Cannabis Paper 6

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Owing to its psychotropic and medicinal effects, Cannabissativa has been mankind’s friend and foe for millennia 1.

Yet, it was only in the second half of the twentieth centurythat research finally cast light onto some of its mecha-nisms of action. First with the identification of its majorpharmacologically active components, the cannabinoids,and then with the discovery in vertebrates of moleculartargets for some of these cannabinoids. These targetsinclude the G-protein-coupled cannabinoid receptors andtheir endogenous ligands, the endocannabinoids2,3. In thepast 10 years, it became clear that, at least in mammals,the functions of this endocannabinoid signalling system(BOX 1; FIG. 1) are not limited to the brain, but are exertedin the whole organism. Endocannabinoids are generallyconsidered to be released from cells immediately afterbiosynthesis, as no evidence exists for their storagein secretory vesicles, and several of their biosyntheticenzymes are found in the plasma membrane. Moreover,endocannabinoids act on their receptors only locally,

possibly because of their high lipophilicity, and areimmediately inactivated under physiological conditions4.However, the regulation of their levels by biosyntheticand degradative enzymes, and their mode of action,are becoming characterized by an increasing degree ofredundancy of pathways and promiscuity of moleculartargets, respectively 4,5.

The realization of the complexity of endocannabinoidregulation in physiological and pathological conditionscould have discouraged the development of new thera-peutics that target this system, other than preparationsbased on∆9-tetrahydrocannabinol and its synthetic analogues— for example, dronabinol (Elevat/Compassia/Marinol;

Solvay Pharmaceuticals) and nabilone (Cesamet; Eli Lilly)— which directly activate cannabinoid receptors and canalso produce central side effects. However, new genera-tions of endocannabinoid-manipulating drugs designedalmost entirely in silico or screened out of libraries ofcompounds have been developed and are either beingtested in clinical trials or already on the market 6,7. Theseinclude compounds that inhibit endocannabinoiddegradation by fatty acid amide hydrolase (FAAH), forexample, SA-47 and URB597, and compounds that act atcannabinoid receptor 1 (CB

1), for example, rimonabant

(Acomplia; Sanofi–Aventis) and taranabant. These newerdrugs are thought to indirectly enhance or directly reducethe functionality of endocannabinoid receptors only inthose tissues and cells in which there is an ongoing syn-thesis, release, action and degradation of endocannabi-noids. So, when it comes to adverse events, they shouldbe more selective and safer than direct agonists. ThisReview article is aimed at critically discussing these new

developments, which are based on the emerging realiza-tion that alterations in the endocannabinoid system canboth counteract and participate in disease symptoms andprogress, thus opening the way to develop both endocan-nabinoid boosters (that is, indirect agonists) and curbers(that is, antagonists) as therapeutics.

Ups and downs of endocannabinoids in diseaseThere are now several examples in almost each of themajor therapeutic areas of interest in which alterations inthe endocannabinoid system are associated with disease.In particular, changes in tissue concentrations of two ofthe most studied endocannabinoids — anandamide and

Endocannabinoid Research

Group, Institute of

Biomolecular Chemistry,

National Research Council

(CNR), Via Campi

Flegrei 34, 80078,

Pozzuoli, Naples, Italy.

e-mail:

[email protected] .it

doi:10.1038/nrd2553

Cannabinoids

Natural lipophilic products

from the flower of Cannabis

sativa, most of which have

a typical bicyclic or tricyclic

structure and a common

biogenetic origin from olivetol.

Cannabinoid receptors

G-protein-coupled receptors

for ∆9-tetrahydrocannabinol,

so far identified in most

vertebrate phyla. Two subtypes

are known: CB1 and CB

2.

Endocannabinoids

Endogenous agonists of

cannabinoid receptors in

animals.

∆9-Tetrahydrocannabinol

The major psychotropic

component of Cannabis sativa,

and one of about 66

‘cannabinoids’ found in the

flowers of this plant.

Targeting the endocannabinoidsystem: to enhance or reduce?Vincenzo Di Marzo

Abstract | As our understanding of the endocannabinoids improves, so does the awareness

of their complexity. During pathological states, the levels of these mediators in tissues

change, and their effects vary from those of protective endogenous compounds to those of

dysregulated signals. These observations led to the discovery of compounds that either

prolong the lifespan of endocannabinoids or tone down their action for the potential futuretreatment of pain, affective and neurodegenerative disorders, gastrointestinal inflammation,

obesity and metabolic dysfunctions, cardiovascular conditions and liver diseases. When

moving to the clinic, however, the pleiotropic nature of endocannabinoid functions will

require careful judgement in the choice of patients and stage of the disorder for treatment.

