CHAPTER 64 Pharmacological Aspects of and 2-Arachidonoyglycerol as Bioactive Lipids M. Alhouayek, G.G. Muccioli Bioanalysis and Pharmacology of Bioactive Lipids Research Group, Louvain Drug Research Institute, Université catholique de Louvain, Brussels, Belgium

SUMMARY POINTS 2-arachidonoylglycerol (2-AG) that do not bind • The endocannabinoids N- receptors. arachidonoylethanolamine (AEA) and • They have long been considered as acting 2-arachidonoylglycerol (2-AG) are involved in via an “,” by protecting many pathophysiological processes. endocannabinoids from degradation, and potentiating their actions; however, they also • Classically, endocannabinoids exert their actions have -specific effects. by activating the CB and CB cannabinoid 1 2 • Among N-acylethanolamines: N- receptors, and their actions are terminated by palmitoylethanolamine (PEA) exerts antiinflammatory, their hydrolysis by specific . analgesic, and neuroprotective effects, and is a • More receptors mediating the effects of AEA and PPAR-α agonist, while N-oleoylethanolamine (OEA) 2-AG have been added over the years. is a GPR119 and a PPAR-α known for its • Endocannabinoids can also be metabolized by anorexigenic properties, and the control of glucagon cyclooxygenase (COX)-2, which leads to the secretion. synthesis of other bioactive lipids. • 2-Oleoylglycerol (2-OG) is a monoacylglycerol related • Endocannabinoids and their COX-2 derived to 2-AG that acts as a GPR119 agonist to increase metabolites can exert different effects in some secretion of glucagon-like peptide 1 (GLP-1). settings, a fact that increases the complexity of endocannabinoid signaling. LIST OF ABBREVIATIONS • Interfering with the of endocannabinoids opens up a lot of therapeutic 2-AG 2-Arachidonoylglycerol opportunities. ABHD6 α/β domain 6 ABHD12 α/β Hydrolase domain 12 AEA N-arachidonoylethanolamine or anandamide

KEY FACTS ABOUT ENDOCANNABINOID- CB1 and CB2 1 and 2 RELATED COMPOUNDS COX-2 Cyclooxygenase-2 • They are bioactive lipids belonging to the families cPLA2 Cytosolic A2 of N-arachidonoylethanolamine (AEA) or CYP450 Cytochrome P450 DAGL Diacylglycerol

Handbook of and Related . http://dx.doi.org/10.1016/B978-0-12-800756-3.00074-0 Copyright © 2017 Elsevier Inc. All rights reserved. 616 Classical metabolism of anandamide and 2-arachidonoylglycerol 617

