro MC, Rezende RM, Bakhle YS, Francischi JN. Endogenous opioids mediate the hypoalgesia induced by inhibitors of cyclooxygenase-2 in rat paws treated with carrageenan. Neuropharmacology 51, 37–43, 2006. Francischi JN, Chaves CT, Moura ACL, Lima AS, Rocha OA, Ferreira-Alves DL, Bakhle YS. Selective inhibitors of cyclo-oxygenase-2 (COX-2) induce hypoalgesia in a rat paw model of inflammation. British Journal of Pharmacology 37, 837–844, 2002. Gutstein HB, Akil H. Opioid analgesics (Chapter 23). In: Brunton, LL, Lazo, JS, Parker, KL (Eds.), The Pharmacological Basis of Therapeutics, 11e. The McGraw-Hill companies, United States of America, pp. 569–620, 2006. Hara N, Minami T, Okuda-Ashitaka E, Sugimoto T, Sakai M, Onaka M, Mori H, Imanishi T, Shingu K, Ito S. Characterization of nociceptin hyperalgesia and allodynia in conscious mice. British Journal of Pharmacology 121, 401–408, 1997. Hargreaves K, Dubner R, Brown F, Flores C, Joris J. A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain 32 (1), 77–88, 1988.
956
J.D. Correa et al. / Life Sciences 86 (2010) 951–956
Hauser KF, Aldrich JV, Anderson KJ, Bakalkin G, Christie MJ, Hall ED, Knapp PE, Scheff SW, Singh IN, Vissel B, Woods AS, Yakovleva T, Shippenberg TS. Pathobiology of dynorphins in trauma and disease. Frontiers in Bioscience 10, 216–235, 2005. Le Bars D, Gozariu M, Cadden SW. Animal models of nociception. Pharmacological Reviews 53 (4), 597–652, 2001. Mansikka H, Sheth RN, DeVries C, Lee H, Winchurch R, Raja SN. Nerve injury-induced mechanical but not thermal hyperalgesia is attenuated in neurokinin-1 receptor knockout mice. Experimental Neurology 162, 343–349, 2000. Pacheco DF, Reis GML, Castro MSA, Francischi JN, Duarte IDG. Delta-opioid receptor agonist SNC80 elicits peripheral antinociception via d1 and d2 receptors and activation of the L-arginine/nitric oxide/cGMP pathway. Life Sciences 78, 54–60, 2005. Penning TB, Talley JJ, Bertenshaw SR, Carter JS, Collins PW, Docter S, Graneto MJ, Lee LF, Malecha JW, Miyashiro JM, Rogers RS, Rogier DJ, Yu SS, Anderson GD, Burton EG, Cogburn JN, Gregory SA, Koboldt CM, Perkins WE, Seibert K, Veenhuizen AW, Zhang YY, Isakson PC. Synthesis and biological evaluation of the 1, 5-diarylpyrazole class of cyclooxygenase-2 inhibitors: identification of 4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-y1] benzenesulfonamide (SC-58635, Celecoxib). Journal of Medicinal Chemistry 40, 1347–1365, 1997. Resende MA, Reis WGP, Pereira LSM, Ferreira W, De Lima ME, Santoro MM, Francischi JN. Hyperalgesia and edema responses induced by rat peripheral blood mononuclear cells incubated with carrageenan. Inflammation 25, 277–285, 2001. Rezende RM, Dos Reis WGP, Duarte ID, Lima PP, Bakhle YS, Francischi JN. The analgesic actions of centrally administered celecoxib are mediated by endogenous opioids. Pain 142 (1–2), 94–100, 2009. Rezende RM, França DS, Menezes GB, Dos Reis WGP, Bakhle YS, Francischi JN. Different mechanisms underlie the analgesic actions of paracetamol and dipyrone in a rat model of inflammatory pain. British Journal of Pharmacology 153, 760–768, 2008.
Sandkuhler J. Models and mechanisms of hyperalgesia and allodynia. Physiological Reviews 89, 707–758, 2009. Sarton E, Teppema L, Dahan A. Naloxone reversal of opioid-induced respiratory depression with special emphasis on the partial agonist/antagonist buprenorphine. Advances in Experimental Medicine and Biology 605, 486–491, 2008. Schepers RJ, Mahoney JL, Gehrke BJ, Shippenberg TS. Endogenous kappa-opioid receptor systems inhibit hyperalgesia associated with localized peripheral inflammation. Pain 138, 423–439, 2008. Silverman SM. Opioid induced hyperalgesia: clinical implications for the pain practitioner. Pain Physician 12, 679–684, 2009. Stein C, Schäfer M, Machelska H. Attacking pain at its source: new perspectives on opioids. Natural Medicines 9, 1003–1008, 2003. Tan-No K, Takahashi H, Nakagawasai O, Niijima F, Sakurada S, Bakalkin G, Terenius L, Tadano T. Nociceptive behavior induced by the endogenous opioid peptides dynorphins in uninjured mice: evidence with intrathecal N-ethylmaleimide inhibiting dynorphin degradation. International Review of Neurobiology 85, 191–205, 2009. Vane JR. Inhibition of prostaglandin synthesis as a mechanism of action for aspirin like drugs. Nature: New Biology 231, 232–235, 1971. Vinegar R, Truax JF, Selph JL, Johnston PR, Venable AL, McKenzie KK. Pathway to carrageenan-induced inflammation in the hind limb of the rat. Federation Proceedings 46, 118–126, 1987. Zimmermann M. Ethical guidelines for investigations of experimental pain in conscious animals. Pain 6, 109–110, 1983. Zöllner C, Shaquira MA, Bopaiah CP, Mousa S, Stein C, Schäfer M. Painful inflammationinduced increase in μ-opioid receptor binding and G-protein coupling in primary afferent neurons. Molecular Pharmacology 64, 202–210, 2003.