The endocannabinoid system may serve important functions in the central and peripheral regulation of pain. In the present study, we investigated the effects of the endocannabinoid transport inhibitor AM404 [N-(4-hydroxyphenyl)-eicosa5,8,11,14-tetraenamide] on rodent models of acute and persistent nociception (intraplantar formalin injection in the mouse), neuropathic pain (sciatic nerve ligation in the rat), and inflammatory pain (complete Freund’s adjuvant injection in the rat). In the formalin model, administration of AM404 (1–10 mg/kg i.p.) elicited dose-dependent antinociceptive effects, which were prevented by the CB1 cannabinoid receptor antagonist rimonabant (SR141716A; 1 mg/kg i.p.) but not by the CB2 antagonist SR144528 (1 mg/kg i.p.) or the vanilloid antagonist capsazepine (30 mg/kg i.p.). Comparable effects were observed with UCM707 [N-(3-furylmethyl)-eicosa-5,8,11,14-tetraenamide], another anandamide transport inhibitor. In both the chronic constriction injury and complete Freund’s adjuvant model, daily treatment with AM404 (1–10 mg/kg s.c.) for 14 days produced a dose-dependent reduction in nocifensive responses to thermal and mechanical stimuli, which was prevented by a single administration of rimonabant (1 mg/kg i.p.) and was accompanied by decreased expression of cyclooxygenase-2 and inducible nitric-oxide synthase in the sciatic nerve. The results provide new evidence for a role of the endocannabinoid system in pain modulation and point to anandamide transport as a potential target for analgesic drug development. The endocannabinoids, anandamide and 2-arachidonoylglycerol (2-AG), are removed from the extracellular space by a high-affinity transport system present both in neural and non-neural cells (Beltramo et al., 1997; Hillard et al., 1997). The molecular identity of this putative transporter is still unknown, but some of its biochemical and pharmacological properties have been characterized (for review, see Hillard and Jarrahian, 2003). These include stereoselective substrate recognition and saturation at 37°C, independence from ion gradients, and pharmacological inhibition by agents such as AM404, UCM707, and LY2183240 (Beltramo et al., 1997; Piomelli et al., 1999; Lopez-Rodriguez et al., 2001; Moore et al., 2005). After reuptake, anandamide is hydrolyzed by fatty acid amide hydrolase (FAAH) (Cravatt et al., 1996), an intracellular membrane-bound serine hydrolase whose activity is selectively inhibited by the compounds URB597 and OL135 (Kathuria et al., 2003; Lichtman et al., 2004) as well as by a variety of nonselective agents (for review, see Piomelli, 2005). Intracellular 2-AG is hydrolyzed by monoacylglycerol lipase (Stella et al., 1997; Dinh et al., 2002), which is inhibited by the compound URB602 (Hohmann et al., 2005; Makara et al., 2005). Pharmacological blockade of FAAH activity elicits modest but significant antinociceptive effects in rats (Kathuria et al., 2003; Lichtman et al., 2004), whereas mutant mice in which This work was supported by the National Institute on Drug Abuse (Grants DA-12447 and DA-3412), by the University of California Discovery Program (to D.P.), and by Ministero dell’Istruzione, Università e Ricerca (to A.C.). M.B. was a National Institute on Drug Abuse INVEST fellow. 1 These authors contributed equally to this work. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.105.100792. ABBREVIATIONS: 2-AG, 2-arachidonoylglycerol; AM404, N-(4-hydroxyphenyl)-eicosa-5,8,11,14-tetraenamide; UCM707, N-(3-furylmethyl)-eicosa-5,8,11,14-tetraenamide; LY2183240, 5-biphenyl-4-ylmethyl-tetrazole-1-carboxylic acid dimethylamide; FAAH, fatty acid amide hydrolase; URB597, cyclohexylcarbamic acid 3 -carbamoylbiphenyl-3-yl ester; OL-135, 1-oxo-1-[5-(2-pyridyl)oxazol-2-yl]-7-phenylheptane; URB602, (1,1 biphenyl)-3-yl-carbamic acid, cyclohexyl ester; SR141716A, rimonabant; CCI, chronic constriction injury; CFA, complete Freund’s adjuvant; iNOS, inducible nitric-oxide synthase; Cox-2, cyclooxygenase-2; PBS, phosphate-buffered saline; s.c., subcutaneous; SR144528, N-[(1S)-endo-1,3,3trimethylbicycloheptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide. 0022-3565/06/3173-1365–1371$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 317, No. 3 Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics 100792/3113920 JPET 317:1365–1371, 2006 Printed in U.S.A.

