Free
Education  |   October 2003
Inhibition of Inflammatory Hyperalgesia by Activation of Peripheral CB2Cannabinoid Receptors
Author Affiliations & Notes
  • Aline Quartilho, B.S.
    *
  • Heriberto P. Mata, B.S.
  • Mohab M. Ibrahim, M.S.
    *
  • Todd W. Vanderah, Ph.D.
  • Frank Porreca, Ph.D.
    §
  • Alexandros Makriyannis, Ph.D.
  • T. Philip Malan, Ph.D., M.D.
    §
  • * Graduate Student, Interdisciplinary Graduate Program in Pharmacology and Toxicology, † Research Specialist, Department of Anesthesiology, ‡ Assistant Professor, § Professor, Departments of Pharmacology and Anesthesiology, The University of Arizona. ∥ Professor, Departments of Medicinal Chemistry and Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut.
  • Received from the Departments of Anesthesiology and Pharmacology, The University of Arizona, Tucson, Arizona.
Article Information
Education
Education   |   October 2003
Inhibition of Inflammatory Hyperalgesia by Activation of Peripheral CB2Cannabinoid Receptors
Anesthesiology 10 2003, Vol.99, 955-960. doi:
Anesthesiology 10 2003, Vol.99, 955-960. doi:
THE identification of cannabinoid receptors in the periphery has led to the concept that it may be possible to develop cannabinoid receptor agonists that act selectively outside the central nervous system (CNS) to produce pain relief without undesirable CNS effects. CB2cannabinoid receptors are not found in the CNS, 1–4 but are primarily located on immune cells in the periphery. 1,5–7 It has recently been shown that CB2receptor–selective agonists produce peripheral antinociception, but do not cause CNS effects produced by nonselective cannabinoid receptor agonists, 8,9 suggesting that selective activation of CB2receptors may achieve the goal of peripheral pain relief without CNS effects.
Increased sensory sensitivity produced by peripheral inflammatory processes is an important component of many pain states. Cannabinoid receptor agonists inhibit inflammatory hyperalgesia in animal models. 10 Significantly, peripheral cannabinoid receptors may be capable of inhibiting inflammatory hyperalgesia, as shown by the observation that the endogenous cannabinoid receptor agonist anandamide exhibits antihyperalgesic actions when injected locally into the inflamed hind paw of the rat. 11 The effects of anandamide were reversed by the CB1receptor–selective antagonist SR141716A, suggesting that they were mediated, at least in part, by CB1receptors. Recently, the ability of CB2cannabinoid receptors to inhibit inflammatory pain responses has begun to be studied using newly developed CB2receptor–selective agonists. In this regard, systemic administration of the CB2receptor–selective agonist GW405833 partially prevented carrageenan-induced inflammatory mechanical hyperalgesia. 12 
The current studies test the hypothesis that selective activation of peripheral CB2receptors can inhibit inflammation and inflammatory hyperalgesia. We tested the ability of the CB2receptor–selective agonist AM1241 to reverse carrageenan-induced edema and inflammatory hyperalgesia. The ability of peripheral CB2receptors to reverse inflammatory hyperalgesia was tested by site-specific drug administration. Peripheral CB2receptors may be an appropriate target for eliciting relief of inflammatory pain without the CNS effects of nonselective cannabinoid receptor agonists.
Methods
Animals
All procedures were approved by The University of Arizona Animal Care and Use Committee and conformed to the United States Public Health Service Policy on Humane Care and Use of Laboratory Animals and the guidelines of the International Association for the Study of Pain. 13 Male Sprague-Dawley rats (Harlan, Indianapolis, IN), weighing 250–300 g at the time of testing, were maintained in a climate-controlled room on a 12-h light–dark cycle and allowed food and water ad libitum  . For all studies, animals were randomly assigned to treatment groups, and measurements were made by a single observer. The observer was not masked to the treatment administered.
Drug Administration
AM1241 is a cannabinoid receptor agonist with 70-fold selectivity for the CB2receptor in vitro  (Ki= 3.4 nm for binding to CB2receptors in mouse spleen tissue and Ki= 239 nm for binding to CB1receptors in rat brain tissue). AM630 is a CB2receptor–selective antagonist with 70- to 165-fold selectivity for binding to the CB2receptor in vitro  . 14,15 AM251 is a 300-fold-selective CB1receptor antagonist. 16,17 All cannabinoid drugs were synthesized in the laboratory of one of the authors (Dr. Makriyannis). Cannabinoid drugs were dissolved in dimethyl sulfoxide and injected subcutaneously in the dorsal surface of the hind paw (intrapaw, 50 μl) or intraperitoneally (0.5 ml). Measurements were taken 25 min after intrapaw injection or 15 min after intraperitoneal injection. In preliminary experiments, these were the times of maximal drug effect. Carrageenan and capsaicin were purchased from Sigma (St. Louis, MO). Carrageenan was dissolved in water, and capsaicin was dissolved in 7% Tween 80.
Carageenan-induced Thermal Hyperalgesia and Paw Edema
Inflammation was induced by injection of 50 μl 2% carrageenan in the dorsal surface of the hind paw. Thermal paw withdrawal latency was measured as described in the Testing of Thermal Withdrawal Latency section. In separate experiments, hind paw edema was assessed by measuring paw volume using a plethysmometer (model 7140; Stoelting Company, Wood Dale, IL). Measurements were made before carrageenan administration and 3 h after carrageenan administration. Cannabinoid test drugs were then administered, and measurements were again made 15 min after intraperitoneal drug administration or 25 min after intrapaw drug administration.
Capsaicin-induced Flinching and Hyperalgesia
Capsaicin (20 μg in 20 μl) was injected into the dorsal surface of the hind paw. Flinches of the hind paw were counted for 5 min. In separate experiments, thermal responses were measured before capsaicin administration and 10 min after capsaicin administration. Cannabinoid test drugs were administered to separate groups of animals in the paw 15 min before capsaicin administration.