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2-arachidonoylglycerol

(2-AG). The second most-

studied endocannabinoid after

anandamide. It is thought to be

the most selective endogenous

agonist of cannabinoid 1 and 2

(CB1 and CB

2) receptors, and

the one most often involved in

CB1-mediated retrograde

signalling.

Hyperphagia

A state characterized by an

exaggerated drive for food

consumption and subsequent

enhanced food-intake.

Gliosis

Proliferation of astrocytes in

damaged areas of the central

nervous system, often

associated with anoxic injury

and neuronal death, and found

in certain brain regions during

various neurodegenerative

disorders.

2-arachidonoylglycerol (2-AG) —have been observed inmany disorders. These include pain and inflammation8,9;immunological (autoimmune and allergic) disorders10;

neurological and neuropsychiatric conditions11; obesityand metabolic12,13, and cardiovascular14 disorders; cancer15;and gastrointestinal16 and hepatic17 disorders.

Importantly, apart from being affected by strictlyexperimental variables (such as the time and methodused for tissue preparation and the analytical conditions),the measured amounts of endocannabinoids in tissuesdo not necessarily reflect extracellular, and hence can-nabinoid receptor-active, levels (intracellular 2-AG, forexample, is an intermediate in phospholipid and glyceridemetabolism)4. Therefore, changes in endocannabinoidconcentrations associated with a given pathological con-dition in a certain tissue should be evaluated (and, hence,their biological significance determined) as changesfrom, rather than percent of, control physiological levelsin that tissue. In fact, it is conceivable that with mediatorsthat are not stored in secretory vesicles — as is the casewith endocannabinoids — disease-associated changes of

their tissue levels reflect corresponding changes in theirrelease from cells, and, therefore, in cannabinoid receptoractivation.

Intriguingly, for the same pathological condition,there are often reports of both positive and negativechanges, and of both protective and worsening effects ofendocannabinoid receptor activation in the tissues andorgans involved in the disorder4,18. Additionally, changesof endocannabinoid tone in the same direction oftenaccompany disorders with opposing symptoms; or levelsof anandamide and 2-AG change in different or evenopposing ways during the same condition. These appar-ently contradictory observations cannot be explained bythe use of different experimental protocols alone (suchas different animal models and species, different cohortsof patients, different analytical procedures). Instead, it isbecoming increasingly clear that, within a certain tissue,the endocannabinoid system is affected in more than justone way by a given stressful or pathological stimulus,depending on the nature and duration of this stimulus,and subsequently leading to more than one functionaloutcome. As this has recently been the subject of specificcomprehensive reviews4,18, I shall mention here onlysome of the best-established examples (that is, arisingfrom work replicated in different laboratories) of this phe-nomenon, to give a general overview of endocannabinoidregulation and function.

Eating disorders. In rodents, hypothalamic endocannabi-noid (particularly 2-AG) levels first increase after ~18 hoursfood deprivation and then decrease after food consump-tion19. The physiological meaning of these changes isclear if one remembers that endocannabinoids act aslocal pro-orexigenic mediators in the hypothalamus13.This function is so important that rodents undergoingprolonged dietary restrictions also exhibit reducedhypothalamic 2-AG levels, possibly to better cope withthe lack of food20, or, given the antilipolytic action ofCB

1 receptors21, to allow the utilization of reserve energy

provided by fat. By contrast, obese rodents with defective

leptin signalling exhibit higher hypothalamic endocan-nabinoid levels that — given the anorexic effects of CB

1

antagonists like rimonabant (see below) and the food-intake stimulatory effects of intra-hypothalamic endo-cannabinoids22,23 — are likely to contribute to hyperphagia.

In humans, blood levels of endocannabinoids are higherboth in obese female subjects with a binge-eating dis-order and in anorexic female subjects, possibly becausein both cases endocannabinoids escape from the tonicinhibitory action of leptin24. Thus, changes in endocan-nabinoid levels seem to represent either an adaptiveresponse to induce the intake of food (or to cope withthe lack thereof) or a disrupted orexigenic mechanismthat participates in hyperphagia, fat accumulation andobesity 12,13. As will be discussed later, this latter phenom-enon is currently being exploited for the development ofCB

1 antagonists (for example, rimonabant and tarana-

bant) as new anti-obesity drugs, although there may besome potential complications with this strategy.

