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Home , ABHD12, ABHD6, AM404, Cannabis, Taranabant, URB597

Neuropharmacology 56 (2009) 235–243

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Neuropharmacology

journal homepage: www.elsevier.com/locate/neuropharm

Review The as a target for the treatment of dependence

Jason R. Clapper a, Regina A. Mangieri a, Daniele Piomelli a,b,c,* a Department of Pharmacology, The University of California, Irvine 3101 Gillespie NRF, Irvine, CA 92697, USA b Department of Biological Chemistry, The University of California, Irvine 3101 Gillespie NRF, Irvine, CA 92697, USA c Unit of Drug Discovery and Development, Italian Institute of Technology, Genoa 16136, Italy article info abstract

Article history: The endocannabinoid system modulates neurotransmission at inhibitory and excitatory synapses in Received 7 May 2008 brain regions relevant to the regulation of pain, emotion, motivation, and cognition. This signaling Received in revised form 2 July 2008 system is engaged by the active component of cannabis, D9- (D9-THC), which exerts Accepted 7 July 2008 its pharmacological effects by activation of G protein-coupled type-1 (CB1) and type-2 (CB2) receptors. During frequent cannabis use a series of poorly understood neuroplastic changes occur, which Keywords: lead to the development of dependence. Abstinence in cannabinoid-dependent individuals elicits withdrawal symptoms that promote relapse into drug use, suggesting that pharmacological strategies 2-Arachidonoylglycerol Fatty-acid amide aimed at alleviating cannabis withdrawal might prevent relapse and reduce dependence. Cannabinoid URB597 replacement therapy and CB1 antagonism are two potential treatments for cannabis depen- Depression dence that are currently under investigation. However, abuse liability and adverse side-effects may limit the scope of each of these approaches. A potential alternative stems from the recognition that (i) frequent cannabis use may cause an adaptive down-regulation of brain endocannabinoid signaling, and (ii) that genetic traits that favor hyperactivity of the endocannabinoid system in humans may decrease suscep- tibility to cannabis dependence. These findings suggest in turn that pharmacological agents that elevate brain levels of the endocannabinoid , anandamide and 2-arachidonoylglycerol (2-AG), might alleviate cannabis withdrawal and dependence. One such agent, the fatty-acid amide hydrolase (FAAH) inhibitor URB597, selectively increases anandamide levels in the brain of rodents and primates. Preclinical studies show that URB597 produces , anxiolytic-like and -like effects in rodents, which are not accompanied by overt signs of abuse liability. In this article, we review evidence suggesting that (i) cannabis influences brain endocannabinoid signaling and (ii) FAAH inhibitors such as URB597 might offer a possible therapeutic avenue for the treatment of cannabis withdrawal. Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction limited to psychotherapeutic interventions which, while moder- ately effective, require the aid of much needed pharmacotherapies Nearly 160 million people world-wide used cannabis in 2005 (Nordstrom and Levin, 2007). (UNODC, 2007), with approximately 10% of first-time users and 50% A major obstacle slowing the development of pharmacological of daily users developing dependence (Hall and Degenhardt, 2007). treatments for cannabis abuse is the continued belief, even among Among adolescents, cannabis abuse is common and positively the scientific community, that cannabis does not produce depen- correlates with continued use later in life. Accordingly, 63% of the dence. On the contrary, recent studies have unequivocally docu- 2.1 million first-time users in 2006 were under the age of 18 mented the occurrence of a cannabis dependence syndrome by (SAMHSA, 2007); of those who used cannabis 5 times, half demonstrating (i) that the major psychotropic constituent of continued to use the drug 10 years later (Perkonigg et al., 2008). cannabis, D9-THC, possesses reinforcing properties in non-human While the prevalence of cannabis abuse is striking by epidemio- primates and (ii) that abstinence from the drug causes withdrawal logical measurements, the approved options for treatment are in humans (Budney et al., 2003, 2004; Fattore et al., 2008; Tanda and Goldberg, 2003). Indeed, the animal self-administration studies of Justinova and colleagues have demonstrated drug- seeking behavior for D9-THC in drug-naı¨ve squirrel monkeys with * Corresponding author. Department of Pharmacology, The University of Cal- similar acquisition and responding rates as other drugs of abuse ifornia, Irvine 3101 Gillespie NRF, Irvine, CA 92697, USA. Tel.: þ1 949 824 6180; fax: þ1 949 824 6305. (Justinova et al., 2003). Furthermore, the notion of a cannabis E-mail address: [email protected] (D. Piomelli). withdrawal syndrome ensuing from interruption of frequent drug