Author(s): La Rana, G; Russo, R; D'Agostino, G; Sasso, O; Mattace Raso, G; Iacono, A; Meli, R; Piomelli, D; Calignano, A

Neuropathic pain consequent to peripheral nerve injury has been associated with local inflammation. Following noxious stimulation afferent fibres release substance P (SP) and calcitonin-gene related peptide (CGRP), which are closely related to oedema formation and plasma leakage. The effect of the anandamide transport blocker AM404 has been studied on plasma extravasation after chronic constriction injury (CCI) which consists in a unilateral loose ligation of the rat sciatic nerve (Bennett and Xie, 1988). AM404 (1–3–10mgkg−1) reduced plasma extravasation in the legated paw, measured as μg of Evans Blue per gram of fresh tissue. A strong effect on vascular permeability was also produced by the synthetic cannabinoid agonist WIN 55,212-2 (0.1–0.3–1mgkg−1). Using specific antagonists or enzyme inhibitors, we demonstrate that cannabinoids act at several levels: data on the 3rd day suggest a strong involvement of substance P (SP) and calcitonin gene-related peptide (CGRP) in the control of vascular tone, whereas at the 7th and 14th days the major role seems to be played by prostaglandins (PGs) and nitric oxide (NO). Capsaicin injection in ligated paws of AM404- or WIN 55,212-2-treated rats resulted in an increase of Evans Blue extravasation, suggesting the involvement of the cannabinergic system in the protective effect of C fibres of ligated paws. Taken together, these data demonstrate the efficacy of cannabinoids in controlling pain behaviour through the modulation of several pain mediators and markers of vascular reactivity, such as SP, CGRP, PGs and NO.

The endocannabinoid system may serve important functions in the central and peripheral regulation of pain. In the present study, we investigated the effects of the endocannabinoid transport inhibitor AM404 [N-(4-hydroxyphenyl)-eicosa-5,8,11,14-tetraenamide] on rodent models of acute and persistent nociception (intraplantar formalin injection in the mouse), neuropathic pain (sciatic nerve ligation in the rat), and inflammatory pain (complete Freund's adjuvant injection in the rat). In the formalin model, administration of AM404 (1-10 mg/kg i.p.) elicited dose-dependent antinociceptive effects, which were prevented by the CB 1 cannabinoid receptor antagonist rimonabant (SR141716A; 1 mg/kg i.p.) but not by the CB 2 antagonist SR144528 (1 mg/kg i.p.) or the vanilloid antagonist capsazepine (30 mg/kg i.p.). Comparable effects were observed with UCM707 [N-(3-furylmethyl)-eicosa-5,8,11,14-tetraenamide], another anandamide transport inhibitor. In both the chronic constriction injury and complete Freund's adjuvant model, daily treatment with AM404 (1-10 mg/kg s.c.) for 14 days produced a dose-dependent reduction in nocifensive responses to thermal and mechanical stimuli, which was prevented by a single administration of rimonabant (1 mg/kg i.p.) and was accompanied by decreased expression of cyclooxygenase-2 and inducible nitric-oxide synthase in the sciatic nerve. The results provide new evidence for a role of the endocannabinoid system in pain modulation and point to anandamide transport as a potential target for analgesic drug development.

Palmitoylethanolamide (PEA), the naturally occurring amide of palmitic acid and ethanolamine, reduces pain and inflammation through an as-yet-uncharacterized mechanism. Here, we identify the nuclear receptor peroxisome proliferator-activated receptor-alpha (PPAR-alpha) as the molecular target responsible for the anti-inflammatory properties of PEA. PEA selectively activates PPAR-alpha in vitro with an EC(50) value of 3.1 +/- 0.4 microM and induces the expression of PPAR-alpha mRNA when applied topically to mouse skin. In two animal models, carrageenan-induced paw edema and phorbol ester-induced ear edema, PEA attenuates inflammation in wild-type mice but has no effect in mice deficient in PPAR-alpha. The natural PPAR-alpha agonist oleoylethanolamide (OEA) and the synthetic PPAR-alpha agonists GW7647 and Wy-14643 mimic these effects in a PPAR-alpha-dependent manner. These findings indicate that PPAR-alpha mediates the anti-inflammatory effects of PEA and suggest that this fatty-acid ethanolamide may serve, like its analog OEA, as an endogenous ligand of PPAR-alpha.