Testing of Thermal Withdrawal Latency
Withdrawal latency to thermal stimuli was tested, as described by Hargreaves et al.  , 18 by using equipment purchased from the Department of Anesthesiology of the University of California, San Diego. Rats were allowed to acclimate within Plexiglas enclosures on a clear glass plate maintained at 30°C using a warming mechanism driven by a thermocouple. A radiant heat source, a projector bulb, was focused onto the plantar surface of the hind paw. The intensity of the heat source was adjusted to yield a baseline paw withdrawal latency of 20 s. The temperature of the paw at the time of withdrawal was not directly measured. Activation of the heat source activated a timer that stopped when withdrawal of the paw was detected with a photodetector. A maximal cutoff of 40 s was used to prevent tissue damage.
Data Analysis
Groups were compared using ANOVA followed by pair-wise comparisons using the Student t  test with Bonferroni correction. Repeated-measures ANOVA was used when repeated measurements were made on the same animals. Significance was defined as P  less than 0.05.
Results
Carrageenan-induced inflammation decreased the paw withdrawal latency to thermal stimuli by 48% to 63%, when measured 3 h after carrageenan injection (figs. 1 and 2).
Fig. 1. Reversal by systemic (intraperitoneal) AM1241 of carrageenan-induced thermal hypersensitivity. Antagonism of the effects of AM1241 by the CB2receptor–selective antagonist AM630 (100 μg/kg, intraperitoneal). Lack of antagonism by the CB1receptor–selective antagonist AM251 (300 μg/kg, intraperitoneal). Data are expressed as mean ± SEM. Groups were compared using ANOVA followed by pair-wise comparisons using the Student t  test with Bonferroni correction. *P  < 0.05 compared with precarrageenan baseline; +P  < 0.05 compared with AM1241 alone; n = 6 per group. BL = precarrageenan baseline; CG = postcarrageenan value.
Fig. 1. Reversal by systemic (intraperitoneal) AM1241 of carrageenan-induced thermal hypersensitivity. Antagonism of the effects of AM1241 by the CB2receptor–selective antagonist AM630 (100 μg/kg, intraperitoneal). Lack of antagonism by the CB1receptor–selective antagonist AM251 (300 μg/kg, intraperitoneal). Data are expressed as mean ± SEM. Groups were compared using ANOVA followed by pair-wise comparisons using the Student t 
	test with Bonferroni correction. *P 
	< 0.05 compared with precarrageenan baseline; +P 
	< 0.05 compared with AM1241 alone; n = 6 per group. BL = precarrageenan baseline; CG = postcarrageenan value.
Fig. 1. Reversal by systemic (intraperitoneal) AM1241 of carrageenan-induced thermal hypersensitivity. Antagonism of the effects of AM1241 by the CB2receptor–selective antagonist AM630 (100 μg/kg, intraperitoneal). Lack of antagonism by the CB1receptor–selective antagonist AM251 (300 μg/kg, intraperitoneal). Data are expressed as mean ± SEM. Groups were compared using ANOVA followed by pair-wise comparisons using the Student t  test with Bonferroni correction. *P  < 0.05 compared with precarrageenan baseline; +P  < 0.05 compared with AM1241 alone; n = 6 per group. BL = precarrageenan baseline; CG = postcarrageenan value.
×
Fig. 2. (A  ) Reversal by peripheral (intrapaw) AM1241 of carrageenan-induced thermal hypersensitivity. (B  ) Antagonism of the effects of AM1241 by the CB2receptor–selective antagonist AM630 (100 μg/kg, intrapaw), but not by the CB1receptor–selective antagonist AM251 (300 μg/kg, intrapaw). Data are expressed as mean ± SEM. Groups were compared using ANOVA followed by pair-wise comparisons using the Student t  test with Bonferroni correction. *P  < 0.05 compared with preinflammation baseline; #P  < 0.05 compared with postcarrageenan value; +P  < 0.05 compared with ipsilateral administration of AM1241 alone; n = 6 per group. BL = precarrageenan baseline; CG = postcarageenan value.
Fig. 2. (A 
	) Reversal by peripheral (intrapaw) AM1241 of carrageenan-induced thermal hypersensitivity. (B 
	) Antagonism of the effects of AM1241 by the CB2receptor–selective antagonist AM630 (100 μg/kg, intrapaw), but not by the CB1receptor–selective antagonist AM251 (300 μg/kg, intrapaw). Data are expressed as mean ± SEM. Groups were compared using ANOVA followed by pair-wise comparisons using the Student t 
	test with Bonferroni correction. *P 
	< 0.05 compared with preinflammation baseline; #P 
	< 0.05 compared with postcarrageenan value; +P 
	< 0.05 compared with ipsilateral administration of AM1241 alone; n = 6 per group. BL = precarrageenan baseline; CG = postcarageenan value.
Fig. 2. (A  ) Reversal by peripheral (intrapaw) AM1241 of carrageenan-induced thermal hypersensitivity. (B  ) Antagonism of the effects of AM1241 by the CB2receptor–selective antagonist AM630 (100 μg/kg, intrapaw), but not by the CB1receptor–selective antagonist AM251 (300 μg/kg, intrapaw). Data are expressed as mean ± SEM. Groups were compared using ANOVA followed by pair-wise comparisons using the Student t  test with Bonferroni correction. *P  < 0.05 compared with preinflammation baseline; #P  < 0.05 compared with postcarrageenan value; +P  < 0.05 compared with ipsilateral administration of AM1241 alone; n = 6 per group. BL = precarrageenan baseline; CG = postcarageenan value.
×
Systemic (intraperitoneal) administration of the CB2cannabinoid receptor–selective agonist AM1241 dose-dependently reversed the inflammation-induced decrease in thermal withdrawal latency and prolonged thermal withdrawal latency beyond preinflammation baseline values (fig. 1). The CB2receptor–selective antagonist AM630 (100 μg/kg, intraperitoneal) completely reversed the effect of AM1241 on thermal withdrawal latency, whereas the CB1receptor–selective antagonist AM251 (300 μg/kg, intraperitoneal) had no effect (fig. 1). This dose of AM630 blocked the antinociception produced by the CB2receptor–selective agonist AM1241, but not the CNS effects produced by the mixed CB1–CB2receptor agonist WIN55,212–2, suggesting that this dose is selective for the CB2receptor. 9 This dose of AM251 blocked the CNS effects produced by the mixed CB1–CB2receptor agonist WIN55,212–2, but not the antinociception produced by the CB2receptor–selective agonist AM1241, suggesting that this dose is selective for the CB1receptor. 9 These doses of AM630 and AM251 had no effect when administered alone. The paw withdrawal latency after carrageenan was 11.1 ± 1.3 s. After AM251, it was 10.5 ± 1.7 s, and after AM630, it was 9.7 ± 10.9 s.
