General Introduction

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Chapter 1

General Introduction

1.1 Overview of Endocannabinoid System

Endocannabinoid system (ECs) is a new and basically not completely detected neuromodulator system discovered in the first part of 1990th, and since its birth has been attracted more and more studies and researches too. The term “endocannabinoid” was originally coined after the discovery of membrane receptors for the psychoactive principle in Cannabis, Δ9-tetrahydrocannabinol, and their endogenous ligands.

Figure 1.1 Overview of endocannabinoid system (Alhouayek e Muccioli, 2012).

In particular, the ECs is composed from various parts: few ligands, whose N-arachidonoylethanolamide (AEA) and 2-Arachidonylglycerol (2-AG) are the most

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potent, the 2 main receptors CB1r and CB2r, and all the carriers, enzymes and membrane transporters involved in endocannabinoid synthesis, regulation and degradation.

Apparently, ECs seems to be involved in an ever-increasing number of diseases, in maintenance of normal health condition and homeostasis, modulating and protecting other systems (especially the nervous system). (Devane et al.,1988; Mechoulam et al., 1995; Sugiura et al., 1995; Cravat et al., 1996; Pertwee, 1997; Murillo-Rodriguez et al., 2011; Dinh et al., 2002a; Saario, 2006; Di Marzo et al., 2004).

1.2 How ECs works

Cannabinoids AEA and 2-AG are synthesized <<on demand>> and the release is induced by either depolarization or activation of Gq-coupled receptors that induce an increase of the intracellular levels of Ca2+ (Hashimotodani et al., 2005). The release is retrograde in neurons – retrograde signaling means that the messenger is diffusible (or bound by a carrier molecule), and after release from the postsynaptic element travels "backwards" across the synaptic cleft, where it activates receptors on the presynaptic terminals. Immediately after the receptors activation, the endocannabinoids are rapidly removed from the extracellular space by uptake and degraded. The retrograde signaling can inhibit presynaptic release of neurotransmitters (transient and long-lasting reduction). Presynaptic inhibition of neurotransmitter release is associated with the inhibitory action of endocannabinoids on presynaptic calcium channels via the activation of cannabinoid receptors. It can happen through two different forms of short-term synaptic plasticity, depending on the involvement of GABA or Glutamate transmission, respectively: depolarization-induced suppression of inhibition = DSI (sending messenger to GABA terminal) and depolarization-induced suppression of excitation = DSE (glutamate terminal). DSI means that depolarization of postsynaptic neurons induces a transient suppression of inhibitory synaptic transmission. So, finally, a transient suppression of either the presynaptic inhibitory or the excitatory input is induced. The fact that neurons are able to control the efficacy of their own synaptic input by retrograde synaptic

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signaling is functionally very important. The physiological sense of DSE and DSI could be the coordination of neural networks inside the hippocampus and the cerebellum, which are involved in important physiological processes, such as memory or motor coordination. This was the short-term synaptic plasticity. The long term-synaptic plasticity, also modulating the synaptic transmission, can be long-term potentiation (LTP) and long-term depression (LTD). Both forms of synaptic plasticity involve long-term changes in the efficacy of synaptic transmission in glutamatergic neurons (major influence on consolidation and remodelling of the synapses). Activation of the cannabinoid receptors prevents the induction of LTP in the hippocampal synapses and a facilitation of LTD in the striatum and the nucleus accumbens (Ohno-Shosaku et al., 2001; Alger, 2002; Howlett et al., 2002; Freund et al., 2003; Rodriguez et al., 2005; Kano et al., 2009; Savinainen et al., 2012).