EET Epoxyeicosatrienoic acid modulators of endocannabinoid signaling, as well as the EET-EA Epoxyeicosatrienoic acid-ethanolamide potential therapeutic opportunities of such modulators. EET-G Epoxyeicosatrienoic acid-glycerol ester FAAH fatty acid amide hydrolase GDE1 Glycerophosphodiester OVERVIEW OF THE EFFECTS 1 OF ANANDAMIDE AND HETE Hydroxyeicosatetraenoic acid 2-ARACHIDONOYLGLYCEROL HETE-EA Hydroxyeicosatetraenoic acid- ethanolamine These endocannabinoids have several beneficial effects HETE-G Hydroxyeicosatetraenoic acid-glycerol that could be used in potential treatments. For instance, ester endocannabinoids have antiinflammatory effects, which LOX Lipoxygenase can be beneficial in diseases such as inflammatory bowel MAGL diseases and arthritis (Alhouayek & Muccioli, 2012; Mecs, NAAA N-acylethanolamine-hydrolyzing acid Tuboly, Toth, Nagy, & Nyari, 2010). They are also valuable amidase in neurodegenerative diseases such as multiple sclerosis, NAE N-acylethanolamine or following cerebral trauma, due to their antiinflam- NAPE N-acylphosphatidylethanolamine matory and neuroprotective properties (Lourbopoulos, NAPE-PLD N-acylphosphatidylethanolamine Grigoriadis, Lagoudaki, Touloumi, & Polyzoidou, 2011; Panikashvili, Shein, Mechoulam, Trembovler, & Kohen, PG-EA -ethanolamine or 2006). Endocannabinoids also exert analgesic effects prostamide in many settings (Guindon & Hohmann, 2009), modu- PG-G Prostaglandin-glycerol ester late the stress response, reduce anxiety and depression PPAR Peroxisome proliferator activated (Ruehle et al., 2012), promote sleep (Murillo-Rodriguez, receptor Poot-Ake, Arias-Carrion, Pacheco-Pantoja, & Fuente- TRPV1 Transient receptor potential vanilloid 1 Ortegon, 2011), and decrease nausea and vomiting (Sharkey, Darmani, & Parker, 2014). Generally, increas- ing endocannabinoid levels is thought to be beneficial, INTRODUCTION therefore endocannabinoid degradation con- stitute interesting targets, and many inhibitors of these The concept of bioactive lipids stems from the recog- enzymes have been developed. However, the endocan- nition that lipids are not only involved in cell membrane nabinoid system also exerts some unwanted effects. As structure and energy storage. Indeed, some lipids bind an example, cannabis use is associated with psychotropic to specific receptors, leading to signal transduction and effects that are reproduced following a chronic increase biological effects. Accordingly, variations in the levels of 2-AG levels in animals (Long, Li, Booker, Burston, & of such lipids lead to pathophysiological consequences. Kinsey, 2009a; Schlosburg, Blankman, Long, Nomura, & These are considered bioactive lipids. Eicosanoids, such Pan, 2010). Moreover, cannabis use is associated with im- as the -derived , are com- paired memory, and this is also observed with adminis- monly known bioactive lipids. tration of cannabinoid receptor agonists to mice. Finally, The endocannabinoids N-arachidonoylethanolamine endocannabinoids have been shown to exert deleterious (anandamide or AEA) and 2-arachidonoylglycerol effects in some settings, such as obesity. Indeed, AEA and (2-AG) are bioactive lipids that are involved in many 2-AG are orexigenic and adipogenic agents (Bisogno, physiological processes, such as the regulation of food Mahadevan, Coccurello, Chang, & Allara, 2013; Muccioli, intake, the control of anxiety, and the modulation of pain Naslain, Backhed, Reigstad, & Lambert, 2010). In this and inflammation (Borrelli & Izzo, 2009; Ruehle, Rey, context, inhibitors of endocannabinoid biosynthesis seem Remmers, & Lutz, 2012; Guindon & Hohmann, 2009; to be interesting therapeutic tools. Alhouayek & Muccioli, 2012). As for many bioactive lip- ids, the activity of endocannabinoids is regulated by their tissue levels and, therefore by the balance between their CLASSICAL METABOLISM biosynthesis and their degradation (Muccioli, 2010). In OF ANANDAMIDE AND this context, pharmacological modulation of endocan- 2-ARACHIDONOYLGLYCEROL nabinoid levels represents an attractive therapeutic av- enue for many diseases. Although both 2-AG and AEA are arachidonic acid This chapter presents an overview of endocannabinoid derivatives synthetized from membrane phospholipids, metabolism, with many of the intricacies that character- their biosynthetic and degradation routes are quite dif- ize it, in order to describe the available pharmacological ferent, with diverse enzymes implicated.

V. Pharmacology and cellular activities of and endocannabinoids 618 64. Pharmacological Aspects of Anandamide and 2-Arachidonoyglycerol as Bioactive Lipids

The most described biosynthetic route for 2-AG and diacylglycerol (DAGLα and DAGLβ) to pro- (Fig. 64.1A) is through the sequential actions of an duce 2-AG. However, DAGLs do not constitute the only activity-dependent -β (releasing diacyl- biosynthesis pathway for 2-AG, as this bioactive lipid is glycerols from phosphatidylinositols-4,5-bisphosphate) synthetized in DAGL-knockout mice (Aaltonen, Riera,

FIGURE 64.1 Biosynthesis of the endocannabinoids 2-AG and AEA. (A) 2-AG is produced through the sequential action of either a phos- pholipase C (PLC) and (sn-1-DAGL), or a phospholipase A (PLA1) and a C (LysoPLC). Diacylglycerol (DAG) levels are also regulated by a phosphatidic acid hydrolase (PA hydrolase) and a diacylglycerol kinase (DAG kinase); (B) N-acylphosphati- dylethanolamine (NAPE), the key intermediate in the synthesis of AEA, is synthesized from phosphatidylcholine (PC) and phosphatidylethanol- amine (PE) by a calcium-dependent N-acyltransferase activity (NAT). Several pathways are responsible for the production of AEA from NAPE: (1) directly via a N-acylphosphatidylethanolamine preferring phospholipase D (NAPE-PLD); (2) or through a phosphate intermediate (P-NAE) generated by a phospholipase C (PLC) and hydrolyzed by the PTPN22; (3) or through a lyso-N-acylphosphatidylethanolamine (lyso-NAPE), produced by ABHD4, and which in turn can be hydrolyzed into AEA by a lyso-phospholipase D (lyso-PLD), or converted into the glycerophosphate intermediate (GP-NAE) before its conversion into AEA by glycerophosphodiester phosphodiesterase 1 (GDE1).