Neurodegenerative disorders. In animals with lesions

of nigrostriatal dopaminergic neurons leading toimpaired striatal dopamine signalling and locomotionsimilar to Parkinson’s disease (PD), both increases25–27 and decreases28,29 of striatal endocannabinoid levelshave been reported, even when using the same animalmodel26,29. In my opinion, these opposing changes mightbe explained by the impairment of possible tonic oppos-ing effects (inhibition and stimulation, respectively) ofdopamine D

1 and D

2 receptors on the level of endocan-

nabinoids. The subsequent increased and decreased CB1

activity might contribute to locomotor impairment byoccurring at the level of medium spiny neurons partici-pating in different ways in locomotor control by the basalganglia (reviewed in REF. 11) (FIG. 2). This might explainwhy inhibitors of endocannabinoid degradation26,29 andCB

1 receptor antagonists27,28,30,31, despite their opposing

effects on CB1 activity, can both restore locomotion in

PD models.Other examples of neurodegenerative conditions in

which endocannabinoid signalling can undergo oppos-ing changes and participate in contrary or similar waysto the aetiology or symptoms are β-amyloid-inducedcytotoxicity, multiple sclerosis and amyotrophic lateralsclerosis. In β-amyloid-induced cytotoxicity, hippo-campal 2-AG levels are increased and anandamide levelsdecreased 12 and 20 days following β-amyloid peptideinjection, respectively 32. Moreover, both CB

1 and CB

2

receptor activation might either counteract or con-tribute to β-amyloid-induced gliosis, neuronal damageand memory-retention loss33–35. These findings mightbe relevant to the possible role of the endocannabi-noid system in Alzheimer’s disease (AD) and, togetherwith the finding of increased CB

2 receptor and FAAH

expression in the post-mortem brain of patients withAD36, suggest that both the symptoms and progress ofthis disorder (depending on disease progression) mightbe treated with either enhancers or blockers of the sys-tem. In rats and mice with acute experimental allergicencephalomyelitis (EAE; a well-established model formultiple sclerosis), brain endocannabinoid levels are

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Retrograde signalling

A mechanism whereby a

chemical signal is released from

the postsynaptic neuron, travels

in the synaptic space and

activates presynaptic receptors

to modulate the release of

neurotransmitters, thereby

influencing synaptic plasticity.

either reduced37, unchanged, because of impaired bio-synthesis38, or enhanced39, although in all these casesendocannabinoids are suggested to exert protectiveeffects against excitotoxicity and autoimmune reactions

via CB1 and CB

2 receptors, respectively 40,41. In a model

of amyotrophic lateral sclerosis, endocannabinoid levelsincrease with time in both the brain and spinal cord ofsuperoxide dismutase 1(SOD1)-mutant mice. Moreover,genetic knockout not only of FAAH but also of CB

1

receptors, ameliorates symptoms and survival42, thussuggesting, together with pharmacological experiments,protective and counterprotective roles of CB

2 and CB

1

receptors, respectively 43,44.

Anxiety and depression. CB1agonists and antagonists

can produce both anxiogenic- and anxiolytic-like,or anti-depressant- and pro-depressant-like effects.These effects are dependent on the animal species, itsstarting emotional state, the tests used to investigateanxiety-like and depression-like behaviours, and thedose of compounds used. Little is known on whetherendocannabinoid levels change during anxiogenic-and depressive-like conditions in limbic areas of thebrain, although it is clear that, at least in rodents, thesystem is activated during conditioned fear (in the amyg-dala and hippocampus45,46) and chronic stress (in thehypothalamus47). That is, conditions that might lead toanxiety and depression, respectively. A reduction (in thehypothalamus47) or enhancement (in the periaqueductalgrey 48) of endocannabinoid levels is also observed fol-lowing various acute stressors, and endocannabinoidsparticipate in some of the effects of long-term treatmentwith tricyclic antidepressants49. However, inhibition ofendocannabinoid inactivation in some cases reducesanxiety 49–51 and depression-like behaviour52 in rodents,

and in some cases it does not53, and CB1 receptor antago-nists can produce neurochemical effects that are similarto those observed with antidepressant drugs (reviewedin REF. 54). Although anxiolytic and antidepressantdrugs used in the clinic are always taken as referencecompounds in these animal models, a note of caution isalways required when extrapolating the results of thesestudies — in which acute stressors are mostly used — tohuman anxiety and, particularly, depression, which areinstead chronic conditions. In this sense, the studies thatare probably more clinically relevant are those that sug-gest a protective function of CB

1 receptors against the

consequences of stress55,56 and in the adaptation to newstressful environmental conditions57, which both play amajor role in human affective disorders.