1.3 Endocannabinoids

Through the time, more and more chemical compounds are being classified as endogenous ligands of the cannabinoid receptors. The two most famous, explored and plentiful are N-arachidonoylethanolamide (AEA) and 2-Arachidonoylglycerol (2-AG). AEA was the first introduced and proved by Devane et al. (1992), isolated from porcine brain, whereas 2-AG was isolated from canine intestines (Mechoulam et al., 1995; Sugiura et al., 1995). AEA occurs mostly in brain tissue while 2-AG was, except of nervous system, found also in e.g. heart, liver, spleen, lung, kidney, plasma, colon, small intestine and even in human milk (Sugiura et al., 2002; Sugiura et al., 2006). Chemically, major endocannabinoids AEA and 2-AG are structural derivatives of arachidonic acid, conjugated with ethanolamine or glycerol respectively (De Petrocellis et al., 2004; Rodriguez et al., 2005). 2-AG is a full agonist at both cannabinoid receptors, while anandamide behaves like a partial agonist at both receptors (Gonsiorek et al., 2000; Steffens et al., 2005; Sugiura et al., 2006; Sugiura, 2009). Moreover, AEA also activates vanilloid receptor TRPV1 (transient receptor potential vanilloid-type-1) and is thus being involved in the process of pain sensation in a study of patients with chronic pain (De Petrocellis and

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Di Marzo, 2009; Savinainen et al., 2012; Xia et al., 2011). Afterwards, more compounds have been examined and considered as endogenous ligands of cannabinoid receptors: 2-Arachidonyl-glycerylether (2-AGE), O-arachidonoyl-ethanolamine (Virodhamine), N-arachidonoyl-dopamine (NADA), Palmitoyl ethanolamine (PEA) and Oleamide (OA) (De Petrocelis et al., 2004; Rodriguez et al., 2005; Saario et al., 2005) (see Fig 1.2). These putative endocannabinoids, however, are not so well studied as 2-AG and AEA. 2-AGE was discovered and isolated from porcine brain by Hanus et al., (2001), being found in the whole brain, mainly in thalamus and hippocampus (Fezza et al., 2002). In particular it acts as full agonist at CB1r and CB2r (Hanus et al., 2001; Shoemaker et al., 2005; Steffens et al., 2005). Virodhamine has been known since 2002, first described by Porter et al., (2002), and was identified from human and rat brains/peripheral tissues. It is a partial agonist at the CB1r and a full agonist at the CB2r (Kozlowska et al., 2008). In the peripheral tissues, the concentration of Virhodamine is up to nine times higher than that of anandamide. In brain Virhodamine occurs too (Porter et al., 2002). NADA was discovered in 2002 as an endogenous molecule in brain (Huang and Walker, 2006) and it impacts the endocannabinoid and vanilloid system as well (due to its agonistic effect at CB1r and TRPV1 receptors). PEA and OEA are synthesized together with AEA and they inhibit the FAAH hydrolysis of AEA (Schuel et al., 2002), with PEA which enhances the effect of AEA on cannabinoid and vanilloid receptors (Naccarato et al., 2010), whereas OEA also acts on CB1r (Leggett et al., 2004) but only in high concentrations (Martinez-Gonzalez et al., 2004).

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Figure 1.2 Chemical structures of biologically active endocannabinoids.

1.4 Endocannabinoid receptors and their synthetic ligands

Two cannabinoid receptors (CB1r and CB2r) have been cloned so far. GPR55, a third putative cannabinoid receptor has been shown to possess higher potency to bind lysophosphatidylinositol than AEA or 2-AG. Furthermore, there is no genetic similarity compared with CB1r or CB2r (Pertwee et al., 2010; Savinainen et al., 2012). Both the receptors belong to the family of G protein coupled receptors, with the typical structure that passes the cell membrane seven times. After bound with endocannabinoid (or other activating ligand), the receptor conformation is changed, thereby leading to signal transduction through the inhibitory class of G proteins. Endocannabinoid receptors have been found not only in humans, but also in other mammals (cat, dog, bovine, primates), fishes (puffer fish), amphibians (newt), birds (finch) and reptiles (Onaivi et al., 2002).