AM1241 dose-dependently reversed the carrageenan-induced decrease in thermal withdrawal latency and prolonged thermal withdrawal latency beyond preinflammation baseline values when administered into the inflamed paw, but it had no effect when administered into the contralateral paw (fig. 2). The CB2receptor–selective antagonist AM630 (100 μg/kg, intrapaw) completely reversed the effect of intrapaw AM1241 (2 mg/kg), whereas the CB1receptor–selective antagonist AM251 (300 μg/kg, intrapaw) had no effect (fig. 2). These doses of AM630 and AM251 had no effect when administered alone. The paw withdrawal latency after carrageenan was 12.8 ± 1.1 s. After AM251, it was 11.6 ± 2.2 s, and after AM630, it was 9.2 ± 2.6 s.
Hind paw carrageenan injection increased hind paw volume to 230% of normal (fig. 3). At the dose used (2 mg/kg), intrapaw AM1241 reversed inflammation-induced paw edema by 63%. AM630 (100 μg/kg intrapaw) completely reversed the effect of AM1241, whereas AM251 (300 μg/kg intrapaw) had no effect.
Fig. 3. Reversal by peripheral (intrapaw) AM1241 of carrageenan-induced hind paw edema. Antagonism of the effects of AM1241 by the CB2receptor–selective antagonist AM630 (100 μg/kg), but not by the CB1receptor–selective antagonist AM251 (300 μg/kg). Data are expressed as mean ± SEM. Groups were compared using ANOVA followed by pair-wise comparisons using the Student t  test with Bonferroni correction. *P  < 0.05 compared with precarrageenan baseline; #P  < 0.05 compared with postcarrageenan value; +P  < 0.05 compared with AM1241 alone; n = 6 per group. BL = precarrageenan baseline; CG = postcarageenan value.
Fig. 3. Reversal by peripheral (intrapaw) AM1241 of carrageenan-induced hind paw edema. Antagonism of the effects of AM1241 by the CB2receptor–selective antagonist AM630 (100 μg/kg), but not by the CB1receptor–selective antagonist AM251 (300 μg/kg). Data are expressed as mean ± SEM. Groups were compared using ANOVA followed by pair-wise comparisons using the Student t 
	test with Bonferroni correction. *P 
	< 0.05 compared with precarrageenan baseline; #P 
	< 0.05 compared with postcarrageenan value; +P 
	< 0.05 compared with AM1241 alone; n = 6 per group. BL = precarrageenan baseline; CG = postcarageenan value.
Fig. 3. Reversal by peripheral (intrapaw) AM1241 of carrageenan-induced hind paw edema. Antagonism of the effects of AM1241 by the CB2receptor–selective antagonist AM630 (100 μg/kg), but not by the CB1receptor–selective antagonist AM251 (300 μg/kg). Data are expressed as mean ± SEM. Groups were compared using ANOVA followed by pair-wise comparisons using the Student t  test with Bonferroni correction. *P  < 0.05 compared with precarrageenan baseline; #P  < 0.05 compared with postcarrageenan value; +P  < 0.05 compared with AM1241 alone; n = 6 per group. BL = precarrageenan baseline; CG = postcarageenan value.
×
Hind paw capsaicin injection decreased thermal paw withdrawal latency by 74% (fig. 4). Intraperitoneal injection of AM1241 dose-dependently reversed the capsaicin-induced decrease in paw withdrawal latency and increased paw withdrawal latency beyond precapsaicin values (fig. 4). AM630 (100 μg/kg, intraperitoneal) completely reversed the effect of intraperitoneal AM1241 (300 μg/kg), whereas AM251 (300 μg/kg, intraperitoneal) had no effect. Hind paw capsaicin injection produced a flinching response of 17 ± 1 flinches/min. Intraperitoneal injection of AM1241 produced a dose-dependent decrease in capsaicin-induced hind paw flinches (fig. 4). AM630 (100 μg/kg, intraperitoneal) completely reversed the effect of intraperitoneal AM1241 (300 μg/kg), whereas AM251 (300 μg/kg, intraperitoneal) had no effect.
Fig. 4. (A  ) Reversal by systemic (intraperitoneal) AM1241 of capsaicin-induced thermal hypersensitivity. (B  ) Reversal by peripheral (intraperitoneal) AM1241 of capsaicin-induced flinches of the hind paw. Antagonism of the effects of AM1241 by the CB2receptor–selective antagonist AM630 (100 μg/kg, intraperitoneal), but not by the CB1receptor–selective antagonist AM251 (300 μg/kg, intraperitoneal). Data are expressed as mean ± SEM. Groups were compared using ANOVA followed by pair-wise comparisons using the Student t  test with Bonferroni correction. *P  < 0.05 compared with precapsaicin baseline; +P  < 0.05 compared with AM1241 alone; n = 6 per group. BL = precapsaicin baseline; CS = postcapsaicin value.
Fig. 4. (A 
	) Reversal by systemic (intraperitoneal) AM1241 of capsaicin-induced thermal hypersensitivity. (B 
	) Reversal by peripheral (intraperitoneal) AM1241 of capsaicin-induced flinches of the hind paw. Antagonism of the effects of AM1241 by the CB2receptor–selective antagonist AM630 (100 μg/kg, intraperitoneal), but not by the CB1receptor–selective antagonist AM251 (300 μg/kg, intraperitoneal). Data are expressed as mean ± SEM. Groups were compared using ANOVA followed by pair-wise comparisons using the Student t 
	test with Bonferroni correction. *P 
	< 0.05 compared with precapsaicin baseline; +P 
	< 0.05 compared with AM1241 alone; n = 6 per group. BL = precapsaicin baseline; CS = postcapsaicin value.
Fig. 4. (A  ) Reversal by systemic (intraperitoneal) AM1241 of capsaicin-induced thermal hypersensitivity. (B  ) Reversal by peripheral (intraperitoneal) AM1241 of capsaicin-induced flinches of the hind paw. Antagonism of the effects of AM1241 by the CB2receptor–selective antagonist AM630 (100 μg/kg, intraperitoneal), but not by the CB1receptor–selective antagonist AM251 (300 μg/kg, intraperitoneal). Data are expressed as mean ± SEM. Groups were compared using ANOVA followed by pair-wise comparisons using the Student t  test with Bonferroni correction. *P  < 0.05 compared with precapsaicin baseline; +P  < 0.05 compared with AM1241 alone; n = 6 per group. BL = precapsaicin baseline; CS = postcapsaicin value.