Pain and inflammation. Endocannabinoid levels inthe skin, spinal cord or peripheral nerves of rodents areusually elevated following treatment with irritant andinflammatory stimuli58. Endocannabinoid levels are alsoincreased in different models of neuropathic pain59–61

and following the development of allergic contact der-matitis62. These alterations probably represent adaptivereactions aimed at reducing pain and inflammation, assuggested by the fact that pharmacological or geneticinactivation of endocannabinoid degradative enzymesusually counteract pain and inflammation62–64. However,

depending on which of the tissues involved in pain trans-mission is analysed, decreases in endocannabinoid levelshave also been observed61,63. Emerging evidence alsosuggests that CB

1 and CB

2 receptor antagonists can exert

analgesic65,66 and anti-inflammatory 67,68 actions, possiblydue also to the fact that endocannabinoids can behave asboth pro- and anti-inflammatory mediators58,67.

Liver diseases and osteoporosis. These two types of disor-ders have been grouped together because both representtypical examples of how CB

1 and CB

2 receptors, targeted

by elevated endocannabinoid levels, can be detrimentaland beneficial, respectively 17,69.

Box 1 | Biosynthesis, action and inactivation of anandamide and 2-AG

Several pathways might exist for the formation and catabolism of anandamide and

2-arachidonoylglycerol (2-AG) (FIG. 1). Anandamide originates from a phospholipid

precursor,N-arachidonoyl-phosphatidyl-ethanolamine (NArPE), which is formed from

the N-arachidoylation of phosphatidylethanolamine via both Ca2+-sensitive and

Ca2+-insensitive N-acyltransferases (NATs). NArPE is then transformed into anandamide

by four possible alternative pathways, the most direct of which (that is, direct

conversion) is catalysed by a N-acyl-phosphatidylethanolamine-selectivephosphodiesterase (NAPE-PLD)4,199. Other fatty-acid ethanolamides that do not

necessarily bind to cannabinoid receptors with high affinity and act on different

receptors — for example, the anti-inflammatory compound palmitoylethanolamide

and the anorexic mediator oleoylethanolamide — can also be formed through these

pathways5. 2-AG, when serving as an endocannabinoid, is produced almost exclusively

by the hydrolysis of diacylglycerols (DAGs) via sn-1-selective DAG lipases (DAGLs)

α and β. DAGL-α is more abundant in adult nervous tissues, and DAGL-β is moreabundant in developing nervous tissues4. However, redundancy might exist regarding

the routes through which DAGs serving as 2-AG precursors are obtained, although

the one catalysed by phospholipase Cβ seems to be the most widely used4.After their cellular re-uptake, anandamide is metabolized via fatty acid amide

hydrolase (FAAH), and 2-AG via monoacylglycerol lipase (MAGL)4. 2-AG is also

metabolized to some extent by other recently identified lipases, theαβ-hydrolases 6(ABH6) and 12 (ABH12), as well as FAAH200. The cellular re-uptake mechanism of

anandamide and 2-AG is yet to be characterized and is still controversial; however, itappears to also mediate the release of de novo biosynthesized endocannabinoids164,165.

In FIG. 1, it is denoted as ‘endocannabinoid membrane transporter’ (EMT), even though

it might not necessarily be uniquely mediated by plasma-membrane proteins.

Both anandamide and 2-AG, possibly under conditions in which the activity of MAGL

or FAAH is suppressed, might become substrates for cyclooxygenase 2 (COX2) and

give rise to the corresponding hydroperoxy derivatives. The anandamide and 2-AG

hydroperoxy derivates can then be converted to prostaglandin ethanolamides

(prostamides) and prostaglandin glycerol esters, respectively, by various prostaglandin

synthases201. These metabolites are inactive at cannabinoid receptors but appear to act

at new binding sites, for which pharmacological, but no molecular, evidence exists201,202.

However, the physiological relevance of these pathways is still not fully understood.

Anandamide also interacts with several non-cannabinoid receptors150, the best

established of which is the transient receptor potential, vanilloid subtype 1 (TRPV1)

channel, to which the endocannabinoid binds at an intracellular site151. Recently,

anandamide and 2-AG were reported by some authors203, but not by others204,to activate GPR55, an orphan G-protein-coupled receptor. Evidence for interaction of

endocannabinoids with peroxisome-proliferator-activating receptors (PPARs)α and γ ,although at high concentrations, has also been recently reviewed205. However,cannabinoid 1 receptor (CB

1) and CB

2 are certainly the most-studied molecular targets

for anandamide and 2-AG, which activate them with different affinity. Anandamide has

the highest affinity in both cases, whereas 2-AG has the highest efficacy in both cases.

Importantly, in the brain CB1 receptors are often expressed in presynaptic terminals so

that endocannabinoids synthesized from postsynaptic neurons can travel backwards

(retrograde signalling) and inhibit neurotransmitter release3,4,7. Apart from cannabinoid

receptor antagonists, so far specific blockers have only been developed for FAAH,

MAGL, DAGLs and the putative EMT (see main text and REF. 206).