After a first pharmacological characterization by radioligand binding at rat brain, CB1r was cloned in 1990 by Matsuda et al. from human and rat brain and was found

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to be widely distributed in the CNS, being clearly more abundant than dopamine or other receptors.

CB1r is located mostly in the presynaptic terminals of nerve cells (central and peripheral neurons and glial cells) – mediating inhibition of ongoing release of a number of different excitatory and inhibitory neurotransmitters. CB1r reaches highest density in the basal ganglia, cerebellum, hippocampus and cortex, but is also present in the peripheral nervous system (PNS) ad several peripheral organs as reproductive system, urinary bladder, heart, lungs and bone marrow. Due to its main distribution in CNS, activation of theme can affect processes such as cognition and memory, alter the control of motor function, and induce analgesia. Furthermore, the large physiological importance of CB1r in mammals is supported by the fact that even 99% of the CB1r amino acid sequences among human, mouse and rat are identical (molecular weight estimated from Western blot analysis is about 56-58 kDa for CB1r) (Matsuda et al., 1990; Munro et al., 1993; Pertwee, 1997; Rodriguez et al., 2005; Pertwee et al., 2010; Gerard et al., 1991; Facci et al., 1995; Galiegue et al., 1995; Pertwee et al., 1997; Martin et al., 2000; Carlisle et al., 2002; Di Marzo et al., 2004; Savinainen, 2005; Bagavandoss and Grimshaw, 2010; Ruiz-Llorente et al., 2003; Hungund et al., 2004).

Development of CB1r selective agonists as target for drugs have been not investigated deeply so far because of the unwanted central effects. Anyway, this class of agonists express their important role in study either in vitro or in vivo to better understand and split the effects of activation of CB1r from CB2r. Among these are reported the arachidonoil-cloro-ethanolammide and arachidonoil-cyclopropylammide (structures not reported). Generation of CB1r selective antagonist and/or inverse agonist have been much more studied in the last decade because the anti-obesity activity and its implication in cardiovascular, gastrointestinal and respiratory functions. Unfortunately, drugs developed from CB1r antagonist/inverse agonist displayed also psychiatric collateral effects as anxiety and depression given by the binding withcentral receptors, therefore it is currently under investigation a scaffold not capable to pass through the Hematoencephalitic Barrier. SR141716A (Rimonabant) and AM-251 represent two exponents of this class of compounds (see Fig. 1.3)

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SR141716A AM-251

Fig. 1.3 Chemical structures of CB1r antagonist/inverse agonist compounds

CB2r has been known since 1993 (Munro et al. cloned it from human pro-myelocytic leukemic cell line HL-60) and it is genetically about 44% identical with CB1r, with the molecular weight estimated from Western blot about 46 kDa (Filppula et al., 2004).

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CB2r is mainly present in cells and organs related to immune system, such as lymphoid tissues (tonsils, spleen), mast cells, macrophages and white blood cells. In addition, it seems to be present in human ovary and microglial cells as well. After activation, CB2r can modify migration of immune cells and cytokine release in brain and outside. Moreover, there are some evidences that show how CB1r is present also in non-neuronal cells, including immune cells, and vice versa CB2r is expressed by central neurons system (Matsuda et al., 1990; Gerard et al., 1991; Munro et al., 1993; Facci et al., 1995; Galiegue et al., 1995; Pertwee , 1997; Martin et al., 2000; Carlisle et al., 2002; Di Marzo et al., 2004; Rodriguez, 2005; Bagavandoss and Grimshaw, 2010; Pertwee et al., 2010).

Currently, the research of molecules that may interact with endocannabinoid system is mainly focused on CB2r compared with CB1r, avoiding the psychiatric collateral effects given by bound with CB1r in the brain. Few selective agonists for CB2r have been synthetized and tested so far with different chemical scaffolds. The most important are: HU-308, JWH-015, JWH-133, JWH-139 and AM-1241 (see Fig. 1.4).