V. Pharmacology and cellular activities of cannabinoids and endocannabinoids Classical metabolism of anandamide and 2-arachidonoylglycerol 619

Lehtonen, Savinainen, & Laitinen, 2014). 2-AG is then N-acyltransferase (to generate N-acylphosphatidyleth- inactivated through its hydrolysis by several lipases into anolamines or NAPEs), and a NAPE-preferring phos- arachidonic acid and glycerol (Fig. 64.2). Monoacylglyc- pholipase D (NAPE-PLD) that leads to the production erol lipase (MAGL) was the first described as of N-acylethanolamines (NAEs), including AEA hydrolyzing 2-AG. This enzyme plays a prevalent role in (Muccioli, 2010). While NAPE-PLD was first described the brain, where it controls around 80% of 2-AG hydro- as responsible for the transition from NAPEs to NAEs, lysis (Blankman, Simon, & Cravatt, 2007). Two addition- other less direct routes have been described over the al α/β-hydrolases—α/β-hydrolase domain 6 (ABHD6) years (Fig. 64.1B). AEA is hydrolyzed by two enzymes and 12 (ABHD12)—were also shown to hydrolyze 2-AG (Fig. 64.3), the fatty acid amide hydrolase (FAAH) (Marrs, Blankman, Horne, Thomazeau, & Lin, 2010; and the more recently described N-acylethanolamine- Navia-Paldanius, Savinainen, & Laitinen, 2012). These hydrolyzing acid amidase (NAAA), which differ in three enzymes exhibit different subcellular localiza- particular by their catalytic properties and tions, which led to the hypothesis that they have ac- specificity (Ueda, Tsuboi, & Uyama, 2010). FAAH is a cess to different pools of 2-AG (Savinainen, Saario, & hydrolase active at neutral and alkaline pH, and Laitinen, 2012). However, contrary to MAGL which only has the highest reactivity against AEA, while NAAA hydrolyzes monoacylglycerols, ABHD6 and ABHD12 is a cysteine amidase active at acidic pH, inactive at also hydrolyze other bioactive lipids, such as lysoglyc- alkaline pH, and is more efficient at hydrolyzing erophospholipids (Thomas, Betters, Lord, Brown, & another NAE, N-palmitoylethanolamine (Ueda et al., Marshall, 2013; Blankman, Long, Trauger, Siuzdak, & 2010; Muccioli, 2010). However, recent studies point to a Cravatt, 2013). potential differential role for these enzymes in the regu- The primary biosynthetic route for AEA (Fig. 64.1B) lation of NAE levels, depending on the tissue, or even is through the sequential actions of a Ca2+-dependent the condition (Alhouayek & Muccioli, 2014b).

FIGURE 64.2 2-AG is at the center of a bioactive lipid network. 2-AG can be either hydrolyzed, by MAGL, ABHD6, or ABHD12, into arachidonic acid (AA) or oxidized, by 15-lipoxygenase (15-LOX), cyclooxygenase-2 (COX-2), or cytochrome P450 (CYP450) enzymes, to gener- ate 2-AG-derived bioactive lipids. Thus, 15-LOX will generate 15-hydroxyeicosatetraenoic acid-glycerol ester (15-HETE-G), CYP450 will gener- ate epoxyeicosatrienoic acid-glycerol esters (EET-Gs), and COX-2 will produce several prostaglandin-glycerol esters (PG-Gs), depending on the prostaglandin synthase expressed. The AA produced upon 2-AG hydrolysis can also be metabolized through the same pathways. The molecular targets identified so far for these bioactive lipids are presented in green.

V. Pharmacology and cellular activities of cannabinoids and endocannabinoids 620 64. Pharmacological Aspects of Anandamide and 2-Arachidonoyglycerol as Bioactive Lipids

FIGURE 64.3 AEA is at the center of a bioactive lipid network. AEA can be either hydrolyzed, by FAAH or NAAA, into arachidonic acid (AA) or oxidized, by lipoxygenases (LOXs), cyclooxygenase-2 (COX-2), or cytochromes P450 (CYP450) enzymes, to generate AEA-derived bioac- tive lipids. Thus, LOXs will generate hydroxyeicosatetraenoic acid-ethanolamines (HETE-EAs), CYP450 will generate epoxyeicosatrienoic acid- ethanolamide (EET-EAs), and COX-2 will produce several prostaglandin-ethanolamines or prostamides (PG-EAs), depending on the prostaglan- din synthase expressed. The AA produced upon AEA hydrolysis can also be metabolized through the same pathways. The molecular targets identified so far for these bioactive lipids are presented in green.