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TRPV1

NATs

Anandamide

Phosphatidic acidPhosphatidylethanolamine

Prostamide F2α Prostaglandin E2 glycerol ester

N-arachidonoyl-phosphatidylethanolamine

sn-1-arachidonate-containingphospholipid

sn-2-lysophosphatidic acid

sn-1-lysophospholipidArachidonoyl-CoA

2-AG

2-AG

NAPE-PLD

ABH4 × 2PLC sPLA2

lyso-PLD

Phospho-diesteraseGDE1

PTPN22

EMT EMTEMT EMT

Anandamide

FAAH MAGL, ABH6,

ABH12, FAAH

MAGL, ABH6,

ABH12, FAAH

FAAH

COX2

PGF2αsynthase

COX2

Phospholipid

PA phospho-hydrolase PLCβ PLA1

sn-1 DAG lipases Lyso-PLC

PGE2synthase

CB1, CB2, GPR55? CB1, CB2, GPR55?

Prostamideendoperoxide

Prostaglandin glycerolester endoperoxide

Arachidonate

+ ethanolamine

Arachidonate

+ glycerol

sn-2-arachidonate-containing diacylglycerol

Phosphoanandamide

2-lyso-N-arachi-donoyl-phosphatidyl-ethanolamide

Glycerophosphoanandamide

Extracellular

Figure 1 | Biosynthesis, action and inactivation of anandamide and 2-arachidonoylglycerol (2-AG):

new targets for drug development. The biosynthetic pathways for anandamide and 2-AG are shown in blue, degradative

pathways are shown in pink. Thick arrows denote movement or action. For more information please refer to BOX 1.

ABH4/6/12,αβ-hydrolase 4/6/12; CB1/2, cannabinoid receptor 1/2; COX2, cyclooxygenase 2; DAG, diacylglycerol;EMT, ‘endocannabinoid membrane transporter’; FAAH, fatty acid amide hydrolase; GDE1, glycerophosphodiester

phosphodiesterase 1; GPR55, G protein-coupled receptor 55; MAGL, monoacylglycerol lipase; NAPE-PLD, N-acyl-

phosphatidylethanolamine-selective phosphodiesterase; NATs, N-acyltransferases; PA, phosphatidic acid; (s)PLA1/2,

(soluble) phospholipase A1/2; PLC, phospholipase C; PLCβ, phospholipase Cβ; PLD, phospholipase D; PTPN22, proteintyrosine phosphatase, non-receptor type 22; TRPV1, transient receptor potential, vanilloid subtype 1 receptor.

Superoxide dismutase 1

(SOD1). One of the enzymes

that converts the superoxide

anion in oxygen and hydrogen

peroxide. Gain-of-function

mutations in the Cu,Zn-SOD1

gene are implicated in

progressive motor neuron

death and paralysis in one

form of inherited amyotrophiclateral sclerosis.

Conditioned fear

An animal defensive behaviour

(for example, immobility or

‘freezing’) that is induced by

exposure to aversive stimuli

(for example, a non-noxious

electrical shock) coupled to a

non-aversive one (for example,

a light or an acoustic tone).

This behaviour can later

be reinstated by simply

re-exposing the animal to

the non-aversive stimulus.

Several hepatic pathological conditions, such asnon-alcoholic steatosis70,71, fibrosis72,73 and ischaemia/reperfusion injury 74, cause a chronic upregulation ofendocannabinoid levels. The subsequent activation of CB

1

receptors then contributes to the symptoms (for example,ectopic fat formation, fibrogenesis, hepatocyte death)70,73.

By contrast, activation of CB2 receptors, which maybecome overexpressed during these disorders, oftencounteracts these effects71,72,74. In the case of osteoporosis,evidence is accumulating for a role of CB

2 receptors in

enhancing endocortical osteoblast number and activity,and restraining trabecular ostoeoclast formation75, whichwould together reinforce bone structure. By contrast,CB

1 receptors reduce bone mass and contribute to

ovariectomy-induced bone loss in mice76. However, a largepart of the available data on the role of cannabinoid recep-tors in bone formation was obtained in CB

1 or CB

2 receptor

knockout mice with different genetic backgrounds, whichhas given rise to some discrepant results75.