JWH-139 HU-308

Figure 1.5 Chemical structures of some selective CB2r agonist compounds

O

O OH

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As reported for CB1r, CB2r selective agonists express an important role in studying either in vitro or in vivo different effects of activation of the two receptors as well. Furthermore, CB2r agonists may represent a source of <<templates>> for analgesic and anti-inflammatory drugs. Recently, some strong selective agonists have been reported in literature to have analgesic, anti-inflammatory and anti-cancer properties (Manera et al., 2008; Pasquini et al., 2008). Most strong selective ligands that block CB2r activity are AM-630 and SR144528 (see Fig. 1.6). Both the compounds display a more relevant inverse agonism behavior in vivo affecting opposite results in tissues that express CB2r. To date, a completely selective CB2r antagonist has not been obtained.

Figure 1.6 Chemical structures of some selective CB2r antagonist/inverse agonist

compounds

Finally are reported some of the most important non-selective agonists for endocannabinoid receptors: CP-55,940, WIN-55,512-2 and HU-210 (see Fig. 1.7). They are used mainly in current experiments as controls.

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Figure 1.7 Chemical structures of some of the most important non-selective agonists for

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1.5 MAGL, ABHDs, FAAH, COX-2 and EMT

Several biosynthesis and degradative processes govern the subtle balance between the production and the degradation of endocannabinoids (Liu et al., 2008; Wang and Ueda, 2009).

Figure 1.8 Pathways of synthesis and degradation of most important endocannabinoids (De

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1.5.1 Mono Acyl Glycerol lipase (MAGL)

The first evidence of the presence, in the adipose tissue, of a lipase activity specific for monoacylglycerols was established more than forty years ago (Cravatt et al., 2001), and in 1976 the first thorough purification of monoacylglycerol lipase was achieved, starting from the adipose tissue of 100 rats (Tornqvist et al., 1976). Since its first identification in the adipose tissue, MAGL localization has extended to many other tissues and is now known to constitute an ubiquitously expressed enzyme. In the brain, MAGL transcript is present in the cortex, hippocampus, cerebellum, thalamus and striatum (Dinh et al., 2002). MAGL completes the hydrolysis of triacylglycerols in the adipocyte and it behaves as a selective 2-monoacylglycerol hydrolase. This observation turned out to be important when the endocannabinoid system was brought to light. Indeed, besides its original role in the mobilization of fat in the adipocyte, the discovery of 2-arachidonoylglycerol as a key messenger of the endocannabinoid system, marked a turning point in the study of MAGL: several studies have highlighted the physiological relevance of MAGL in 2-AG degradation (Labar et al., 2010). Blankman and colleagues also contributed to the elucidation of the enzymatic pathways governing 2-AG catabolism (Blankman et al., 2007). After that, mouse brain was mapped for the enzymes involved in 2-AG degradation. This allowed to assign about 85% of the 2-AG catabolism to MAGL and to limit the FAAH involvement in the degradation of 2-AG to ~1%. The study of Blankman allowed the identification of two additional hydrolases as well, the hydrolase domain-containing 6 and 12 (ABHD-6 and ABHD-12), which were previously uncharacterized and are together responsible for ~13 % of 2-AG degradation. Finally, treatment of mice with JZL-184, one of the most potent and selective MAGL inhibitor, resulted in a ~90% inhibition of 2-AG hydrolysis and a substantial increase of 2- AG brain levels (Long et al., 2009-b).