ANANDAMIDE AND not only free fatty acids, but also their amide and ester 2-ARACHIDONOYLGLCEROL derivatives (Rouzer & Marnett, 2011). This COX-2 me- AT THE CENTER OF A NETWORK diated metabolism of the endocannabinoids will lead to OF BIOACTIVE LIPIDS ester (prostaglandin-glycerol esters or PG-Gs for 2-AG) and amide (prostaglandin-ethanolamides, also known The role of MAGL in hydrolyzing 2-AG in the brain as prostamides or PG-EAs for AEA) derivatives of the was reinforced with the studies by Nomura, Morrison, prostaglandins. These PG-Gs and PG-EAs are bioactive Blankman, Long, and Kinsey (2011), showing that lipids in their own right that exert biological actions dif- MAGL inhibition in the brain leads to a drastic increase ferent from those of endocannabinoids and prostaglan- in 2-AG levels, but also to a reduction in arachidonic dins (Alhouayek & Muccioli, 2014a). acid levels. This study also demonstrated that inhibit- PGF2α-EA is probably the most studied of these metab- ing MAGL affects bioactive lipids levels at a larger scale olites. It has been used, along with its more stable analogs than endocannabinoid hydrolysis, by reducing the levels such as , for the treatment of high intraocu- of arachidonic acid, a major bioactive lipid implicated lar pressure (Woodward, Wang, & Poloso, 2013). More in inflammation (Nomura et al., 2011). But arachidonic recently, PGF2α-EA was also shown to negatively regu- acid is not the only connection between the endocan- late adipogenesis (Silvestri, Martella, Poloso, Piscitelli, & nabinoid and eicosanoid systems. Seminal studies by Capasso, 2013), and induce hyperalgesic effects in mice the Marnett lab put forth the ability of COX-2 to metabo- (Gatta, Piscitelli, Giordano, Boccella, & Lichtman, 2012). lize the endocannabinoids, similarly to arachidonic acid. AEA, on the other hand, has analgesic effects, and stim- This is due to the arachidonoyl moiety shared by 2-AG ulates adipogenesis (Muccioli et al., 2010; Guindon & and AEA, and to the ability of COX-2 to accommodate Hohmann, 2009). This example is a perfect illustration of

V. Pharmacology and cellular activities of cannabinoids and endocannabinoids Tools and consequences of endocannabinoid levels modulation 621 the differential effects exerted by endocannabinoids and AEA, are also agonists of the cannabinoid receptors or their COX-2-derived metabolites. the TRPV1 receptor (Fig. 64.3) (Rouzer & Marnett, 2011; Adding to the complexity of this system is the fact Alhouayek & Muccioli, 2014a). Similarly, CYP450- that all PG-Gs and PG-EAs do not exert the same effects derived metabolites of AEA and 2-AG exhibit high affin-

(Alhouayek & Muccioli, 2014a). PGE2-EA and PGD2-G ity at cannabinoid receptors, while a 15-LOX derived me- exhibit antiinflammatory effects (Alhouayek, Masquelier, tabolite of 2-AG is a PPARα agonist (Figs. 64.2 and 64.3) Cani, Lambert, & Muccioli, 2013; Brown, Davidson, & (Rouzer & Marnett, 2011; Alhouayek & Muccioli, 2014a).

Rotondo, 2013), while PGE2-G and PGF2α-G exert pro- Moreover, the receptors mediating the effects of the inflammatory and hyperalgesic effects (Hu, Bradshaw, endocannabinoid-derived prostaglandins remain elusive

