The endogenous cannabinoid 2-arachidonoylglycerol (2-AG) plays an important role in a variety of physiologic processes, but its rapid breakdown by monoacylglycerol lipase (MAGL) results in short-lived actions. Initial MAGL inhibitors were limited by poor selectivity and low potency. In this study, we tested JZL184 [4-nitrophenyl 4-[bis(2H-1,3-benzodioxol-5-yl)(hydroxy)methyl]piperidine-1-carboxylate] and MJN110 [2,5-dioxopyrrolidin-1-yl 4-(bis(4-chlorophenyl)methyl)piperazine-1-carboxylate], MAGL inhibitors that possess increased selectivity and potency, in mouse behavioral assays of neuropathic pain [chronic constriction injury (CCI) of the sciatic nerve], interoceptive cannabimimetic effects (drug-discrimination paradigm), and locomotor activity in an open field test. MJN110 (1.25 and 2.5 mg/kg) and JZL184 (16 and 40 mg/kg) significantly elevated 2-AG and decreased arachidonic acid but did not affect anandamide in whole brains. Both MAGL inhibitors significantly reduced CCI-induced mechanical allodynia with the following potencies [ED50 (95% confidence limit [CL]) values in mg/kg: MJN110 (0.43 [0.30-0.63]) > JZL184 (17.8 [11.6-27.4])] and also substituted for the potent cannabinoid receptor agonist CP55,940 [2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]-5-(2-methyloctan-2-yl)phenol] in the drug-discrimination paradigm [ED50 (95% CL) values in mg/kg: MJN110 (0.84 [0.69-1.02]) > JZL184 (24.9 [14.6-42.5])]; however, these compounds elicited differential effects on locomotor behavior. Similar to cannabinoid 1 (CB1) receptor agonists, JZL184 produced hypomotility, whereas MJN110 increased locomotor behavior and did not produce catalepsy or hypothermia. Although both drugs substituted for CP55,940 in the drug discrimination assay, MJN110 was more potent in reversing allodynia in the CCI model than in producing CP55,940-like effects. Overall, these results suggest that MAGL inhibition may alleviate neuropathic pain, while displaying limited cannabimimetic effects compared with direct CB1 receptor agonists.

In recent years, lipids have come to the foreground as signaling mediators in the central nervous system (CNS) 1,2 . While classical neurotransmitters are stored in synaptic vesicles and released on fusion with the plasma membrane of neurons, due to their lipophilic nature, lipids readily diffuse through membranes and are not stored in vesicles. It is, therefore, generally accepted that signaling lipids are produced 'on demand' and are rapidly metabolized to terminate their biological action 3 . In particular, NAEs, including N-palmitoylethanolamine (PEA), N-oleoylethanolamine (OEA) and the endocannabinoid anandamide (N-arachidonoylethanolamine, AEA) have emerged as key lipid signaling molecules. Genetic deletion or pharmacological inhibition of the main NAE hydrolytic enzyme, fatty acid amide hydrolase (FAAH), revealed elevated anandamide, PEA and OEA levels in brain and implicated these molecules in the modulation of various physiological processes such as pain, stress, anxiety, appetite, cardiovascular function and inflammation [4][5][6][7] . The physiological effects resulting from perturbation of the production of anandamide and other NAEs in living systems are, however, poorly studied, partly because of a lack of pharmacological tools to modulate their biosynthetic enzymes 8 . NAPE-PLD is generally considered a principal NAE biosynthetic enzyme 9,10 . Biochemical and structural studies have demonstrated that NAPE-PLD is a membrane-associated, constitutively active zinc hydrolase with a metallo-β-lactamase fold 11 . The enzyme generates a broad range of NAEs by hydrolysis of the phosphodiester bond between the phosphoglyceride and the NAE in N-acylphosphatidylethanolamines (NAPEs) 12 . Knockout (KO) studies have shown that the Ca 2+ -dependent conversion of NAPE to NAEs bearing both saturated and polyunsaturated fatty acyl groups are fivefold reduced in brain lysates from mice that genetically lack Napepld 13 . In accordance, reduced levels of saturated and mono-unsaturated NAEs were observed in the brains of NAPE-PLD KO mice [13][14][15] . Anandamide levels were not reduced in the transgenic model reported by Leung et al., which suggested the presence of compensatory mechanisms 13 . Indeed, multiple alternative biosynthetic pathways for anandamide have been discovered since 10 .

Although opioids are highly efficacious analgesics, their abuse potential and other untoward side effects diminish their therapeutic utility. The addition of non-opioid analgesics offers a promising strategy to reduce required antinociceptive opioid doses that concomitantly reduce opioid-related side effects. Inhibitors of the primary endocannabinoid catabolic enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) show opioid-sparing effects in preclinical models of pain. As simultaneous inhibition of these enzymes elicits enhanced antinociceptive effects compared with single enzyme inhibition, the present study tested whether the dual FAAH-MAGL inhibitor SA-57 [4-[2-(4-chlorophenyl)ethyl]-1-piperidinecarboxylic acid 2-(methylamino)-2-oxoethyl ester] produces morphine-sparing antinociceptive effects, without major side effects associated with either drug class. SA-57 dose-dependently reversed mechanical allodynia in the constriction injury (CCI) of the sciatic nerve model of neuropathic pain and carrageenan inflammatory pain model. As previously reported, SA-57 was considerably more potent in elevating anandamide (AEA) than 2-arachidonyl glycerol (2-AG) in brain. Its anti-allodynic effects required cannabinoid (CB)1 and CB2 receptors; however, only CB2 receptors were necessary for the anti-edematous effects in the carrageenan assay. Although high doses of SA-57 alone were required to produce antinociception, low doses of this compound, which elevated AEA and did not affect 2-AG brain levels, augmented the antinociceptive effects of morphine, but lacked cannabimimetic side effects. Because of the high abuse liability of opioids and implication of the endocannabinoid system in the reinforcing effects of opioids, the final experiment tested whether SA-57 would alter heroin seeking behavior. Strikingly, SA-57 reduced heroin-reinforced nose poke behavior and the progressive ratio break point for heroin. In conclusion, the results of the present study suggest that inhibition of endocannabinoid degradative enzymes represents a promising therapeutic approach to decrease effective doses of opioids needed for clinical pain control, and may also possess therapeutic potential to reduce opioid abuse.