0028-3908/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2008.07.018 236 J.R. Clapper et al. / Neuropharmacology 56 (2009) 235–243 use, has been validated by Budney and colleagues, who have shown localized exclusively in immune cells, but recent work has sug- that this syndrome is characterized by craving, irritability, anxiety, gested that low levels of these receptors are also present in the depressed mood, decreased appetite and sleep difficulties, and brainstem (Van Sickle et al., 2005) and possibly in other brain displays similar scope and severity to the withdrawal associated regions (Gong et al., 2006). with use (Budney et al., 2003, 2004; Vandrey et al., 2005, The activation of CB1 receptors by endogenous anandamide is 2008). Prompted by these findings, clinical studies have examined rapidly terminated through carrier-mediated uptake into neurons a of treatment options for the affective symptoms of and glia, followed by intracellular hydrolysis (Fig. 1). The cannabis withdrawal. These investigations have primarily included molecular entity(ies) that transports anandamide into cells has anxiolytic and antidepressant drugs and have, thus far, yielded not been molecularly identified, but has been characterized mixed results (McRae et al., 2003; Nordstrom and Levin, 2007). But, pharmacologically (Beltramo et al., 1997; Cravatt et al., 1996; Di despite their limitations, the studies have suggested that treating Marzo et al., 1994; Hillard et al., 1997; Ligresti et al., 2004). the symptoms of cannabis withdrawal may improve the likelihood Internalization of anandamide in neural cells is a rapid, temper- an individual will remain abstinent. This possibility has received ature sensitive, saturable process that is independent of anan- further support by two recent reports, which indicate that the damide hydrolysis (Beltramo et al., 1997; Kathuria et al., 2003) symptoms associated with cessation of cannabis use strongly and susceptible to stereoselective pharmacological inhibition contribute to relapse. A telephone survey of daily cannabis and (Fegley et al., 2004; Piomelli et al., 1999). Inside neural cells, tobacco users has shown that the discomfort associated with anandamide deactivation is completed by the activity of fatty- withdrawal symptoms contributes considerably to relapse to the acid amide hydrolase (FAAH), a membrane-associated use of either drug. Interestingly, cannabis users reported that hydrolase that belongs to the amidase signature family of craving contributed less to relapse than did affective and cognitive (Cravatt et al., 1996; De´ sarnaud et al., 1995; Giang and symptoms (Budney et al., in press). Furthermore, a study of Cravatt, 1997; Hillard et al., 1995; Patricelli et al., 1999; Ueda et al., adolescents and young adults with major depressive disorder and 1995). Genetic deletion of the FAAH or pharmacological comorbid cannabis dependence has indicated that withdrawal inhibition of intracellular FAAH activity each impair anandamide symptoms such as craving, irritability, restlessness, anxiety, and hydrolysis, resulting in elevated CNS levels of this transmitter depression prevent prolonged abstinence from the drug (Cornelius (Cravatt et al., 2001; Fegley et al., 2005; Kathuria et al., 2003). As et al., in press). Though more work is clearly needed, the results discussed below, anandamide deactivation mechanisms have available thus far do suggest that pharmacotherapies targeting provided two useful opportunities to elevate endogenous levels withdrawal symptoms may be useful to treat cannabis dependence. of anandamide and, thus, indirectly activate CB1 receptors. In this article, we have four main objectives. First, we briefly Like anandamide’s, the deactivation of 2-AG in neurons and glia review the properties of the endocannabinoid system. Second, we is thought to proceed through a two-step process. First, an uptake discuss the possible role of CB1 receptor modulation (with either process clears 2-AG from the extracellular space (Beltramo and or antagonist drugs) in the treatment of cannabis depen- Piomelli, 2000; Piomelli et al., 1999). 