Chen, Tan, & Walker, 2008; Valdeolivas, Pazos, Bisogno, for the most part. PGF2α-EA was shown to act through a Piscitelli, & Iannotti, 2013; Alhouayek et al., 2013; Ligresti, heterodimer of the PGF2α receptor, and one of its splice Martos, Wang, Guida, & Allara, 2014). So, while 2-AG ex- variants (FP-FPalt4) (Woodward et al., 2013). PGE2-EA erts antiinflammatory effects, two of its COX-2-derived can bind to all four of the PGE2 receptors, but it is not metabolites are proinflammatory, and one is antiinflam- clear yet whether they mediate its effects (Ross, Craib, matory. Therefore, it becomes important to determine Stevenson, Pertwee, & Henderson, 2002). Receptors for under which conditions these PG-Gs are formed, and in the other PG-Gs and PG-EAs have not been described yet, 12,14 which tissues, in order to determine if 2-AG levels should although 15-deoxy-∆ -PGJ2-G, a metabolite of PGD2- be increased or decreased, and which enzyme(s) to target. G, was shown to be a PPARγ agonist (Raman, Kaplan, These few examples highlight the intricacy of endocan- Thompson, Vanden Heuvel, & Kaminski, 2011). nabinoid metabolism, and the importance of using the These numerous targets of the endocannabinoids right tools, at the right moment, in the right conditions. and their metabolites also add to the complexity of the And this is only the tip of the iceberg. Other enzymes of the . As interfering with endocan- eicosanoid system, such as cytochromes P450 (CYP450) nabinoid levels also affects a network of related bioac- and lipoxygenases (LOX), are also able to metabolize the tive lipids, it can be expected that, in some situations, the endocannabinoids, leading to glycerol esters and etha- effects of administering cannabinoid receptor agonists nolamide derivatives of epoxyeicosatrienoic acid (EETs) will not have the same effects as increasing the levels of and hydroxyeicosatetraenoic acid (HETEs), respectively endocannabinoids. (Rouzer & Marnett, 2011; Alhouayek & Muccioli, 2014a). As the number of endocannabinoid-hydrolyzing en- zymes increases, it becomes very important, from a drug TOOLS AND CONSEQUENCES discovery standpoint, to understand where and under OF ENDOCANNABINOID which circumstances each enzyme is responsible for con- LEVELS MODULATION trolling 2-AG or AEA levels. This entails the availability of potent and selective inhibitors for these enzymes, in Interfering with endocannabinoid metabolism of- order to dissect their contributions. fers an array of potential therapeutic applications in the fields of pain, inflammation, anxiety, and obesity, to cite a few. Inhibitors are available to interfere with en- RECEPTORS AND TARGETS docannabinoid biosynthesis or degradation. However, OF ANANDAMIDE, similarly to FAAH which hydrolyzes AEA, the enzymes 2-ARACHIDONOYLGLYCEROL, implicated in the metabolism of 2-AG—that is, DAGL, AND THEIR DERIVATIVES MAGL, ABHD6, and ABHD12—are all serine hydrolas- es. This made the identification of selective inhibitors of Classically, endocannabinoids exert their actions by ac- these enzymes more challenging. Considerable effort is tivating two G protein-coupled receptors, the cannabinoid being done to obtain more selective inhibitors. The most receptors 1 and 2 (CB1 and CB2). However, over the years, studied of these inhibitors, and the latest developments, several other targets have been identified. These include are summarized in Tables 64.1 and 64.2 and Fig. 64.4. the peroxisome proliferator activated receptors (PPARs) In this section, we will try to highlight the benefits and for both AEA (PPARα and PPARγ) and 2-AG (PPARγ), and drawbacks of inhibiting the different enzymes respon- the transient potential vanilloid receptor TRPV1 for AEA sible for the metabolism of endocannabinoids. (Pertwee, Howlett, Abood, Alexander, & Di Marzo, 2010). The complexity of endocannabinoid targets and bio- Inhibitors of the Metabolism of 2-AG logical effects is increased, when considering the oxida- tive derivatives of these compounds through enzymes Inhibitors for several enzymes involved in the metab- such as COX-2, LOX, and CYP450s. Indeed, some of olism of 2-AG have been synthetized and characterized, these derivatives, such as LOX-derived metabolites of such as inhibitors for DAGLs, MAGL, and ABHD6.

V. Pharmacology and cellular activities of cannabinoids and endocannabinoids 622 64. Pharmacological Aspects of Anandamide and 2-Arachidonoyglycerol as Bioactive Lipids

TABLE 64.1 selected Modulators of 2-AG Metabolism

ABHD6 Other serine DAGLα DAGLβ

Inhibitor Structure MAGL (IC50) (IC50) hydrolases (IC50) (IC50)

MAGL JZL184 8 nM mouse 3.3 µM FAAH: 4 µM — — 262 nM rat ABHD12: no 4 nM human significant inhibition

JJKK-048 0.3 nM mouse 229 nM FAAH: 2 µM — — 0.2 nM rat ABHD12: no 0.4 nM human significant inhibition

MJN110 9.5 nM mouse 260 nM FAAH: no — — 9 nM human significant inhibition

JZL195 4 nM 50 nM FAAH: 2 nM — —

OMDM169 1.5 µM mouse — FAAH: 3 µM 2.8 µM — 0.9 µM human

URB602 30–200 µM — FAAH: 17 µM — —

ABHD6 WWL70 No inhibition 70–85 nM FAAH: no — — up to inhibition 1–10 µM at 10 µM; ABHD12: no inhibition at 1 µM

WWL123 No significant 0.43 µM FAAH: no — — inhibition significant inhibition; ABHD12: no significant inhibition

V. Pharmacology and cellular activities of cannabinoids and endocannabinoids Tools and consequences of endocannabinoid levels modulation 623

TABLE 64.1 selected Modulators of 2-AG Metabolism (cont.)