×
Discussion
These results show that selective activation of peripheral CB2cannabinoid receptors reverses inflammatory hyperalgesia. Local administration of AM1241 in the inflamed hind paw reversed the decrease in thermal withdrawal latency produced by carrageenan injection. The actions of intrapaw AM1241 were local to the paw and not the result of systemic uptake and spread to distant sites, as shown by the lack of effect of equivalent doses of AM1241 administered into the contralateral paw. Local peripheral effects of AM1241 are consistent with our finding that administration of AM1241 in the tested hind paw produced antinociception to thermal stimuli in otherwise untreated animals, whereas administration in the contralateral paw did not. 9 
The effects of AM1241 seem to be mediated by CB2receptors. They were fully reversed by coadministration of the CB2receptor–selective antagonist AM630, whereas coadministration of the CB1receptor–selective antagonist AM251 had no effect. These results are consistent with our previous demonstration that the antinociceptive effects of AM1241 are mediated by the CB2receptor. 9 In addition to reversing the inflammation-induced decrease in thermal paw withdrawal latency, CB2receptor activation prolonged thermal withdrawal latencies beyond preinflammation values, consistent with the thermal antinociception produced by CB2receptor activation. 9 
AM1241 was also active when administered systemically (i.e.  , intraperitoneally). The effects of systemic AM1241 seem to be mediated at peripheral CB2receptors. We have previously shown that the antinociceptive effects of systemic AM1241 were reversed by injection of the CB2receptor–selective antagonist AM630 in the tested hind paw. 9 AM1241 was more potent when administered systemically than when injected into the dorsal surface of the ipsilateral paw. We have hypothesized that intraperitoneal AM1241 potency may be high as a result of efficient systemic absorption through the large surface area of the peritoneum, leading to distribution through the circulation to the peripheral site of action. 9 We have also hypothesized that the potency of intrapaw AM1241 may be low because AM1241 does not efficiently penetrate the tissue of the paw from the dorsal surface, where the drug was injected, to the plantar surface, where the test stimulus was applied. 9 Drug administration in the dorsal hind paw was necessary because injection of the vehicle (i.e.  , dimethyl sulfoxide) into the plantar surface prolonged withdrawal latency. We have also hypothesized that after subcutaneous injection into the paw, systemic absorption of the vehicle (dimethyl sulfoxide) in excess of AM1241 may cause AM1241 to precipitate, thereby resulting in local deposition of the drug and diminished availability of the drug to receptors at the site of action. 9 This hypothesis is supported by the qualitative observation that at necropsy, a white precipitate was frequently observed subcutaneously at the site of injection. The observation that AM1241 is more potent when administered intraperitoneally than when administered locally seems to argue against a local, peripheral effect of AM1241. However, comparison of peripheral and intraperitoneal administration of drugs is complicated by issues of drug distribution, such as those discussed in this paragraph. Therefore, comparison of ipsilateral and contralateral administration of drugs is a much more direct test of a local site of action than is local versus  systemic administration. The observation that AM1241 is active when administered in the ipsilateral paw, but has no effect when administered contralaterally, argues strongly for a local site of action.
Previous investigators have observed hyperalgesia after administration of the CB1receptor–selective antagonist SR141716A or antisense oligodeoxynucleotide-mediated knockdown of spinal CB1receptor expression, suggesting the presence of endogenous cannabinoid tone leading to constitutive activation of CB1receptors. 19,20 In contrast, in our study, intraperitoneal administration of the CB1receptor–selective antagonist AM251 alone did not result in hyperalgesia. This difference may have been caused by differences in the route of administration or in the drug used. Similarly, administration of the CB2receptor–selective antagonist AM630 alone did not result in hyperalgesia, suggesting the absence of an endogenous CB2receptor–mediated cannabinoid tone.
The hypothesis that activation of CB2receptors inhibits inflammatory hyperalgesia came from studies of the endogenous fatty acid ethanolamide, palmitoylethanolamide. Among other actions, palmitoylethanolamide inhibited inflammation-induced edema, carrageenan-induced hyperalgesia of the hind paw, and a referred hyperalgesia caused by inflammation of the bladder. 21,22 The antinociceptive effects of palmitoylethanolamide were blocked by the CB2receptor–selective antagonist SR144528, but not by the CB1receptor–selective antagonist SR141716A, suggesting that the actions of palmitoylethanolamide were mediated by the CB2receptor. However, palmitoylethanolamide has no significant affinity for CB1or CB2receptors expressed in cultured cells or for CB2receptors in rat spleen slices. 23,24 Therefore, it has been proposed that palmitoylethanolamide may indirectly activate CB2receptors, perhaps by inhibiting the inactivation of other endocannabinoids that are direct CB2receptor ligands. 25 Alternatively, it has been suggested that palmitoylethanolamide may act at an as-yet uncharacterized, possibly cannabinoid-like, receptor. 26 Because of these uncertainties, direct testing of the effects of CB2receptor activation awaited synthesis of direct CB2receptor–selective ligands.
In a preliminary report, the CB2receptor–selective agonist, 1-(2-(4-morpholinyl)ethyl)-2-methyl-3-(4-bromo-1-naphylcarbonyl)-7-methoxyindole suppressed Freund's adjuvant-induced hypersensitivity of the flexor reflex to mechanical, touch, and pinch stimuli. 27 In a published report, the CB2receptor–selective agonist GW405833 prevented carrageenan-induced mechanical hyperalgesia by as much as 50%. 12 This effect was not increased when higher doses were used. Our results significantly extend these findings in two ways. First, we observed a complete reversal of inflammatory thermal hyperalgesia by AM1241, whereas Clayton et al.  12 observed only a partial inhibition of inflammatory mechanical hyperalgesia by GW405833. It is not clear whether the differences in maximal effect are the result of differences in the drugs tested, of the different sensory modalities tested, or of the use of a prevention protocol when testing GW405833 and a reversal protocol when testing AM1241. Second, we used site-specific drug injection to show that activation of CB2receptors in the periphery is sufficient to reverse inflammatory hyperalgesia.
