N-arachidonyl maleimide potentiates the pharmacological and biochemical effects of the endocannabinoid 2-arachidonylglycerol through inhibition of monoacylglycerol lipase

Burston, J. J., Sim-Selley, L. J., Harloe, J. P., Mahadevan, A., Razdan, R. K., Selley, D. E. and Wiley, J. L. 2008. N-arachidonyl maleimide potentiates the pharmacological and biochemical effects of the endocannabinoid 2-arachidonylglycerol through inhibition of monoacylglycerol lipase. Journal of Pharmacology and Experimental Therapeutics 327 (2) , pp. 546-553. 10.1124/jpet.108.141382

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Abstract

Inhibition of the metabolism of the endocannabinoids, anandamide (AEA) and 2-arachidonyl glycerol (2-AG), by their primary metabolic enzymes, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively, has the potential to increase understanding of the physiological functions of the endocannabinoid system. To date, selective inhibitors of FAAH, but not MAGL, have been developed. The purpose of this study was to determine the selectivity and efficacy of N-arachidonyl maleimide (NAM), a putative MAGL inhibitor, for modulation of the effects of 2-AG. Our results showed that NAM unmasked 2-AG activity in a tetrad of in vivo tests sensitive to the effects of cannabinoids in mice. The efficacy of 2-AG (and AEA) to produce hypothermia was reduced compared with Δ9-tetrahydrocannabinol; however, 2-AG differed from AEA by its lower efficacy for catalepsy. All tetrad effects were partially CB1 receptor-mediated because they were attenuated (but not eliminated) by SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-H-pyrazole-3-carboxamide HCl] and in CB1-/- mice. In vitro, NAM increased endogenous levels of 2-AG in the brain. Furthermore, NAM raised the potency of 2-AG, but not AEA, in agonist-stimulated guanosine 5′-O-(3-[35S]thio)triphosphate binding assay, a measure of G-protein activation. These results suggest that NAM is an MAGL inhibitor with in vivo and in vitro efficacy. NAM and other MAGL inhibitors are valuable tools to elucidate the biological functions of 2-AG and to examine the consequences of dysregulation of this endocannabinoid. In addition, NAM's unmasking of 2-AG effects that are only partially reversed by SR141716A offers support for the existence of non-CB1, non-CB2 cannabinoid receptors. The endocannabinoid system is comprised of two main receptors and various endogenous ligands. The CB1 cannabinoid receptor is found in both the CNS and periphery and is believed to interact with and modulate various neurotransmitter systems (Howlett, 2002; Szabo and Schlicker, 2005). The CB2 cannabinoid receptor is found principally in the immune system (Pertwee, 1997), although recent reports suggest that it may also be present in the brain stem (Van Sickle et al., 2005). To date, the two main cannabinoid ligands that have been isolated from the brain are anandamide (AEA) and 2-arachidonylglycerol (2-AG) (Hillard, 2000). Discovery and isolation of these ligands have led to significant advances in the cannabinoid field, ranging from the possible therapeutic application of endocannabinoids to the physiological role of the endocannabinoid system. Despite these advances, understanding the full role of these ligands has proven difficult because of their extremely short biological half-life, which is mediated by degradation enzymes (Laine et al., 2002). The enzymes primarily responsible for inactivation of AEA and 2-AG are fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively (Basavarajappa, 2007). To study these endocannabinoid inactivation pathways, significant work has been undertaken to develop selective enzyme inhibitors. To date, there has been some success with developing potent and selective inhibitors of FAAH. For example, the use of the FAAH inhibitor URB597 has revealed a potential role for AEA degradation inhibitors in the treatment of chronic pain (Jayamanne et al., 2006). In contrast, the development of inhibitors of MAGL has been slower, in part because of the fact that most previous research focused on AEA, the first endocannabinoid to be discovered (Devane et al., 1992). However, recent research has indicated the importance of 2-AG in various physiological processes, including appetite regulation, energy balance, and stress-induced opioid-independent analgesia (Hohmann et al., 2005; Cota, 2007). Other studies have shown that 2-AG levels may be altered in pathological conditions such as celiac disease (D'Argenio et al., 2007). These converging lines of research have prompted renewed interest in developing inhibitors of 2-AG synthesis and inactivation. This increased focus on 2-AG may aid in the understanding of its physiological properties and in the discovery of potential therapeutic indications for 2-AG modulation. Currently, two main compounds have been shown to inhibit 2-AG degradation: URB602 and methyl arachidonyl fluorophosphonate (MAFP) (Savinainen et al., 2003; Makara et al., 2005). Both of these compounds have significant limitations. In addition to inhibiting MAGL, MAFP inhibits FAAH, directly activates CB1 receptors, and has noncannabinoid targets (Lio et al., 1996). Although URB602 is far more selective for MAGL than MAFP, the main limitations to use of this compound in vivo are low potency and solubility. The IC50 of URB602 for MAGL in mice is 28 μM, and its maximal solubility is approximately 1 mg/ml (Makara et al., 2005). These two factors prevent effective systemic administration of this compound. Recent research with N-arachidonyl maleimide (NAM) is more promising. NAM prevented cerebellar membrane-mediated degradation of 2-AG at a relatively low concentration (IC50, 140 nM) (Saario et al., 2005). Despite these initial results, there have been no reports of the effect of NAM on 2-AG action within in vivo systems. However, very recently, Blankman et al. (2007) showed that NAM inhibited up to 80% of 2-AG degradation, thus confirming the results of Saario et al. (2005). Based on this research and the fact that there is little information on the in vivo effects of NAM, the aims of this study were to examine NAM modulation of the tetrad effects of 2-AG (a four-factor test that includes suppression of spontaneous activity, antinociception, hypothermia, and catalepsy) (Martin et al., 1991), to determine the effects of NAM on CB1 receptor binding and activation, to assess the selectivity of NAM for 2-AG versus AEA, and to examine the effect of NAM on endogenous 2-AG levels.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Medicine
Publisher:	American Society for Pharmacology and Experimental Therapeutics (ASPET)
ISSN:	0022-3565
Last Modified:	11 Jul 2018 13:00
URI:	https://orca.cardiff.ac.uk/id/eprint/112078

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