Antinociceptive activity of the endogenous fatty acid amide, palmitylethanolamide

Abstract

The endogenous fatty acid ethanolamide, palmitylethanolamide, alleviated, in a dose-dependent manner, pain behaviors elicited in mice by injections of formalin (5%, intraplantar), acetic acid (0.6%, 0.5 ml per animal, intraperitoneal, i.p.), kaolin (2.5 mg per animal, i.p.), and magnesium sulfate (120 mg per kg, i.p.). The antinociceptive effects of palmitylethanolamide were prevented by the cannabinoid CB₂ receptor antagonist SR144528 [N-([1s]-endo-1.3.3-trimethylbicyclo[2.3.1]heptan-2-yl)-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide], not by the cannabinoid CB₁ receptor antagonist SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide·HCl]. By contrast, palmitylethanolamide had no effect on capsaicin-evoked pain behavior or thermal nociception. The endogenous cannabinoid, anandamide (arachidonylethanolamide), alleviated nociception in all tests (formalin, acetic acid, kaolin, magnesium sulfate, capsaicin and hot plate). These effects were prevented by the cannabinoid CB₁ receptor antagonist SR141716A, not the cannabinoid CB₂ receptor antagonist SR141716A. Additional fatty acid ethanolamides (oleylethanolamide, myristylethanolamide, palmitoleylethanolamide, palmitelaidylethanolamide) had little or no effect on formalin-evoked pain behavior, and were not investigated in other pain models. These results support the hypothesis that endogenous palmitylethanolamide participates in the intrinsic control of pain initiation. They also suggest that the putative receptor site activated by palmitylethanolamide may provide a novel target for peripherally acting analgesic drugs.

Introduction

Cannabinoid drugs exert potent analgesic effects in animals by interacting with cannabinoid CB₁ receptor-type receptors located in various pain-processing areas of the central nervous system (see Walker et al., 1999 for a review). In addition to these centrally mediated effects, cannabinoids may also act at peripheral sites to reduce pain and hyperalgesia evoked by irritant chemicals such as formalin, turpentine and carrageenan Calignano et al., 1998, Jaggar et al., 1998, Ko and Woods, 1999, Richardson et al., 1998. Pharmacological, genetic and anatomical evidence indicates that cannabinoid CB₁ receptors expressed in nociceptive neurons Hohmann and Herkenham, 1999, Ahluwalia et al., 2000 are involved in the peripheral actions of cannabinoid drugs Calignano et al., 1998, Richardson et al., 1998, Zimmer et al., 1999. In addition, cannabinoids inhibit the release of calcitonin gene-related peptide (CGRP) in isolated skin preparations (Richardson et al., 1998), suggesting that one mechanism by which these drugs may modulate pain is the inhibition of neuropeptide release from peripheral sensory terminals.

An important question raised by these findings is whether endogenous cannabinoid (endocannabinoid) compounds, generated spontaneously or as a result of tissue injury, participate in the control of peripheral pain signaling. The endocannabinoids, which include anandamide (arachidonylethanolamide) and 2-arachidonylglycerol, are a class of lipid compounds that are produced in the brain and other tissues through stimulus-dependent cleavage of membrane lipid precursors (see Piomelli et al., 2000 for a review). In the case of anandamide, this precursor is represented, in all likelihood, by a minor N-acylated species of phosphatidylethanolamine, termed N-arachidonyl phosphatidylethanolamine Di Marzo et al., 1994, Cadas et al., 1996, Cadas et al., 1997. Depolarization or activation of cell surface receptors may trigger the enzymatic hydrolysis of N-arachidonyl phosphatidylethanolamine by an unknown phospholipase D activity and result in the extracellular release of anandamide (Giuffrida et al., 1999). Some peripheral tissues, such as the skin, may contain relatively high levels of anandamide, suggesting that under appropriate circumstances, this compound can attain tissue concentrations that are sufficient to activate local cannabinoid CB₁ receptors (Calignano et al., 1998). In agreement with this possibility, intraplantar application of the selective cannabinoid CB₁ receptor antagonist SR141716A in mice (Rinaldi-Carmona et al., 1994) was found to enhance pain behaviors elicited by formalin injection in the same paw (Calignano et al., 1998). This response appears, however, to be species-specific or environment-dependent, as no effect of SR141716A on formalin-evoked pain could be demonstrated in rats (Beaulieu et al., 2000).

The anandamide precursor, N-arachidonyl phosphatidylethanolamine, belongs to a family of N-acylated phosphatidylethanolamines that differ in the fatty acid moiety linked to the primary amino group of phosphatidylethanolamine. Thus, catalytic hydrolysis of N-acyl phosphatidylethanolamines by phospholipase D activity gives rise to various saturated and monounsaturated fatty acid ethanolamides, including palmitylethanolamide and oleylethanolamide (Cadas et al., 1997). Palmitylethanolamide, which was isolated from tissues more than three decades ago (Bachur et al., 1965), was shown to have marked anti-inflammatory Benvenuti et al., 1968, Facci et al., 1995, Mazzari et al., 1996 and antinociceptive effects when administered as a drug Calignano et al., 1998, Jaggar et al., 1998. Although palmitylethanolamide does not bind to either cannabinoid CB₁ or CB₂ receptors Devane et al., 1992, Griffin et al., 2000, its antinociceptive actions may be prevented by the selective cannabinoid CB₂ receptor antagonist SR144528. These results have led to suggestions that palmitylethanolamide-evoked antinociception may be mediated by an uncharacterized receptor with cannabinoid CB₂ receptor-like pharmacology Calignano et al., 1998, Calignano et al., 2000. The cellular localization of this putative receptor and its possible structural relationship with the cloned cannabinoid CB₂ receptor subtype, which is primarily expressed in immune cells (Munro et al., 1993), remain unknown. As is the case with anandamide, endogenous palmitylethanolamide may serve important pain-modulating functions in peripheral tissues. Indeed, biochemical analyses have revealed that nonstimulated skin contains concentrations of palmitylethanolamide in the high nanomolar range (Calignano et al., 1998). Furthermore, administration of the cannabinoid CB₂ receptor antagonist SR144528, was found to enhance formalin-induced pain behaviors in mice, suggesting that locally generated palmitylethanolamide may participate in modulating peripheral nociception (Calignano et al., 1998, but see Beaulieu et al., 2000 for discordant results in rats). The biological effects of oleylethanolamide, if any, are still undetermined. Biochemical studies in vitro have shown, however, that oleylethanolamide can inhibit anandamide inactivation by interfering with both transport and hydrolysis of this lipid molecule Désarnaud et al., 1995, Di Tomaso et al., 1996, Piomelli et al., 1999.

In the present study, we extended our previous investigations on the antinociceptive properties of palmitylethanolamide in mice. Our results show that palmitylethanolamide alleviates pain behaviors in several animal models, and that these antinociceptive effects are blocked by the cannabinoid CB₂ receptor antagonist SR144528.