Our data confirm previous findings that CB2receptor activation inhibits tissue inflammation and extend them by showing that CB2receptor activation not only prevents but also reverses inflammation-induced edema. Peripheral (intrapaw) AM1241 reversed the increase in paw volume produced by injection of carrageenan in the hind paw. The inhibitory effect of AM1241 was completely blocked by the CB2receptor–selective antagonist AM630, whereas the CB1receptor–selective antagonist AM251 had no effect. Potential antiinflammatory effects of CB2receptor activation were initially suggested by the presence of CB2receptors on inflammatory cells. 1,5–7 Antiinflammatory effects of CB2receptors were shown when the CB2receptor–selective agonist HU-308 inhibited arachidonic acid–induced ear edema 8 and when the CB2receptor–selective agonist GW405833 prevented carrageenan-induced paw edema. 12 GW405833 administered 30 min before carrageenan produced approximately 45% inhibition of paw edema that did not increase with further increases in dosage. 12 HU-308 administered 60 min before arachidonic acid 8 or AM1241 administered 3 h after carrageenan also produced only partial inhibition of ear or paw edema. Because only a single dose of each was used, however, it is not known whether these are the maximal effects possible.
It is not known how activation of local, peripheral CB2receptors inhibits inflammatory hyperalgesia. One possibility is that CB2receptors on the peripheral terminals of primary afferent neurons inhibit transduction or conduction of the pain signal. However, evidence regarding the expression of CB2receptors on primary afferent neurons is conflicting. A recent preliminary report showed labeling of medium-diameter cells in trigeminal ganglia by an antibody directed against the CB2receptor. 28 However, CB2receptor mRNA was not detected in dorsal root ganglia or trigeminal ganglia by in situ  hybridization, although the presence of CB1receptor mRNA was clearly shown. 28,29 
Alternatively, CB2receptor agonists could inhibit pain responses by an indirect mechanism. The CB2receptor is present on immune and mast cells. 1,5–7 Activation of CB2receptors on mast or immune cells could inhibit the release of molecules that sensitize the peripheral nociceptor. In particular, nerve growth factor (NGF) seems to play an essential role in the production of inflammatory hyperalgesia. 30,31 Other mediators, such as interleukin-1β and tumor necrosis factor-α, are also essential for the production of inflammatory hyperalgesia, but seem to contribute to the hyperalgesia primarily by increasing tissue NGF content. 32,33 NGF seems to have a direct action on primary afferent neurons, 34,35 and these actions have been proposed to be mediated by the VR1 vanilloid receptor. 36 NGF and bradykinin have recently been shown to remove phosphatidylinositol-4,5-bisphosphate–mediated inhibition of the VR1 receptor. 37 Similarly, we have hypothesized that AM1241 may produce acute antinociception by inhibiting constitutive release of NGF or other sensitizing substances from mast or immune cells. 9 
Because VR1 vanilloid receptors have been proposed to mediate inflammatory thermal hyperalgesia, 30 we tested the ability of AM1241 to inhibit sensory responses to capsaicin, an agonist of the VR1 receptor. AM1241 inhibited capsaicin-induced inflammatory hyperalgesia and flinching. Because capsaicin acts directly on vanilloid receptors of primary afferent neurons, AM1241 could be exerting its effects by activating CB2receptors on primary afferent neurons. Alternatively, as described earlier, CB2receptor activation could indirectly inhibit neuronal responses to capsaicin, by inhibiting the release of pronociceptive substances, such as NGF, from inflammatory and immune cells. Although cannabinoid compounds structurally related to anandamide can exert effects at VR1 receptors, it is unlikely that AM1241 does so, because it is structurally dissimilar from anandamide-like compounds. In addition, its actions are blocked by the CB2receptor–selective antagonist AM630, a compound unlikely to act at VR1 receptors because it is not structurally similar to anandamide.
The findings presented in this article show that activation of peripheral CB2receptors is sufficient to reverse inflammatory thermal hyperalgesia. Because CB2receptors are not found in the CNS 1–4 and CB2receptor–selective agonists do not produce the CNS effects typical of nonselective cannabinoid receptor agonists, 8,9 CB2receptor–selective agonists may have a significant clinical advantage over nonselective cannabinoid compounds. Although it has not been shown that CB2receptor–selective agonists are free of CNS effects in the presence of peripheral inflammation, it is likely that they will be. CB2receptor agonists are predicted to be effective in treating inflammatory pain without the central side effects of cannabinoids retaining activity at the CB1receptor.