ABHD6 Other serine DAGLα DAGLβ

Inhibitor Structure MAGL (IC50) (IC50) hydrolases (IC50) (IC50) KT185 No significant 1.3 nM FAAH: no — No inhibition significant significant inhibition; inhibition ABHD12: no at 1 µM significant inhibition

DAGLβ KT109 No significant 16 nM FAAH: no 2300 nM 82 nM inhibition significant inhibition; ABHD12: no significant inhibition

KT172 5 µM 5 nM FAAH: no 140 nM 71 nM significant inhibition; ABHD12: no significant inhibition

DAGLα RHC80267 No significant No FAAH: 70 µM; 30–50 µMa inhibition significant ABHD12: no inhibition significant inhibition

THL No significant No FAAH: no 60 nM 100 nM inhibition significant significant inhibition inhibition; ABHD12: 80 nM

OMDM- No significant — FAAH: no 16 nM — 188 inhibition significant inhibition

O-7460 Partial Partial FAAH: no 690 nM — inhibition at inhibition significant 10 µM at 10 µM inhibition

a Not an IC50 value, concentrations typically used to inhibit DAGLs activities. The structures and pharmacological properties of several tools useful to modulate 2-AG hydrolysis and biosynthesis are summarized here.

V. Pharmacology and cellular activities of cannabinoids and endocannabinoids 624 64. Pharmacological Aspects of Anandamide and 2-Arachidonoyglycerol as Bioactive Lipids

TABLE 64.2 selected Modulators of AEA Metabolism

Inhibitor Structure FAAH (IC50) NAAA (IC50) MAGL (IC50)

FAAH URB597 IC50 = 5 nM No significant No significant −1 −1a Kinact/Ki = 1590 M .s inhibition inhibition

−1 −1 PF 3845 Kinact/Ki = 12600 M .s — No significant inhibition

b PF 04457845 IC50 = 7–50 nM — No significant −1 −1 Kinact/Ki = 40300 M .s inhibition

NAAA (S)-OOPP No inhibition IC50 = 420 nM No significant inhibition

ARN077 No inhibition IC50 = 50– — (URB913) 130 nMb

14q No inhibition IC50 = 7 nM —

AM9023 No inhibition IC50 = 600 nM No significant inhibition

a Kinact/Ki values are the more accurate way to characterize irreversible inhibitors potency. b Depending on preincubation time. The structures and pharmacological properties of several tools useful to modulate AEA hydrolysis are summarized here.

DAGL inhibition has been proposed as a promising anti- expected that the levels of prostaglandins and PG-Gs will obesity strategy, as reducing 2-AG levels is expected to re- also be reduced. The reduction of arachidonic acid levels duce food intake and adipogenesis (Bisogno et al., 2013). is interesting during inflammation, or in the context of However, as 2-AG is at the center of a network of bioac- activation, where a decrease in prostaglan- tive lipids, the levels of other lipids can also be affected. din levels is desired. It remains to be seen whether this DAGLβ inhibition in peritoneal , for in- decrease in arachidonic acid and prostaglandins will stance, leads to a decrease in 2-AG and arachidonic acid have the same gastric and cardiovascular side effects as levels (Hsu, Tsuboi, Adibekian, Pugh, & Masuda, 2012). nonsteroidal antiinflammatory drugs NS( AIDs). How- As these two COX-2 substrates are decreased, it is also ever, as discussed further, several elements point to a

V. Pharmacology and cellular activities of cannabinoids and endocannabinoids Tools and consequences of endocannabinoid levels modulation 625