Cancer and gastrointestinal inf lammation. Recentdata indicate a protective function of upregulatedendocannabinoid levels in cancer versus non-trans-formed cells, and in gastrointestinal tissues duringexperimental colitis15,16,77,78. Both CB

1 and CB

2 agonists

were found to counteract carcinogenesis79, and the

growth and invasiveness of several types of cancer cells,by modulating processes ranging from mitosis andapoptosis to angiogenesis, cancer-cell migration andmetastasis15,77. However, examples of pro-proliferativeeffects of endocannabinoids have also been reported,although in most cases they do not seem to be mediatedby cannabinoid receptors. Indeed, the CB

1 antagonist

rimonabant was also shown to inhibit cancer-cell pro-liferation by as yet unidentified molecular targets80. Ingastrointestinal inflammation, endocannabinoids exerta homeostatic function via CB

1 and CB

2 receptors at the

level of visceral perception, gut motility, inflammationand endothelial damage78,81,82. However, rimonabant

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a Periaqueductal grey

Glutamate GABA

Antinociception

c Prefrontal cortex

Glutamate GABA

Anxiety

d Cortex

Striatum

SnR GPe

GABA

Glutamate

Directpathway

Indirectpathway

Glutamate

GABA

Locomotion

b Hippocampus

Glutamate GABA

Excitotoxicity

Acetylcholine

Memory

TRPV1 CB1

CB1 CB1

CB1 CB1

CB1TRPV1

CB1 CB1

CB1

CB1 CB1

Non-alcoholic steatosis

Also known as non-alcoholic

fatty liver disease, this is the

inflammatory accumulation of

fat in the liver when this is not

due to excessive alcohol use.

It is related to insulin resistance.

Osteoblasts and osteoclasts

Osteoblasts are mononucleate

cells that are responsible

for bone formation. They

produce osteoid, which is

composed mainly of type I

collagen, and are responsible

for mineralization of the

osteoid matrix. Bones are

constantly being reshaped by

osteoblasts, which build bone,

for example in its endocortical

region, and osteoclasts, which

resorb bone, for example in its

trabecular region.

Figure 2 | Examples of the physiological roles of endocannabinoids and of the potential consequences

of their pathological dysregulation in central neurons. Endocannabinoids are normally produced to act onlyon a selected population of neurons, usually by being released only from certain postsynaptic neurons to inhibitneurotransmitter release from given presynaptic cannabinoid 1 receptor (CB

1)-expressing neurons3,4,7. Under

pathological conditions, such as acute nociception, excitotoxicity and anxiety, endocannabinoids might act solely

on GABA (γ -aminobutyric acid)ergic CB1-expressing interneurons of the periaqueductal grey (a), in the former case85,

or on glutamatergic CB1-expressing terminals of hippocampal (b) and prefrontal cortex principal neurons (c), in the

latter two cases45,50,84,134. This causes descending analgesia, neuroprotection and reduced anxiety, respectively. In thesecases, enhancers of endocannabinoid action produce beneficial effects. However, with prolonged pathological stimuli,

endocannabinoid action might spread to glutamatergic or GABAergic terminals, respectively, thereby producingopposite and undesired effects on the disorder. When this occurs, CB

1 antagonists might produce beneficial effects.

In the hippocampus (b), a similar switch towards activation of CB1

receptors on cholinergic terminals might contributeto memory retention loss during Alzheimer’s disease32,33. During Parkinson’s disease (PD) (d), endocannabinoids might

be initially produced by neurons postsynaptic to GABAergic medium spiny neurons (MSNs) of the ‘indirect’ pathway(the former of which are located in the external layer of the globus pallidus (GPe)) to counteract GABA release. They mightalso be produced from these MSNs to retrogradely counteract glutamate release from upstream corticostriatal terminals.This would help restoring locomotion in both cases11. In an advanced phase of the disorder, endocannabinoids might

spread also to the GABAergic MSNs of the ‘direct’ pathway, whose output terminals are located in the substantia nigrareticulata (SnR). This second subset of MSNs are often very near to the aforementioned neurons of the ‘indirect pathway’,and are also innervated by upstream corticostriatal terminals. However, unlike neurons of the indirect pathway, thesedirect pathway MSNs are coupled to initiation of locomotion. Thus, spreading of endocannabinoid retrograde inhibitory

action to these neurons would produce effects opposite to those mentioned above for the indirect pathway, andcontribute to locomotor impairment11. Thus, inhibitors of endocannabinoid degradation might be beneficial in the earlyphase of Parkinson’s disease, whereas CB

1 antagonists might be beneficial in the late phases of this disease. In some

brain areas (a,c), activation of transient receptor potential, vanilloid subtype 1 (TRPV1) channels by anandamide might

occur and produce effects on glutamate release opposite to those of CB1.