Palmitoylethanolamide (PEA), the naturally occurring amide of ethanolamine and palmitic acid, is an endogenous lipid that modulates pain and inflammation. Although the anti-inflammatory effects of PEA were first characterized nearly 50 years ago, the identity of the receptor mediating these actions has long remained elusive. We recently identified the ligand-activated transcription factor, peroxisome proliferator-activated receptor-alpha (PPAR-α), as the receptor mediating the anti-inflammatory actions of this lipid amide. Here we outline the history of PEA, starting with its initial discovery in the 1950s, and discuss the pharmacological properties of this compound, particularly in regards to its ability to activate PPAR-α.

Smoking marijuana or administration of its main active constituent, Δ9-tetrahydrocannabinol (Δ9-THC), may exert potent dilating effects on human airways1,2,3,4. But the physiological significance of this observation and its potential therapeutic value are obscured by the fact that some asthmatic patients respond to these compounds with a paradoxical bronchospasm3,5. The mechanisms underlying these contrasting responses remain unresolved. Here we show that the endogenous cannabinoid anandamide exerts dual effects on bronchial responsiveness in rodents: it strongly inhibits bronchospasm and cough evoked by the chemical irritant, capsaicin, but causes bronchospasm when the constricting tone exerted by the vagus nerve is removed. Both effects are mediated through peripheral CB1 cannabinoid receptors found on axon terminals of airway nerves. Biochemical analyses indicate that anandamide is synthesized in lung tissue on calcium-ion stimulation, suggesting that locally generated anandamide participates in the intrinsic control of airway responsiveness. In support of this conclusion, the CB1 antagonist SR141716A enhances capsaicin-evoked bronchospasm and cough. Our results may account for the contrasting bronchial actions of cannabis-like drugs in humans, and provide a framework for the development of more selective cannabinoid-based agents for the treatment of respiratory pathologies.

This chapter discusses the role for the endogenous cannabinoid system in the peripheral control of pain initiation. The chapter outlines the experimental evidence supporting such a role. To place this evidence into perspective, the pathways of formation and inactivation of the endogenous cannabinoids (endocannabinoids) and the pharmacological properties of the receptors they activate is described. The implication of the endocannabinoid system in the central processing of pain signaling is discussed. The anandamide and 2-AG are released upon demand from neurons and other cells through distinct receptor-dependent mechanisms. This property, along with the short life spans of anandamide and 2-AG, suggests that these endocannabinoid lipids may act near their site of synthesis to modulate the effects of primary neurotransmitters and hormones. The chapter also discusses the antinociceptive functions served by these compounds in peripheral organs and tissues and delineates several unresolved issues.

For centuries cannabis has been used for the management of pain, however, clinical studies have not led to a uniform idea regarding the analgesic effectiveness of cannabis and its constituent Δ9-tetrahydrocannabinol (Δ9-THC). The discovery of anandamide and 2-arachidonylglycerol, two endogenous ligands for cannabinoid receptors, has aroused interest about the possible role for these molecules in modulation of pain perception. The analgesic effects observed are mediated through cannabinoid receptors, localized in the brain, that interact with the noradrenergic and κ-opioid system; in the spinal cord, modulating the nociceptive perception, and on peripheral sensory neurons regulating the afferent stimulations. Anandamide is an effective antinociceptive agent, acting centrally as well as on peripheral neurons, modulating pain perception and initiation.

The endogenous cannabinoid ligand anandamide (arachidonylethanolamide) inhibited the intestinal passage of a charcoal meal when administered s.c. in mice at doses ranging from 0.1 to 50 mg/kg. This effect was prevented by the cannabinoid CB1 receptor antagonist SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-me thyl-1H-pyrazole-3-carboxamide x HCl] (1 mg/kg s.c.), but it was not affected by the anandamide transport inhibitor, N-(4-hydroxyphenyl) arachidonylethanolamide (AM404) (50 mg/kg, s.c.). The results indicate that anandamide modulates intestinal motility in mice by activating cannabinoid CB1 receptors. They also suggest that anandamide transport, which was previously shown to participate in terminating neural and vascular responses to anandamide, does not contribute to anandamide inactivation in intestinal tissue.

The putative endogenous cannabinoid, anandamide (0.2–2 mg/kg i.v.), decreased systemic blood pressure dose-dependently in anesthesized guinea pigs. These effects were prevented by the CB1 cannabinoid receptor antagonist SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide·HCl] at the dose of 0.2 mg/kg i.v. The vasodepressor responses to anandamide were significantly potentiated and prolonged by a novel inhibitor of carrier-mediated anandamide transport, N-(4-hydroxyphenyl) arachidonylethanolamide (AM404) (10 mg/kg, i.v.). These results suggest that anandamide transport participates in terminating the vascular actions of anandamide.