as functional antagonism of cannabinoid receptors (Long et al., 2009a; Schlosburg et al., 2010). Two strat- egies can be proposed to circumvent these side effects: the development of peripherally-restricted MAGL in- hibitors, and the inhibition of another 2-AG hydrolase, such as ABHD6, to increase peripheral 2-AG levels. Indeed, ABHD6 was shown to control less than 5% of 2-AG hydrolysis in mouse brain (Blankman et al., 2007) and ABHD6 inhibitors, while increasing 2-AG levels in FIGURE 64.4 Representative substrate-selective COX-2 inhibi- various tissues did not affect 2-AG levels in the brain tors. R-flurbiprofen and LM4131 preferentially inhibit the oxidative metabolism of endocannabinoids by COX-2, compared to the metabo- (Alhouayek et al., 2013). Also, as ABHD6 controls only lism of arachidonic acid. a fraction of the 2-AG pool, its inhibition does not affect arachidonic acid levels (Alhouayek et al., 2013). There- fore, ABHD6 was proposed as a means for fine-tuning differential regulation of arachidonic acid levels by 2-AG 2-AG levels, and thus avoiding the side effects due to metabolism, or the classical (cPLA2) the drastic increase of 2-AG levels. However, both these pathway, depending on the tissues. strategies lead to a loss of some of the beneficial central Indeed, studies with MAGL inhibitors led to a drastic effects of 2-AG, such as the reduction of nausea, anxiety, increase in 2-AG levels in all the tissues assessed (Long, and depression. Perhaps one way to deal with that is to Nomura, & Cravatt, 2009b; Nomura et al., 2011). However, increase the levels of the other endocannabinoid, AEA. this was accompanied by a decrease in arachidonic acid Indeed, FAAH inhibition is associated with antiemetic levels in some tissues (eg, brain, liver, and lung) but not and antidepressive effects, without all the side effects in others (eg, the gut) (Nomura et al., 2011). Accordingly, observed following MAGL inhibition (Long, Nomura, MAGL inhibition did not induce gastric ulcers (Nomura Vann, Walentiny, & Booker, 2009c). et al., 2011), and actually protects against NSAID-induced ABHD6 inhibition increased 2-AG levels in the liver and gastric ulcers, through a CB1-dependent mechanism lung of mice with lipopolysaccharide (LPS)-induced in- (Kinsey, Nomura, O’Neal, Long, & Mahadevan, 2011b). flammation, and reduced inflammatory parameters (Al- MAGL inhibition was therefore proposed as a potent an- houayek et al., 2013). This effect was partly dependent on tiinflammatory and neuroprotective strategy, devoid of cannabinoid receptors activation, and partly on COX-2 the classical side effects of NSAIDs. Accordingly, MAGL metabolism of 2-AG. Indeed, in macrophages activated inhibition was shown to exert beneficial effects in many with LPS, ABHD6 inhibition led to a decrease of several models of inflammatory diseases, such as IBD and Par- parameters of macrophage activation, and this effect was kinson disease in mice (Nomura et al., 2011; Alhouayek, due to the production of PGD2-G. Moreover, PGD2-G was Lambert, Delzenne, Cani, & Muccioli, 2011). shown to exert antiinflammatory effects in LPS-induced However, things are not so simple, and while MAGL inflammation in mice (Alhouayek et al., 2013). So, in this inhibition increased the levels of 2-AG in a murine case, we have a COX-2-derived metabolite of 2-AG with model of Parkinson disease, and decreased proinflam- antiinflammatory properties, in opposition with PGE2-G matory prostaglandin levels, leading to an overall pro- that was formed in the brain, following MAGL inhibi- tective effect, this was not the case in a murine model tion. It remains to be seen whether this difference in the of Huntington’s disease (Valdeolivas et al., 2013). In this production of PG-Gs following the inhibition of two dif- latter case, MAGL inhibition had a neurotoxic effect. ferent 2-AG hydrolases is due to a different subcellular This was blocked by COX-2 inhibition and replicated localization of the prostaglandin synthases, leading to a by PGE2-G administration, suggesting that the neuro- differential coupling with one enzyme or the other, or to toxic effect is the result of a COX-2 and prostaglandin a different regulation of prostaglandin synthases, in dif- E synthase metabolism of 2-AG. Accordingly, DAGL ferent tissues and different disease settings. inhibition reduced 2-AG levels, and led to a neuropro- Recently, ABHD6 was shown to be more than a tective effect, probably due to the decrease in PGE2-G monoacylglycerol hydrolase, as it is able to hydro- (Valdeolivas et al., 2013). It is therefore crucial to further lyze lysoglycerophospholipids (Thomas et al., 2013). understand the pathways mediating the effects of 2-AG In this context, knockdown of ABHD6 in the liver and in different pathologies. adipose tissue exerted beneficial effects on high fat diet- MAGL inhibition, although very efficient at increas- induced obesity and hepatic steatosis, probably due to ing 2-AG levels and reducing nausea, depression, anxi- the increase in the levels of other substrates than 2-AG ety, pain, and inflammation in some settings, comes with (Thomas et al., 2013). two major drawbacks: psychotropic side effects due to Identification of selectiveinhibitors of ABHD12 proved the drastic increase in 2-AG levels in the brain, as well more difficult. The first identified inhibitor of ABHD12