2-AG transport shows several dence. Third, we outline available experimental evidence for an pharmacological similarities with anandamide transport, but also interrelationship between cannabis dependence and activity of the notable differences. For example, 2-AG and anandamide compete endocannabinoid signaling system. Finally, we describe animal for each other’s internalization, and the transport of both is studies with inhibitors of anandamide deactivation, which provide inhibited by the anandamide analog (and transport inhibitor) a rationale for a further exploration of these agents as medicines for AM404 (Beltramo and Piomelli, 2000; Beltramo et al., 1997; cannabis dependence (for reviews of the endocannabinoid system Piomelli et al., 1999). However, 2-AG uptake in human astrocytoma see Di Marzo, 2008; Freund et al., 2003). cells is also blocked by inhibitors of esterification into phospholipids (for example, triacsin C) whereas anandamide 2. The endocannabinoid system internalization is insensitive to these agents (Beltramo and Piomelli, 2000). Clearly, the molecular characterization of proteins Two endogenous ligands, arach- involved in the transport of anandamide and 2-AG is essential to idonoylethanolamide (anandamide) (Devane et al., 1992; Di Marzo allow progress in this field to occur. et al., 1994) and 2-arachidonoylglycerol (2-AG), have been identi- The second step of 2-AG deactivation is the intracellular fied (Mechoulam et al., 1995; Stella et al., 1997; Sugiura et al., 1995). hydrolysis of this compound to arachidonic acid and glycerol. The These substances meet three key criteria for being considered best characterized route of 2-AG hydrolysis is via the intracellular endocannabinoid neurotransmitters: they are produced in an , monoacylglycerol (also referred to as activity-dependent manner by neurons in the central nervous lipase, MGL) (Dinh et al., 2002, 2004; Karlsson et al., system (CNS); they modulate synaptic transmission (via activation 1997). This protein is heterogeneously expressed throughout the of Gai/o-protein-coupled CB1 receptors); and they are rapidly rodent brain and is specifically localized to presynaptic nerve deactivated (Freund et al., 2003). Anandamide and 2-AG also bind terminals (Dinh et al., 2002; Gulyas et al., 2004). Adenovirus- to and activate CB2 receptors (Munro et al., 1993), but the roles of mediated overexpression of MGL enhances hydrolysis of endoge- this receptor subtype in the CNS are still incompletely understood nous 2-AG, while RNA interference-mediated silencing of MGL (Ishiguro et al., 2007; Onaivi et al., 2006; Van Sickle et al., 2005). elevates 2-AG levels, suggesting that this is a primary The distribution of CB1 receptors in the brain is reflective of the regulator of 2-AG levels in cells (Dinh et al., 2002, 2004). Addi- important functions served by the endocannabinoid signaling tionally, administration of the MGL inhibitor, URB602, to organo- system in the control of pain, emotion, motivation and cognition typic brain slices or to select brain regions (by microinjection) (Piomelli, 2003). In rodents and humans, CB1 receptors are found at elevates the levels of 2-AG without affecting those of anandamide highest concentrations in the hippocampus, neocortex, basal (Hohmann et al., 2005; Makara et al., 2005). Recently, Stella and ganglia, and anterior olfactory nucleus (Glass et al., colleagues identified a novel 2-AG-hydrolyzing activity in the 1997; Herkenham et al., 1991; Matsuda et al., 1993). Moderate mouse microglial cell line, BV-2, which does not express MGL. Such receptor levels are also present in the basolateral amygdala, activity is pharmacologically distinct from that of the cloned MGL, hypothalamus, and the periaqueductal gray matter of the midbrain but has not been molecularly characterized (Muccioli et al., 2007). (Glass et al., 1997; Herkenham et al., 1991; Katona et al., 2001; In a separate study, two additional 2-AG-hydrolyzing , Matsuda et al., 1993). Initially, CB2 receptors were thought to be ABHD6 and ABHD12, were identified in the brain using a functional J.R. Clapper et al. / Neuropharmacology 56 (2009) 235–243 237