As reported in section 1.2, the depolarization of the postsynaptic neuron, and the correlated increase of intracellular level of Ca2+, results in an activation of endocannabinoid biosynthesis pathways in the postsynaptic neuron. Endocannabinoids just synthetized are released in the synaptic space and they bind the cannabinoid receptors on presynaptic neuron. The subtlety of this modulation is

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further consolidated by the intervention of endocannabinoid degradating pathways. Indeed, growing evidence has emerged to assign a key role to MAGL in the endocannabinoid-mediated retrograde signalling (Labar et al., 2010). In the last decades, many compounds have been synthetized in order to find selective and potent MAGL inhibitors and many different approaches of targeting have been used. In particular, the Nucleophilic Serine targeting gave an important MAGL inhibitor as the irreversible inhibitors (MAFP) (Ghafouri et al., 2004; Labar et al., 2007), together with the other two main molecules JZL-184 (irreversible inhibitor with an high selectivity for MAGL) and OMDM-169 (see Fig 1.9).

JZL-184 MAFP OMDM-169

Figure 1.9 Structures of some MAGL inhibitors.

The effect of a suppression of MAGL activity results in a robust increase of the brain levels of 2-AG and a concomitant reduction of arachidonic acid and downstream eicosanoid metabolites (Long et al., 2009; Nomura et al., 2011; Blankman and Cravatt, 2013). Therefore, inhibition of MAGL may lead to a suppression of pro-inflammatory signaling in the nervous system. Recently, it was shown that inhibition of MAGL with JZL-184, the most selective and potent MAGL inhibitor (Long et al., 2009-a), improved synaptic plasticity and memory in a mouse model of Alzheimer's disease (Chen et al., 2012) Furthermore, MAGL KO (MAGL knock-out) mice also exhibited increased synaptic plasticity and memory (Pan et al., 2011), suggesting that disruption of MAGL activity could positively affect higher brain functions.

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1.5.2 α/β Hydrolases (ABHD-6 and ABHD-12)

As reported above, 2-AG degradation is mediated for 85% by MAGL enzyme and for ~13 % by two additional hydrolases, the hydrolase domain-containing 6 and 12 (ABHD-6 and ABHD-12) (Blankman et al., 2007).

ABHD-6 is a newly discovered post-genomic protein and relative little is known on its physiological functions. High expression of ABHD-6 has been reported in certain forms of tumors suggesting that ABHD-6 might serve a new diagnostic marker of these tumors (Li et al., 2009, Max et al., 2009). ABHD-6 appears to be an integral membrane protein localizing to the post-synaptic neuronal membrane (Blankman et al., 2007) and is intracellular strategically positioned to regulate 2-AG levels at the site of its generation (post-synaptic neuron). ABHD-6 was expressed also in many principal glutamatergic neurons, some GABAergic interneurons, as well as astrocytes but not in resident microglia (Marrs et al., 2010).

Like ABHD-6, ABHD-12 is a recently identified postgenomic protein with poorly defined physiological function, and the 2-AG hydrolase activity is the only feature so far potentially linking ABHD-12 to the ECs. ABHD-12 appears to be an integral membrane protein whose active site is predicted to face the lumen/extracellular space (Blankman et al., 2007). Interestingly, ABHD-12 transcripts are highly expressed in various brain regions and specifically in microglia, but are also abundant in different cell types such as macrophages and osteoclasts (Fiskerstrand et al., 2010). Even though ABHD-12 is still poorly characterized, recently developed ABHD-12 KO mice have shed some light to its possible physiological functions. In the study of Blankman and Cravatt (2013), ABHD-12 deficient mice developed age-dependent symptoms that resemble the human neurodegenerative disorder PHARC (polyneuropathy, hearing loss, ataxia, retinosis pigmentosa, cataract), and the authors suggest that the disrupted LPS metabolism and resulting neuroinflammation may form one of the molecular basis for PHARC (Fiskerstrand et al., 2010; Blankman and Cravatt, 2013; Savinainen et al., 2012).