References
Munro S, Thomas KL, Abu-Shaar M: Molecular characterization of a peripheral receptor for cannabinoids. Nature 1993; 365: 61–5Munro, S Thomas, KL Abu-Shaar, M
Griffin G, Wray EJ, Tao Q, McAllister SD, Rorrer WK, Aung MM, Martin BR, Abood ME: Evaluation of the cannabinoid CB2receptor selective antagonist, SR144528: Further evidence of cannabinoid CB2receptor absence in the rat central nervous system. Eur J Pharmacol 1999; 377: 117–25Griffin, G Wray, EJ Tao, Q McAllister, SD Rorrer, WK Aung, MM Martin, BR Abood, ME
Buckley NE, McCoy KL, Mezey E, Bonner T, Zimmer A, Felder C, Glass M, Zimmer A: Immunomodulation by cannabinoids is absent in mice deficient for the cannabinoid CB2receptor. Eur J Pharmacol 2000; 396: 141–9Buckley, NE McCoy, KL Mezey, E Bonner, T Zimmer, A Felder, C Glass, M Zimmer, A
Zimmer A, Zimmer AM, Hohmann AG, Herkenham M, Bonner TI: Increased mortality, hypoactivity, and hypoalgesia in cannabinoid CB1receptor knockout mice. Proc Natl Acad Sci USA 1999; 96: 5780–5Zimmer, A Zimmer, AM Hohmann, AG Herkenham, M Bonner, TI
Galiegue S, Mary S, Marchand J, Dussossoy D, Carriere D, Carayon P, Bouaboula M, Shire D, Le Fur G, Casellas P: Expression of central and peripheral cannabinoid receptors in human immune tissues and leukocyte subpopulations. Eur J Biochem 1995; 232: 54–61Galiegue, S Mary, S Marchand, J Dussossoy, D Carriere, D Carayon, P Bouaboula, M Shire, D Le Fur, G Casellas, P
Facci L, Dal Torso R, Romanello S, Buriani A, Skaper SD, Leon A: Mast cells express a peripheral cannabinoid receptor with differential sensitivity to anandamide and palmitoylethanolamide. Proc Natl Acad Sci USA 1995; 92: 3376–80Facci, L Dal Torso, R Romanello, S Buriani, A Skaper, SD Leon, A
Schatz AR, Lee M, Condie RB, Pulaski JT, Kaminski NE: Cannabinoid receptors CB1and CB2: A characterization of expression and adenylate cyclase modulation within the immune system. Tox App Pharm 1997; 142: 278–87Schatz, AR Lee, M Condie, RB Pulaski, JT Kaminski, NE
Hanu L, Breuer A, Tchilibon S, Shiloah S, Goldenberg D, Horowitz M, Pertwee RG, Ross RA, Mechoulam R, Fride E: HU-308: A specific agonist for CB2, a peripheral cannabinoid receptor. Proc Natl Acad Sci USA 1999; 96: 14228–33Hanu, L Breuer, A Tchilibon, S Shiloah, S Goldenberg, D Horowitz, M Pertwee, RG Ross, RA Mechoulam, R Fride, E
Malan TP, Ibrahim MM, Deng H, Liu Q, Mata HP, Vanderah T, Porreca F, Makriyannis A: CB2cannabinoid receptor-mediated peripheral antinociception. Pain 2001; 93: 239–45Malan, TP Ibrahim, MM Deng, H Liu, Q Mata, HP Vanderah, T Porreca, F Makriyannis, A
Richardson JD: Cannabinoids modulate pain by multiple mechanisms of action. J Pain 2000; 1: 2–14Richardson, JD
Richardson JD, Kilo S, Hargreaves KM: Cannabinoids reduce hyperalgesia and inflammation via interaction with peripheral CB1 receptors. Pain 1998; 75: 111–9Richardson, JD Kilo, S Hargreaves, KM
Clayton N, Marshall FH, Bountra C, O'Shaughnessy CT: CB1and CB2cannabinoid receptors are implicated in inflammatory pain. Pain 2002; 96: 253–60Clayton, N Marshall, FH Bountra, C O'Shaughnessy, CT
Zimmermann M: Ethical guidelines for investigations of experimental pain in conscious animals. Pain 1983; 16: 109–10Zimmermann, M
Hosohata Y, Quock RM, Hosohata K, Makriyannis A, Consroe P, Roeske WR, Yamamura HI: AM630 antagonism of cannabinoid-stimulated [35S]GTPγS binding in the mouse brain. Eur J Pharmacol 1997; 321: R1–3Hosohata, Y Quock, RM Hosohata, K Makriyannis, A Consroe, P Roeske, WR Yamamura, HI
Ross RA, Brockie HC, Stevenson LA, Murphy VL, Templeton F, Makriyannis A, Pertwee RG: Agonist-inverse agonist characterization at CB1and CB2cannabinoid receptors of L759633, L759656, and AM630. Br J Pharmacol 1999; 126: 665–72Ross, RA Brockie, HC Stevenson, LA Murphy, VL Templeton, F Makriyannis, A Pertwee, RG
Gatley SJ, Gifford AN, Volkow ND, Lan R, Makriyannis A:123I-labelled AM251: A radioiodinated ligand which binds in vivo to mouse brain cannabinoid receptors. Eur J Pharmacol 1996; 307: 331–8Gatley, SJ Gifford, AN Volkow, ND Lan, R Makriyannis, A
Gatley SJ, Lan R, Pyatt B, Gifford AN, Volkow ND, Makriyannis A: Binding of the non-classical cannabinoid CP55, 940, and the diarylpyrazole AM251 to rodent brain cannabinoid receptors, Life Sci 1997; 61: PL191–7Gatley, SJ Lan, R Pyatt, B Gifford, AN Volkow, ND Makriyannis, A
Hargreaves KM, Dubner R, Brown F, Flores C, Joris J: A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain 1988; 32: 77–88Hargreaves, KM Dubner, R Brown, F Flores, C Joris, J
Richardson JD, Aanonsen L, Hargreaves KM: SR141716A, a cannabinoid receptor antagonist produces hyperalgesia in untreated mice. Eur J Pharmacol 2997; 319: R3–4Richardson, JD Aanonsen, L Hargreaves, KM
Richardson JD, Aanonsen L, Hargreaves KM: Hypoactivity of the spinal cannabinoid system results in NMDA-dependent hyperalgesia. J Neurosci 1998; 18: 451–7Richardson, JD Aanonsen, L Hargreaves, KM
Mazzari S, Canella R, Petrelli L, Marcolongo G, Leon A: N-(2-hydroxyethyl) hexadecanamide is orally active in reducing edema formation and inflammatory hyperalgesia by down-modulating mast cell activation. Eur J Pharmacol 1996; 300: 227–36Mazzari, S Canella, R Petrelli, L Marcolongo, G Leon, A
Farquhar-Smith WP, Rice AS: Administration of endocannabinoids prevents a referred hyperalgesia associated with inflammation of the urinary bladder. A nesthesiology 2001; 94: 507–13Farquhar-Smith, WP Rice, AS
Showalter VM, Compton DR, Martin BR, Abood ME: Evaluation of binding in a transfected cell line expressing a peripheral cannabinoid receptor (CB2): Identification of cannabinoid receptor subtype selective ligands. J Pharmacol Exp Ther 1996; 278: 989–99Showalter, VM Compton, DR Martin, BR Abood, ME