V. Pharmacology and cellular activities of cannabinoids and endocannabinoids 626 64. Pharmacological Aspects of Anandamide and 2-Arachidonoyglycerol as Bioactive Lipids was tetrahydrolipstatin, which also inhibited DAGL Regarding NAAA inhibition, much fewer inhibi- (Hoover, Blankman, Niessen, & Cravatt, 2008). This tors are available, probably due to the more recent made it difficult to identify the contribution of ABHD12 molecular identification ofN AAA, and the fact that to the metabolism of 2-AG. Perhaps the more recently less is known when it comes to the molecular mecha- described triterpenoid derivatives, having no effect on nisms of cysteine hydrolases and their inhibitors, com- DAGL, will allow for a more thorough understanding pared to serine hydrolases. Among the first described of this role of ABHD12 (Parkkari, Haavikko, Laitinen, NAAA inhibitors, β-lactone derivatives are relatively Navia-Paldanius, & Rytilahti, 2014). However, studies potent, but exhibit limited plasma stability (Ponzano, with ABHD12 knockout mice point to a role for this en- Bertozzi, Mengatto, Dionisi, & Armirotti, 2013). These zyme in controlling the levels of lysophosphatidylser- compounds were mostly tested in local administra- ines, rather than 2-AG (Blankman et al., 2013). tion schemes, where they increased N-palmitoyleth- anolamine levels, and exerted antiinflammatory and Inhibitors of the Metabolism of AEA analgesic effects (Solorzano, Zhu, Battista, Astarita, & Lodola, 2009; Sasso, Moreno-Sanz, Martucci, Realini, Currently, there are no known inhibitors of AEA bio- & Dionisi, 2013). In these cases, the NAAA inhibitor synthesis. Redundancy in the biosynthetic pathways of did not affect AEA levels, or this was not reported (Sol- NAEs might be a reason for this lack of inhibitors, as orzano et al., 2009; Sasso et al., 2013). Therefore, the several enzymes can make the transition from NAPEs to impact of NAAA inhibition on AEA levels remains to NAEs (Fig. 64.1B). In this context, it is interesting to note be elucidated. As N-palmitoylethanolamine is a well- that some NAPE-PLD knockout mice exhibit altered NAE known antiinflammatory bioactive lipid Alhouayek( & levels, while another NAPE-PLD knockout strain does Muccioli, 2014b), NAAA inhibitors are potential thera- not exhibit alterations in AEA levels (Leung, Saghatelian, pies in inflammatory diseases. Simon, & Cravatt, 2006; Tsuboi, Okamoto, Ikematsu, In- oue, & Shimizu, 2011). The same goes for GDE1 knockout Dual Inhibitors of the Metabolism of 2-AG mice in the brain (Simon & Cravatt, 2010). and AEA In the context of interfering with AEA hydrolysis, the most developed and characterized inhibitors are FAAH Following the characterization of the MAGL inhibitor inhibitors (Table 64.2). FAAH and NAAA profoundly JZL184, another related carbamate inhibitor was devel- differ in their catalytic mechanisms (Ueda et al., 2010) oped as a dual FAAH/MAGL inhibitor, JZL195. While which allowed for the development of selective inhibi- increasing the levels of both endocannabinoids seems tors of FAAH, devoid of activity at NAAA, and vice promising to tackle numerous pathologies, the use of versa. FAAH inhibitors were shown to increase AEA this inhibitor was accompanied by similar psychotropic levels in vivo, in physiological and pathological set- side effects as those observed following cannabis use tings. These can be used for the treatment of pain and (Long et al., 2009c). inflammatory diseases such as arthritis, as well as more Another means to increase the levels of both AEA and central effects of the endocannabinoids, such as nau- 2-AG is COX-2 inhibition. Indeed, following the discov- sea and vomiting and mood disorders (Ahn, Johnson, ery that endocannabinoids can be oxidized by COX-2, Mileni, Beidler, & Long, 2009; Kinsey, Naidu, Cravatt, it was hypothesized that some of the effects of NSAIDs Dudley, & Lichtman, 2011a; Bluett, Gamble-George, were not only due to the decreased prostaglandin pro- Hermanson, Hartley, & Marnett, 2014). FAAH inhibi- duction, but also to increased endocannabinoid levels. tion, and the subsequent increase in AEA levels in the This hypothesis was supported by several observations , was not associated with the that COX-2 is able to control AEA levels in the brain, and same psychotropic effects observed with MAGL inhibi- COX inhibitors can increase endocannabinoid levels in tion (Long et al., 2009c). As increasing AEA levels can murine models of inflammation (Telleria-Diaz, Schmidt, be accompanied by a subsequent increase in prostamide Kreusch, Neubert, & Schache, 2010). Moreover, some of levels in inflammatory settings, novel FAAH inhibitors the effects of NSAIDs can be blocked by CB1 antagonists that can double as antagonists of the PGF2α-EA receptor (Telleria-Diaz et al., 2010; Staniaszek, Norris, Kendall, (AGN 211335) are being considered for the treatment of Barrett, & Chapman, 2010). Thus, exploiting the endo- inflammatory pain (Ligresti et al., 2014). Indeed, as we cannabinoid component of COX-2 inhibition might be mentioned earlier, AEA has analgesic and antiinflam- a way to enhance the benefits of NSAIDs, and reduce matory properties, while PGF2α-EA is hyperalgesic and their side effects. Indeed, endocannabinoids are analge- proinflammatory. 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