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Direct and indirect pathways

of locomotor control

Neuronal circuitries in the

basal ganglia involving medium

spiny GABA (γ -aminobutyric

acid)ergic neurons of the dorsal

striatum terminating onto

other GABAergic neurons in

either the substantia nigra

reticulata or external layer

of the globus pallidus, andultimately causing stimulation

or inhibition of locomotion,

respectively.

TRPV1

A six-transmembrane-

domain non-selective cation

channel that is activated by

either physical or chemical

stimuli. Stimuli include

thermosensation, sensory

transduction, taste, flow-

sensing, and the detection

of obnoxious and irritant

compounds.

was also found to exert paradoxical protective effects via both CB

1-mediated and CB

1-independent effects in

gastrointestinal inflammation83.

Summary. The above examples indicate how, within itsgeneral strategy of action of a local modulatory system,endocannabinoid signalling can undergo several types ofchanges during pathological conditions. Initial endocan-nabinoid responses to acute stressful or noxious stimulihelp to restore homeostasis with tight time-specific andspace-specific restrictions. By contrast, the continuationof the pathological state dysregulates this system in sucha way that endocannabinoids act for a longer time, orstart activating the same receptors, or even differentreceptor types, on cell populations that they were notinitially meant to target.

In neurodegenerative disorders (FIG. 2), endocannabi-noids might be upregulated first to target only glutama-tergic CB

1-expressing cells84 and to reduce excitotoxicity,

but then go on to act on neighbouring GABA (γ -amino-butyric acid)ergic neurons (which seem to require higher

concentrations of CB1 receptor ligands85), thus causingthe opposite effects. This might also be true for anxietyand supraspinal descending antinociception, in whichevidence exists for opposing effects of CB

1 receptor acti-

vation in glutamatergic (anxiolytic/pro-nociceptive) orGABAergic (anxiogenic/antinociceptive) terminals8,50,85.In the basal ganglia (FIG. 2), even a slightly spatially dys-regulated endocannabinoid signal might have dramati-cally different outcomes on nearby GABAergic afferentneurons of the ‘direct’ or ‘indirect’ pathways of locomotorcontrol, with opposing effects on movement11. Activationof CB

2 receptors in the brain that are not strongly

expressed under normal conditions but are upregulatedin microglial and glial cells during neuroinflammatoryconditions, might cause both anti-inflammatory effects,by reducing cytokine release, and pro-inflammatoryactions, by recruiting more immune-competent cellsfrom the blood when the blood–brain barrier becomesdisrupted86,87 (FIG. 3). Also, during conditions of peripheralhyper-reactivity, these two effects of endocannabinoidsmight act in opposing ways on the outcome of the inflam-matory response, and the enhancement of CB

2 receptor-

mediated chemotaxis might produce opposing effectsdepending on whether it occurs proximally or distally tothe site of inflammation (FIG. 4).

Conversely, in the liver, elevated endocannabinoidsduring fibrosis might first attempt to counteract the

growth of fibrogenic cells via CB2 receptors72 and thenend up contributing to this process via CB

1 receptors73.

Finally, when the more promiscuous of the two most-studied endocannabinoids, anandamide, is upregulated,other receptor types might also come into play. Thetransient receptor potential, vanilloid subtype 1 (TRPV1)receptor is activated by concentrations of anandamidehigher than those necessary to activate CB

1 receptors.

Yet, this receptor can be sensitized by several noxiousand inflammatory stimuli, and participates in someof the pernicious effects of anandamide, for exampleduring anxiogenic51,88, inflammatory 89,90 or noxioushypotensive91–93 states and neuropathic pain94. Evidence

is emerging for the sensitizing effects of CB1 on TRPV1

receptors95,96 and this might underlie some of the para-doxical neuroprotective and analgesic/anti-inflamma-tory actions observed following chronic administrationof CB

1 receptor blockers, although other mechanisms,

such as adaptive responses to prolonged CB1 receptor

inactivation, might also explain these effects.In summary, it might be difficult to predict whether

indirect agonists or antagonists of the endocannabinoidsystem will be beneficial for a given condition. There existto date only a few clear-cut cases in which the exclusiveuse of either an enhancer or a reducer of endocannabi-noid action can be recommended for future clinical trials.More often than not, studies in as many animal models aspossible, and then in the clinic —especially with animalsand patients that are at different stages of the disorder— are required to provide a conclusive answer.

Therapeutic use of indirect agonists

Inhibitors of catabolism. At least two enzymes thatcatalyse the degradation of anandamide and 2-AG

(BOX 1; FIG.1) have been cloned: FAAH, which recognizesboth compounds as substrates but is more selectivetowards anandamide, and the monoacylglycerol lipases(MAGLs), which are specific for 2-AG. Selective andpotent inhibitors of FAAH only have been developed,and tested in vivo in several animal models of disease sofar (TABLE 1). Three FAAH blockers in particular seempromising for future clinical development: URB-597 (REF. 97), arachidonoylserotonin (AA-5-HT)98 and SA-72(REFS 99,100).