O Extracellular space

HO N H Anandamide

AT AT

O

N OH O H

OH

Arachidonic Acid Intracellular space

FAAH

HO NH2 Ethanolamine

Fig. 1. In the CNS, anandamide is thought to be eliminated through a two-step deactivation process. First, it is carried across neural cell membranes by a transport system (AT), which remains uncharacterized at the molecular level. After internalization, anandamide is hydrolyzed to arachidonic acid and ethanolamine by fatty-acid amide hydrolase (FAAH), a serine hydrolase localized to intracellular membranes.

proteomics approach (Blankman et al., 2007). In agreement with 3.2. Direct modulation of CB1 receptors as a treatment for cannabis previous work (Dinh et al., 2004), these enzymes were shown to dependence account for approximately 15% of the total 2-AG-hydrolyzing activity in the brain, while MGL accounted for the remaining 85% Even though, as we have seen above, direct activation of CB1 (Blankman et al., 2007). receptors may yield variable behavioral responses, low-dosage oral D9-THC has shown promise in the management of human cannabis 3. Direct modulation of the CB1 receptor withdrawal. The rationale for this approach is that controlled replacement of D9-THC for smoked cannabis may reduce the 3.1. Involvement of CB1 receptors in mood regulation severity of withdrawal symptoms and allow a dependent individual to remain abstinent. Additionally, given that dependent subjects 9 Evidence suggests that CB1 receptor signaling is involved in the are experienced with cannabis, and D -THC is administered at low regulation of mood, but the nature of this involvement and the doses, administration of the latter is unlikely to result in the anxiety relationship between the endocannabinoid system and mood responses observed with inexperienced users or high dosages. disorders remains unclear (for a review, see Viveros et al., 2005). In Consistent with this idea, two independent clinical studies have rodents and humans, D9-THC produces dose- and context-depen- shown that low-dose oral D9-THC attenuates withdrawal symptom dent responses that include relaxation and , but also scores and is minimally intoxicating in non-treatment seeking daily anxiety and panic. In general, low doses of D9-THC exert anxiolytic cannabis users (Budney et al., 2007; Haney et al., 2004). In a sepa- and mood enhancing effects, whereas high doses are anxiogenic rate study, however, similar doses of D9-THC exhibited reinforcing and dysforic, however, these dose-dependent effects are also properties in healthy male cannabis users, suggesting that abuse contingent on other factors, including environment and previous liability remains a concern (Hart et al., 2005). 9 experience with the drug. For example, administration of D -THC to The opposite strategy, administration of a CB1 receptor antag- mice exerted anxiolytic effects in the light/dark box, when onist, has been investigated as a means to block the acute phar- administered at an intraperitoneal (i.p.) dose of 0.3 mg kg1;by macological (Gorelick et al., 2006; Huestis et al., contrast, administration of a higher dose (5 mg kg1, i.p.) produced 2001). This approach is attractive, because several structurally anxiogenic effects (Berrendero and Maldonado, 2002; Valjent et al., distinct CB1 antagonists have recently become available for clinical 2002). Likewise, the potent CB1 receptor agonist, HU-210, exerted testing, including and . However, one anxiogenic effects in the rat defensive-withdrawal test after acute potential problem with its application to the management of i.p. administration at a dose of 0.1 mg kg1 (Rodrı´guez de Fonseca cannabis withdrawal is represented by potential adverse effects on et al., 1996), whereas administration of this same dose for 10 days mood associated with brain CB1 receptor blockade. Experiments resulted in anxiolytic-like and antidepressant-like effects in the rat with CB1-null mice and CB1 receptor antagonists suggest that novelty-suppressed feeding and forced-swim tests (Jiang et al., removing CB1 activity heightens anxiety- and depression-related 2005). Similar dose- or context-dependent actions on anxiety- behaviors (Alberich Jorda et al., 2002; Haller et al., 2002, 2004; related behaviors in the elevated plus maze and social interaction Marsicano et al., 2002; Martin et al., 2002; Uriguen et al., 2004). For tests have been reported following treatment with a structurally example, in one study CB1-null mice showed increased anxiety-like distinct cannabinoid agonist, CP-55,940 (Genn et al., 2004; Marco behavior compared to wild-type controls under conditions that et al., 2004). were more stressful to the animals (i.e. high ambient light, 238 J.R. Clapper et al. / Neuropharmacology 56 (2009) 235–243 infrequent handling, novel environment). Additionally, CB1-null A mice displayed increased sensitivity to the development of anhe- O O donia-like symptoms in a chronic unpredictable stress model of N OH N human depression, and showed other behaviors reminiscent of H H O those observed in patients with melancholic depression, such as AM404 UCM707 decreased food intake, decreased responsiveness to rewarding OH stimuli, and hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis (reviewed in Hill and Gorzalka, 2005a). While some OH O studies have reported anxiolytic- and antidepressant-like effects of O OH CB1 receptor antagonists in rodents (Griebel et al., 2005; Haller N NH et al., 2004; Shearman et al., 2003), others have shown that H administration of these agents produces anxiogenic-like effects OMDM1 VDM11 (Are´ valo et al., 2001; McGregor et al., 1996; Navarro et al., 1997; Rodgers et al., 2005). The latter findings are consistent with the B O NH side-effect profile demonstrated by the CB1 antagonist rimonabant 2 in human trials, where anxiety, irritability and depression were N N H among the most frequent adverse events reported (Despres et al., O N O 2005; Gelfand and Cannon, 2006; Pi-Sunyer et al., 2006; Scheen O O et al., 2006; Van Gaal et al., 2005). Though similar side-effects have URB597 OL-135 been reported for taranabant (Medical Week News, Inc., 2008), it is O O important to point out that both rimonabant and taranabant are N N N N inverse at CB1 receptors, and that their ability to alter N N H affective states may be a consequence of this property. In conclu- H N PF-750 PF-622 sion, though still limited, available evidence suggests that CB1 receptor activity modulates affective responses to stress. This Fig. 2. Chemical structures of select (A) anandamide transport inhibitors and (B) FAAH demands that caution be exerted in future studies aimed at testing inhibitors. the effects of CB1 antagonists on cannabis withdrawal in humans.