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1.5.3 Fatty Acid Amide Hydrolase (FAAH)

FAAH is a member of the serine hydrolase family of enzymes and plays an important role in hydrolizing of the amide bond of AEA after being taken up into the cell (Deutsch et al., 1995; Day et al., 2001), as first reported in 1993 (Deutsch and Chin, 1993). Its catalytic mechanism involves the formation of a tetrahedral intermediate, derived from nucleophilic attack of the catalytic Ser241 on the carbonyl group of the substrate The tetrahedral intermediate collapses to release the amine and the enzyme-bound acyl intermediate. Lys142 acts as a general base-general acid, mediating the deprotonation of the Ser241 and subsequent protonation of the leaving group that are shuttled through Ser217. The reaction terminates with a water-mediated de-acylation of the enzyme-bound acyl intermediate and release of the free fatty acid with restoration of the active enzyme (Patricelli and Cravatt, 1999; Patricelli et al., 2000; Patricelli et al., 1999; McKinney and Cravatt, 2003). Although FAAH acts on a wide range of fatty acid amides or esters (Cravatt et al., 1996; Patricelli MP and Cravatt BF, 2001) it preferentially hydrolyzes arachidonoyl and oleoyl substrates (arachidonoyl > oleoyl, 3-fold) and primary amides are hydrolyzed 2-fold faster than ethanolamides (Boger et al., 2000)

A series of recent studies (Fowler et al., 2001) have defined its potential to serve as a new therapeutic target for the treatment of a range of clinical disorders including pain, inflammation and sleep disorders. Both, non-selective and selective inhibitors of the enzyme, have been described so far: Phenylmethylsulfonylfluoride (PMSF), Arachidonoyltrifluoromethylketone (ATMK) represent examples of non-selective inhibitors, while URB-597 is a potent, relatively selective and irreversible carbamate-based inhibitor. Finally the urea-based inhibitors PF-622 and PF-750 displayed a more potent and selective activity on FAAH than URB-597 (Deutsch et al., 1997; Deutsch and Chin, 1993; De Petrocellis et al., 1997; Koutek et al., 1994; Ahn et al., 2007).

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URB-597 PF-622

PF-750

Figure 1.10 Chemical structures of some FAAH inhibitors.

1.5.4 Cyclooxygenase type II (COX-2)

Cyclooxygenase-2 (COX-2) is an enzyme that plays a key role in inflammatory processes. Classically, this enzyme is upregulated in inflammatory situations and is responsible for the generation of prostaglandins (PGs) from arachidonic acid (AA). One lesser-known property of COX-2 is its ability to metabolize the endocannabinoids. The classic inactivation of the endocannabinoids AEA and 2-AG is well established, with the hydrolysis to arachidonic acid mediated by FAAH and MAGL, respectively (as reported above).Endocannabinoid metabolism by COX-2 is not merely a means to terminate their actions. On the contrary, it generates PG analogs, namely PG-glycerol esters (PG-Gs) for 2-AG and PG-ethanolamides (PG-EAs or prostamides) for AEA (Alhouayek and Muccioli, 2014). These

endocannabinoid-derived products are pro-inflammatory (Gatta et al., 2012; Valdeolivas

et al., 2013), whereas some endocannabinoid metabolites such as PGD2-G and PGE2-EA exert beneficial effects (Alhouayek et al., 2013). The work byHermanson et al., reported that rapidly-reversible inhibitors of COX-2 selectively inhibit the

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oxygenation of 2-AG and A-EA with much lower half maximal inhibitory concentrations than for arachidonic acid. This led to substrate-selective inhibition of COX-2 as a possible pharmacological approach for the treatment of anxiety disorders and neuropathic painwithout disrupting prostaglandin synthesis (Urquhart et al., 2014).

Unfortunately all PG-EAs or PG-Gs do not exert the same effects, therefore a COX inhibition would decrease the production of these anti-inflammatory lipid mediators. Because of this, would be interesting to see, depending on the tissue or the pathology, which PG synthase is more expressed and which PG-EA or PG-G would be preferentially formed.