Lambert DM, DiPaolo FG, Sonveaux P, Kanyonyo M, Govaerts SJ, Hermans E, Bueb J, Delzenne NM, Tschirhart EJ: Analogues and homologues of N-palmitoylethanolamide, a putative endogenous CB(2) cannabinoid, as potential ligands for the cannabinoid receptors. Biochem Biophys Acta 1999; 1440: 266–74Lambert, DM DiPaolo, FG Sonveaux, P Kanyonyo, M Govaerts, SJ Hermans, E Bueb, J Delzenne, NM Tschirhart, EJ
Lambert DM, DiMarzo V: The palmitoylethanolamide and oleamide enigmas: Are these two fatty acid amides cannabimimetic? Curr Med Chem 1999; 6: 757–73Lambert, DM DiMarzo, V
Calignano A, La Rana G, Giuffrida A, Piomelli D: Antinociceptive activity of the endogenous fatty acid amide, palmitylethanolamide. Eur J Pharmacol 2001; 419: 191–8Calignano, A La Rana, G Giuffrida, A Piomelli, D
Hong Y, Cao CQ, Payza K, St-Onge S, Salois D, Coupal M, Fleurent M, Godbout C, Dray A, Perkins M: CB2receptor-mediated suppression of hypersensitivity in the rats with persistent inflammation. Society for Neuroscience Annual Meeting Abstracts 2001; 716. 4Hong, Y Cao, CQ Payza, K St-Onge, S Salois, D Coupal, M Fleurent, M Godbout, C Dray, A Perkins, M
Price TJ, Dussor GD, Hargreaves KM, Flores CM: Expression and distribution of cannabinoid receptor subtypes in trigeminal ganglion: Colocalization with nociceptive markers. Soc Neurosci Abst 2001; A716.13Price, TJ Dussor, GD Hargreaves, KM Flores, CM
Hohmann AG, Herkenham M: Localization of central cannabinoid CB1receptor messenger RNA in neuronal subpopulations of rat dorsal root ganglia: A double-label in situ hybridization study. Neuroscience 1999; 90: 923–31Hohmann, AG Herkenham, M
Woolf CJ, Safieh-Garabedian B, Ma QP, Crilly P, Winter J: Nerve growth factor contributes to the generation of inflammatory sensory hypersensitivity. Neuroscience 1994; 62: 327–31Woolf, CJ Safieh-Garabedian, B Ma, QP Crilly, P Winter, J
McMahon SB, Bennett DL, Priestly JV, Shelton DL: The biological effects of endogenous nerve factor on adult sensory neurons revealed by a trkA-IgG fusion molecule. Nat Med 1995; 1: 774–80McMahon, SB Bennett, DL Priestly, JV Shelton, DL
Safieh-Garabedian B, Poole S, Allchorne A, Winter J, Woolf CJ: Contribution of interleukin-1 beta to the inflammation-induced increase in nerve growth factor levels and inflammatory hyperalgesia. Br J Pharmacol 1994; 115; 1265–75Safieh-Garabedian, B Poole, S Allchorne, A Winter, J Woolf, CJ
Woolf CJ, Allchorne A, Safieh-Garabedian B, Poole S: Cytokines, nerve growth factor and inflammatory hyperalgesia: The contribution of tumor necrosis factor alpha. Br J Pharmacol 1997; 121: 417–24Woolf, CJ Allchorne, A Safieh-Garabedian, B Poole, S
Shu X, Mendell LM: Nerve growth factor acutely sensitizes the response of adult rat sensory neurons to capsaicin. Neurosci Lett 1999; 274: 159–62Shu, X Mendell, LM
Shu X, Mendell LM: Acute sensitization by NGF of the response of small-diameter sensory neurons to capsaicin. J Neurophysiol 2001; 86: 2931–8Shu, X Mendell, LM
Shu X, Mendell LM: Neurotrophins and hyperalgesia. Proc Natl Acad Sci USA 1999; 96: 7693–6Shu, X Mendell, LM
Chuang H-h, Prescott ED, Kong K, Shields S, Jordt S-E, Basbaum AI, Chao MV, Julius D: Bradykinin and nerve growth factor release the capsaicin receptor from PtdIns(4, 5)P2-mediated inhibition. Nature 2001; 411: 957–62Chuang, H-h Prescott, ED Kong, K Shields, S Jordt, S-E Basbaum, AI Chao, MV Julius, D
Fig. 1. Reversal by systemic (intraperitoneal) AM1241 of carrageenan-induced thermal hypersensitivity. Antagonism of the effects of AM1241 by the CB2receptor–selective antagonist AM630 (100 μg/kg, intraperitoneal). Lack of antagonism by the CB1receptor–selective antagonist AM251 (300 μg/kg, intraperitoneal). Data are expressed as mean ± SEM. Groups were compared using ANOVA followed by pair-wise comparisons using the Student t  test with Bonferroni correction. *P  < 0.05 compared with precarrageenan baseline; +P  < 0.05 compared with AM1241 alone; n = 6 per group. BL = precarrageenan baseline; CG = postcarrageenan value.
Fig. 1. Reversal by systemic (intraperitoneal) AM1241 of carrageenan-induced thermal hypersensitivity. Antagonism of the effects of AM1241 by the CB2receptor–selective antagonist AM630 (100 μg/kg, intraperitoneal). Lack of antagonism by the CB1receptor–selective antagonist AM251 (300 μg/kg, intraperitoneal). Data are expressed as mean ± SEM. Groups were compared using ANOVA followed by pair-wise comparisons using the Student t 
	test with Bonferroni correction. *P 
	< 0.05 compared with precarrageenan baseline; +P 
	< 0.05 compared with AM1241 alone; n = 6 per group. BL = precarrageenan baseline; CG = postcarrageenan value.
Fig. 1. Reversal by systemic (intraperitoneal) AM1241 of carrageenan-induced thermal hypersensitivity. Antagonism of the effects of AM1241 by the CB2receptor–selective antagonist AM630 (100 μg/kg, intraperitoneal). Lack of antagonism by the CB1receptor–selective antagonist AM251 (300 μg/kg, intraperitoneal). Data are expressed as mean ± SEM. Groups were compared using ANOVA followed by pair-wise comparisons using the Student t  test with Bonferroni correction. *P  < 0.05 compared with precarrageenan baseline; +P  < 0.05 compared with AM1241 alone; n = 6 per group. BL = precarrageenan baseline; CG = postcarrageenan value.
×
Fig. 2. (A  ) Reversal by peripheral (intrapaw) AM1241 of carrageenan-induced thermal hypersensitivity. (B  ) Antagonism of the effects of AM1241 by the CB2receptor–selective antagonist AM630 (100 μg/kg, intrapaw), but not by the CB1receptor–selective antagonist AM251 (300 μg/kg, intrapaw). Data are expressed as mean ± SEM. Groups were compared using ANOVA followed by pair-wise comparisons using the Student t  test with Bonferroni correction. *P  < 0.05 compared with preinflammation baseline; #P  < 0.05 compared with postcarrageenan value; +P  < 0.05 compared with ipsilateral administration of AM1241 alone; n = 6 per group. BL = precarrageenan baseline; CG = postcarageenan value.