4. Role of the endocannabinoid system in mood regulation: animal models. For example, the anandamide transport inhibitor insights from studies with anandamide deactivation AM404 and the FAAH inhibitor URB597 have been shown to inhibitors enhance active stress-coping behaviors in assays for antidepres- sant-like drug activity. Hill and colleagues reported that AM404, at An alternative approach to cannabinoid replacement may be to an i.p. dose of 5 mg kg1, decreases immobility time in the rat potentiate normal endocannabinoid signaling with drugs that forced-swim test (Hill and Gorzalka, 2005b). A separate study has inhibit endocannabinoid deactivation mechanism(s). A number of shown that URB597 (0.1 and 0.3 mg kg1, i.p.) exerts a similar effect pharmacological tools that target events in endocannabinoid and also increases struggling behavior in the mouse tail-suspension deactivation have been developed. Anandamide elimination is test (Gobbi et al., 2005). In the latter study, the enhancement of prevented by two distinct classes of agents: transport inhibitors stress-coping behaviors caused by URB597 was accompanied by such as AM404 (Beltramo et al., 1997), UCM707 (Lopez-Rodriguez elevations of anandamide levels in the prefrontal cortex, hippo- et al., 2003), OMDM-1 and OMDM-2 (Ortar et al., 2003), and VDM11 campus, and midbrain – regions that are implicated in the regula- (De Petrocellis et al., 2000); and FAAH-selective anandamide tion of mood and the processing of emotional stimuli (Berton and hydrolysis inhibitors such as URB597 (Kathuria et al., 2003; Pio- Nestler, 2006). Moreover, the antidepressant-like activities of melli et al., 2006), OL-135 (Boger et al., 2005), and PF-622 and PF- AM404 or URB597 were prevented by administration of a CB1 750 (Ahn et al., 2007)(Fig. 2). , further suggesting that the effects of these 2-AG deactivation has been less extensively investigated, but drugs are mediated by anandamide acting at CB1 receptors. a few inhibitors of 2-AG hydrolysis have been reported. The Symptoms of anxiety are frequently reported in depressed compound URB602 is a non-competitive and partially reversible patients (DSM-IV, 1994), thus it is of interest that blockade of inhibitor of MGL (King et al., 2007). Despite its low potency, the ability anandamide deactivation may also result in anxiolytic-like effects. of URB602 to elevate 2-AG levels in intact neurons without changing While characterizing URB597, we discovered that this compound anandamide levels has been exploited experimentally (Hohmann produces a decrease in isolation-induced ultrasonic vocalizations in et al., 2005; King et al., 2007; Makara et al., 2005). Another interesting rat pups and an increase in time spent on the open arms of an probe,thecysteinetrapN-arachidonylmaleimide, inhibits MGL elevated zero maze (Kathuria et al., 2003). Other groups have without affecting ABHD6 or ABHD12 (Saario et al., 2005). Both subsequently confirmed the anxiolytic-like effects of this drug agents appear to lack, however, the drug-like properties needed for (Naidu et al., 2007; Patel and Hillard, 2006). Similarly, we found systemic administration in vivo. that AM404 dose-dependently reduces isolation-induced ultra- As outlined in the following sections, experiments with the sonic vocalizations in rat pups, and increases the time adult rats FAAH inhibitor URB597 have provided evidence that enhancement spend in the open arms of the elevated plus maze or in the open of anandamide signaling promotes active stress-coping behaviors field during defensive withdrawal (Bortolato et al., 2006). The and exerts anxiolytic and antidepressant drug-like effects in anxiolytic-like effects of URB597 and AM404 are accompanied by rodents. elevations of brain anandamide and are blocked by administration of the CB1 antagonist rimonabant, again providing evidence that 4.1. Anandamide as a modulator of mood-related behavior the effects of these compounds are due to enhanced anandamide in rodents activity at CB1 receptors. It should be noted that the effects of anandamide deactivation Results from our lab and others have shown that anandamide inhibitors on stress-coping behaviors in rodents appear to be highly deactivation inhibitors may modulate stress-related behaviors in sensitive to environmental conditions. For example, Naidu and J.R. Clapper et al. / Neuropharmacology 56 (2009) 235–243 239 colleagues failed to find a reduction of immobility in the tail- victims with a life-time diagnosis of major depression, compared to suspension test, or an increase in the percentage of time spent in subjects who died by accident or natural causes (matched by age, the open arms in the elevated plus maze in FAAH/ mice or in sex, and postmortem interval) (Hungund et al., 2004). Hill and wild-type