Endocannabinoids can also be acted upon by LOX enzymes (linoleate/oxygen oxidoreductase), that represent a family of non-heme iron-containing dioxygenases that catalyze the stereo-specific lipid peroxidation of polyunsaturated fatty acids, and CYP450 that metabolizes both the most important endocannabinoids AEA and 2-AG (all the strategies of endocannabinoid metabolism are reported in Fig. 1.11).

(Urquhart et al., 2014; Hermanson et al., 2013)

Fig. 1.11 COX-2 at the interface of the eicosanoid and endocannabinoid systems

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1.5.5 Endocannabinoid Membrane Transporter (EMT)

When the endocannabinoids AEA and 2-AG complete their actions, are immediately removed from the extracellular space by a process of cellular uptake followed by a rapid metabolism and degradation. Considering the very lipophilic structure of endocannabinoids, appears strange the presence of a transporter to help endocannabinoids to cross the cell membrane. The first detailed characterization of AEA uptake into cortical neurons demonstrated that the observed accumulation of AEA inside the cell was moderately fast (t = 2.5 min), temperature dependent and saturable, leading to the conclusion that the cellular clearance of AEA was consistent with a process of carrier-mediated uptake (Di Marzo et al., 1994). In addition, further experiments demonstrated that uptake was not sodium dependent and this evidence suggested that the uptake was a process of facilitated diffusion (Hillard et al., 1997) . As reported above, in the case of AEA, it was soon clear that uptake by cells occurs via diffusion through the cell membrane, facilitated by a saturable, temperature-dependent and selective transporter. Such a transporter, the “AEA membrane transporter” (AMT – how it was called), has been identified in most of the cells analyzed so far (Hillard and Jarrahian, 2000), and inhibitors capable of enhancing AEA actions in vitro and in vivo have been developed (Beltramo et al., 1997; Di Marzo et al., 1998). In addition, structure activity relationship studies have been carried out on the AMT with a large variety of AEA analogues (reviewed in (Hillard and Jarrahian 2000 ; Piomelli et al., 1999; Di Marzo et al., 2000), and results established that at least one or no less than four cis double bonds in the fatty acyl chain are necessary in order to simply bind to the AMT or also being transported into cells, respectively. Unfortunately, the AMT has not been cloned so far (Fowler, 2013) and it still represents just a putative membrane transporter (Chicca et al., 2012). Furthermore, it is hard to claim if the transporter would bind AEA only, that was already turned out in further studies, or 2-AG as well. By this side, data in literature appear in contrast and a deeper investigation would be necessary to make it more clear (Bisogno et al., 2001). In studies carried out in RBL-2H3 and J774 cells (Rakhshan et al., 2000; Di Marzo et al., 1998; Di Marzo et al., 1999) it was observed that 2-AG does not inhibit efficiently the uptake by cells of [14C]AEA when the two

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substances are present at the same concentration, thus suggesting that the AMT would not recognize 2-AG as a substrate. These data were not confirmed by experiments carried out with human astrocytoma cells (Piomelli et al., 1999) rat cerebellar granule cells (Jarrahian et al., 2000), and human umbilical vein endothelial cells (Maccarrone et al., 2000), where the concentration of radiolabelled AEA used was much lower than the concentration of 2-AG.

Anyway, is out from any contrasts the important role of an endocannabinoid uptake inhibitor, especially blocking 2-AG uptake. It has been already published that the inhibition of AEA uptake leads to an important increase of analgesic effect on neuropathic pain given from the overstay of the endocannabinoid in the synaptic space (La Rana et al., 2006; Maione et al., 2008), even if a blockage of 2-AG uptake would be also better, considering the fact that 2-AG works as a full agonist compared the partial agonist behavior of AEA (Bisogno et al., 2001). To date, some AEA uptake inhibitors have been synthetized and characterized. Among these, the most important are reported in Fig. 1.12.

Figure 1.12 Structures of AEA, the FLAT inhibitor ARN272, and the AEA uptake