Fig. 2. (A 
	) Reversal by peripheral (intrapaw) AM1241 of carrageenan-induced thermal hypersensitivity. (B 
	) Antagonism of the effects of AM1241 by the CB2receptor–selective antagonist AM630 (100 μg/kg, intrapaw), but not by the CB1receptor–selective antagonist AM251 (300 μg/kg, intrapaw). Data are expressed as mean ± SEM. Groups were compared using ANOVA followed by pair-wise comparisons using the Student t 
	test with Bonferroni correction. *P 
	< 0.05 compared with preinflammation baseline; #P 
	< 0.05 compared with postcarrageenan value; +P 
	< 0.05 compared with ipsilateral administration of AM1241 alone; n = 6 per group. BL = precarrageenan baseline; CG = postcarageenan value.
Fig. 2. (A  ) Reversal by peripheral (intrapaw) AM1241 of carrageenan-induced thermal hypersensitivity. (B  ) Antagonism of the effects of AM1241 by the CB2receptor–selective antagonist AM630 (100 μg/kg, intrapaw), but not by the CB1receptor–selective antagonist AM251 (300 μg/kg, intrapaw). Data are expressed as mean ± SEM. Groups were compared using ANOVA followed by pair-wise comparisons using the Student t  test with Bonferroni correction. *P  < 0.05 compared with preinflammation baseline; #P  < 0.05 compared with postcarrageenan value; +P  < 0.05 compared with ipsilateral administration of AM1241 alone; n = 6 per group. BL = precarrageenan baseline; CG = postcarageenan value.
×
Fig. 3. Reversal by peripheral (intrapaw) AM1241 of carrageenan-induced hind paw edema. Antagonism of the effects of AM1241 by the CB2receptor–selective antagonist AM630 (100 μg/kg), but not by the CB1receptor–selective antagonist AM251 (300 μg/kg). Data are expressed as mean ± SEM. Groups were compared using ANOVA followed by pair-wise comparisons using the Student t  test with Bonferroni correction. *P  < 0.05 compared with precarrageenan baseline; #P  < 0.05 compared with postcarrageenan value; +P  < 0.05 compared with AM1241 alone; n = 6 per group. BL = precarrageenan baseline; CG = postcarageenan value.
Fig. 3. Reversal by peripheral (intrapaw) AM1241 of carrageenan-induced hind paw edema. Antagonism of the effects of AM1241 by the CB2receptor–selective antagonist AM630 (100 μg/kg), but not by the CB1receptor–selective antagonist AM251 (300 μg/kg). Data are expressed as mean ± SEM. Groups were compared using ANOVA followed by pair-wise comparisons using the Student t 
	test with Bonferroni correction. *P 
	< 0.05 compared with precarrageenan baseline; #P 
	< 0.05 compared with postcarrageenan value; +P 
	< 0.05 compared with AM1241 alone; n = 6 per group. BL = precarrageenan baseline; CG = postcarageenan value.
Fig. 3. Reversal by peripheral (intrapaw) AM1241 of carrageenan-induced hind paw edema. Antagonism of the effects of AM1241 by the CB2receptor–selective antagonist AM630 (100 μg/kg), but not by the CB1receptor–selective antagonist AM251 (300 μg/kg). Data are expressed as mean ± SEM. Groups were compared using ANOVA followed by pair-wise comparisons using the Student t  test with Bonferroni correction. *P  < 0.05 compared with precarrageenan baseline; #P  < 0.05 compared with postcarrageenan value; +P  < 0.05 compared with AM1241 alone; n = 6 per group. BL = precarrageenan baseline; CG = postcarageenan value.
×
Fig. 4. (A  ) Reversal by systemic (intraperitoneal) AM1241 of capsaicin-induced thermal hypersensitivity. (B  ) Reversal by peripheral (intraperitoneal) AM1241 of capsaicin-induced flinches of the hind paw. Antagonism of the effects of AM1241 by the CB2receptor–selective antagonist AM630 (100 μg/kg, intraperitoneal), but not by the CB1receptor–selective antagonist AM251 (300 μg/kg, intraperitoneal). Data are expressed as mean ± SEM. Groups were compared using ANOVA followed by pair-wise comparisons using the Student t  test with Bonferroni correction. *P  < 0.05 compared with precapsaicin baseline; +P  < 0.05 compared with AM1241 alone; n = 6 per group. BL = precapsaicin baseline; CS = postcapsaicin value.
Fig. 4. (A 
	) Reversal by systemic (intraperitoneal) AM1241 of capsaicin-induced thermal hypersensitivity. (B 
	) Reversal by peripheral (intraperitoneal) AM1241 of capsaicin-induced flinches of the hind paw. Antagonism of the effects of AM1241 by the CB2receptor–selective antagonist AM630 (100 μg/kg, intraperitoneal), but not by the CB1receptor–selective antagonist AM251 (300 μg/kg, intraperitoneal). Data are expressed as mean ± SEM. Groups were compared using ANOVA followed by pair-wise comparisons using the Student t 
	test with Bonferroni correction. *P 
	< 0.05 compared with precapsaicin baseline; +P 
	< 0.05 compared with AM1241 alone; n = 6 per group. BL = precapsaicin baseline; CS = postcapsaicin value.
Fig. 4. (A  ) Reversal by systemic (intraperitoneal) AM1241 of capsaicin-induced thermal hypersensitivity. (B  ) Reversal by peripheral (intraperitoneal) AM1241 of capsaicin-induced flinches of the hind paw. Antagonism of the effects of AM1241 by the CB2receptor–selective antagonist AM630 (100 μg/kg, intraperitoneal), but not by the CB1receptor–selective antagonist AM251 (300 μg/kg, intraperitoneal). Data are expressed as mean ± SEM. Groups were compared using ANOVA followed by pair-wise comparisons using the Student t  test with Bonferroni correction. *P  < 0.05 compared with precapsaicin baseline; +P  < 0.05 compared with AM1241 alone; n = 6 per group. BL = precapsaicin baseline; CS = postcapsaicin value.
×