mice treated with URB597 when the tests were con- colleagues reported reduced serum 2-AG levels in drug-free ducted under normal laboratory lighting (Naidu et al., 2007). women diagnosed with major depression compared to demo- However, after adopting stronger (more stressful) lighting condi- graphically matched controls, with levels of 2-AG negatively tions, those investigators were able to observe anxiolytic and correlated to the duration of the depressive episode (Hill et al., antidepressant-like effects of FAAH deletion or inhibition (Naidu 2008). In the latter study, serum anandamide was not associated et al., 2007). Such sensitivity to environmental stress levels is with major depression, but was negatively correlated with consistent with other findings, which show that the ability of measures of anxiety. While still limited, this work provides URB597 to exert anxiolytic-like effects in the rat elevated plus maze intriguing evidence that endocannabinoid signaling might be varies with the lighting conditions and with the amount of animal altered in specific brain regions (and, perhaps, peripheral tissues) handling prior to the test (Bortolato and Piomelli, 2007). during depression or negative affective states. The pharmacological tests mentioned above are not disease models; rather, they were developed as behavioral screens for 4.3. Anandamide and the modulation of hedonic responses specific classes of antidepressant and anxiolytic drugs. The ability of inhibitors of anandamide degradation to regulate stress-related While biochemical alterations hint at a possible endocannabi- behaviors under pathophysiological conditions should provide noid dysfunction in depressive states, perhaps the most exciting more solid evidence of a role for anandamide signaling in real finding from our chronic mild stress study in rats was that URB597 disease states, such as depression. reversed stress-induced reductions in consumption and body-weight gain (Bortolato et al., 2007). These behavioral effects 4.2. Endocannabinoids and stress-related mood disorders were accompanied by elevations in anandamide levels in the thalamus, midbrain, and striatum – regions involved in mood, The core symptoms of depression include mood and/or loss of reward, and the processing of emotional stimuli (Berton and interest in pleasurable activities (anhedonia) (DSM-IV, 1994). Other Nestler, 2006) – suggesting that increased anandamide signaling characteristics are changes in body weight, sleeping patterns, may underlie the antidepressant-like effect of URB597. Consistent psychomotor behavior, energy level, and cognitive functioning with these data, a study by Rademacher and colleagues found that (DSM-IV, 1994). The degree of overlap between the physiological pretreatment with URB597 attenuates the reductions in sucrose functions altered by depression and those affected by CB1 receptor preference and consumption produced by restraint stress in mice activity is striking, and suggests that activation of endocannabinoid (Rademacher and Hillard, 2007), supporting the notion that signaling may have important effects on the pathophysiology and enhanced anandamide signaling might counteract stress-induced regulation of mood. In fact, prolonged and anhedonia. Moreover, anandamide has been implicated as cannabis withdrawal in humans are often associated with depres- a modulator of normal responses to pleasurable stimuli. Injections sion, though whether cannabis use contributes to the development of anandamide into the shell of the nucleus accumbens have been of this disorder is still a matter of debate (for a review see Degen- shown to increase positive ‘liking’ reactions to intraoral sucrose in hardt et al., 2003). rats (Mahler et al., 2007). These results indicate that anandamide Limited but compelling evidence suggests that the endocanna- signaling in specific brain regions may mediate responses to binoid system is altered during stress-related disease states in both rewarding or pleasurable stimuli, and that increasing anandamide rodents and humans. For example, Hill and colleagues found levels might enhance positive reactions to these stimuli both under a significant reduction in the levels of 2-AG and CB1 receptor normal and pathological conditions. Interestingly, studies in our lab protein in the hippocampus of rats subjected to 3 weeks of suggest that anandamide signaling also might contribute to posi- unpredictable stress (Hill et al., 2005). In that study, stressed tive affective states in humans. Ongoing experiments indicate animals also showed impairment of reversal learning in the Morris indeed that certain pleasurable stimuli may increase plasma water maze, which was corrected by administration of the canna- anandamide levels in healthy individuals (Mangieri et al., unpub- binoid agonist HU-210. Our own work in chronically stressed rats lished observations). similarly implicates endocannabinoid signaling in biochemical and behavioral changes induced by stress (Bortolato et al., 2007). While 5. Endocannabinoids and cannabis dependence we did not find significant changes in anandamide or 2-AG levels in most brain regions examined (with the exception of a small 5.1. Cannabis tolerance increase of 2-AG in the thalamus of stress-exposed rats), we did observe that after 10 weeks of mild stress, CB1 receptor mRNA Chronic exposure to cannabis or cannabinoid receptor agonists levels were increased in the prefrontal cortex and decreased in the causes CB1 receptor desensitization and down-regulation, conse- midbrain, and that these effects were opposed by 5 weeks of quently rendering subjects tolerant to the central and peripheral treatment with URB597 (Bortolato et al., 2007). The discrepancy in effects of the drugs (Gonzalez et al., 2005). Consistent with this idea, the biochemical changes observed by Hill and colleagues and those animals made tolerant to the behavioral effects of seen in our study suggests that the roles of the endocannabinoid display decreased CB1 receptor levels as well as impaired G-protein system in different brain regions may vary as a function of duration receptor coupling (Breivogel et al., 1999; Rubino et al., 2000a; Sim- and/or the severity of the stress. Hill and colleagues used a 3-week Selley and Martin, 2002; Sim et al.,1996). Additional studies suggest unpredictable stress protocol that included restraint stress, that disregulated protein phosphorylation downstream of the CB1 whereas our study was longer, but included less severe stressors receptor may also contribute to the development of tolerance such as cage tilting, floor soiling, and food deprivation (Bortolato (Martin et al., 2004; Rubino et al., 2000b). In humans, a post mortem et al., 2007; Willner, 1997). investigation of brains from chronic cannabis users revealed The preclinical data outlined above are complemented by a regional reduction in [3H]SR141716A (rimonabant) binding sites findings from human studies. Hungund and colleagues found an and decreased CB1 receptor mRNA levels (Villares, 2007). Further- increase in both CB1 receptor mRNA and CB1 receptor-stimulated more, a study of -naı¨ve schizophrenic patients showed [35S]GTPgS binding in the dorsolateral prefrontal cortex of suicide that subjects who used cannabis at least 20 times in their life had 240 J.R. Clapper et al. / Neuropharmacology 56 (2009) 235–243 lower levels of anandamide in their cerebrospinal fluid, compared modalities are currently being considered to treat cannabis to subjects who used cannabis 5 times or less. No such difference dependence, including activation or deactivation of CB1 receptors. was noted in serum (Leweke et al., 2007). The latter findings need to While these strategies show promise in measures of cannabis be independently replicated, but they do suggest that cannabis may withdrawal and abstinence, they may also create problems of abuse not only regulate CB1 receptor activity, but may also influence the liability or adverse emotional effects. An additional approach might availability of endocannabinoid ligands in the brain. be to enhance endogenous anandamide signaling using agents that In this context, it is important to point out the results of recent attenuate the deactivation of this endocannabinoid transmitter. genetic analyses, which documented the interaction of cannabis use Increasing anandamide signaling with deactivation inhibitors, with a single nucleotide of the human FAAH gene. such as the FAAH blocker URB597, potentiates stress-coping This study found that, among subjects who tried cannabis, those behaviors in animals, indicating a role for anandamide in physio- carrying a genetic variation of FAAH (C385A) that causes reduced pathological context of stress-related responses. Similarly, eleva- enzyme expression and activity (Chiang et al., 2004) were signifi- tion of anandamide in specific brain regions opposes the anhedonic cantly less likely to become dependent on the drug (Tyndale et al., effects of stress and promotes normal positive responses to plea- 2007). It is tempting to speculate that individuals who carry the surable stimuli in rodents. It is reasonable to hypothesize that these C385A may have elevated brain anandamide levels, which effects could act to the negative affect and stress, which is might reduce their susceptibility to become dependent on cannabis. common during cannabis withdrawal, thus allowing cannabis dependent individuals to successfully abstain from drug use. 5.2. Anandamide deactivation inhibitors: low tolerance and abuse liability Acknowledgements

Two pharmacological properties make FAAH inhibitors attractive The authors wish to thank their colleagues in the lab for their as potential therapies for cannabis dependence: they do not appear to collaborative research contributions and NIDA for its continuing evoke tolerance following long-term administration, and they do not financial support. JRC and RAM contributed equally to this display significant abuse liability. For example, our lab has demon- manuscript. strated that the effects of the FAAH inhibitor URB597 on brain anandamide levels and its antidepressant-like effects in the tail- suspension test and forced-swim test models are maintained References following a repeated 4-day (once a day) dosing regimen (Gobbi et al., 2005). Additionally, once-daily dosing of URB597 for 5 weeks elicited Ahn, K., Johnson, D.S., Fitzgerald, L.R., Liimatta, M., Arendse, A., Stevenson, T., Lund, E.T., Nugent, R.A., Nomanbhoy, T.K., Alexander, J.P., Cravatt, B.F., 2007. antidepressant effects in chronically stressed animals without Novel mechanistic class of fatty acid amide hydrolase inhibitors with remark- altering CB1 receptor mRNA levels (Bortolato et al., 2007). These able selectivity. Biochemistry 46, 13019–13030. studies indicate that the biochemical and behavioral effects of FAAH Alberich Jorda, M., Verbakel, S.E., Valk, P.J., Vankan-Berkhoudt, Y.V., Maccarrone, M., Finazzi-Agro, A., Lowenberg, B., Delwel, R., 2002. Hematopoietic cells express- inhibitors are maintained during both sub-acute and chronic treat- ing the peripheral cannabinoid receptor migrate in response to the endo- ments. Equally important, URB597 did not produce rewarding effects cannabinoid 2-arachidonoylglycerol. 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