URB-597 is an irreversible inhibitor, still at the pre-clinical stage, with anxiety, depression and pain as themost likely therapeutic targets101. It has potent analgesicactivity in models of neuropathic pain when adminis-tered orally 102, and it has proved efficacious followingsystemic administration in models of inflammatorypain103 and inflammation104. However, high oral doses ofthis compound (10–50 mg per kg) are required to inhibitneuropathic pain102, whereas lower intraperitoneal dosesare not effective against this type of pain103. Importantly,the effects of oral102, but not intraplantar64, URB-597against signs of neuropathic pain are accompanied by ele-

vation of endocannabinoid levels in some of the tissuesinvolved in nociception. Other effects of URB-597administration include reduction of: blood pressure inspontaneously hypertensive rats105; retinal damage afterhigh intraocular pressure-induced ischaemia in rats106;

morphine-6-glucuronide-induced emesis in ferrets107 and lithium-induced nausea in rats108. In addition,reduction of locomotor impairment in a PD model29 and of anxiety- and depression-like signs in animalmodels50,97,109 have been observed. Importantly, and asexpected from the mechanism of action of indirect ago-nists, URB-597 in not self-administered in animals110–112 and has no general reinforcing action on drugs of abuseexcept for the increase of alcohol self-administration inrats113,114. Interestingly, URB-597 is effective at increasinganandamide levels in the brain, but not in the gut andliver115, thus possibly preventing its application in gastro-intestinal and hepatic disorders.

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|

Neuronalinflammationand toxicity

CB2

CB2

CB2

CB2

CB2

CB2

CB2

CB2

CB2

CB2

Microglia

Macrophage

Neuron

B cell

NK cell

Blood–brain barrier

Theiler’s virus

Theiler’s murine encephalo-

myelitis virus (TMEV) is

a single-stranded RNA

picornavirus that persistently

infects the mouse central

nervous system, recently

reclassified into the cardiovirus

group. In the wild it produces

a gastrointestinal infection

that may be complicated by

concomitant infection of the

nervous system.

AA-5-HT, on the other hand, increases gut endocan-nabinoid concentrations, at least following systemicadministration116, and, accordingly, was effective in ananimal model of ulcerative colitis82 and against azoxy-methane-induced precancerous lesions in the mousecolon79. In both cases, colon endocannabinoid levels werealso increased. Furthermore, intratumour administrationof AA-5-HT reduces cancer cell growth in vivo in a modelof thyroid carcinoma117, inhibits both acute peripheraland chronic neuropathic pain in rats and mice 63, and,like URB-597, potentiates stress-induced analgesia118 and attenuates hyperlocomotion in an animal model ofhyperdopaminergia119. Interestingly, despite being lesspotent as a FAAH inhibitor in vitro98, AA-5-HT oftenexhibits analgesic efficacy similar to other inhibitors, aproperty that might be attributed to its capability to alsoantagonize TRPV1 receptors (see below)63.

Finally, less is known about SA-47, which, together

with SA-72, belongs to a series of carbamate FAAHinhibitors described in patents held by Sanofi–Aventis99.Its very high selectivity 100, however, prompts its testingin clinical trials.

Inhibitors of cellular reuptake. Although the mechanismof endocannabinoid cellular reuptake has not yet beencharacterized from a molecular point of view and is stillcontroversial, increasingly selective inhibitors of thisprocess are being developed and found to be beneficialin several preclinical studies (TABLE 2). These compoundscan be divided into two classes: aromatic acylamidederivatives and carbamoyl-tetrazoles.

Aromatic acylamide derivatives include the proto-typical AM404 (REF. 120), from which VDM-11 (REF. 121),OMDM-1 and 2 (REF. 122), UCM-707 (REF. 123) andAM1172 (REF. 124) were later developed. Carbamoyl-tetrazoles are developed by Eli Lilly, of which LY2183240is the most potent in vitro125,126. Apart from LY2183240,which, however, is also a very potent FAAH inhibitor(see below), all uptake inhibitors developed so far exhibitlow potency in vitro (0.5–10 µM). Clearly, the full char-acterization of the putative protein(s) responsible forendocannabinoid reuptake will allow the design anddevelopment of more potent and selective blockers ofthis process.

The potential therapeutic targets for reuptake inhibi-tors can be predicted from animal studies carried outwith the above compounds, which were administeredsystemically. These studies showed beneficial actions inneuropathic and inflammatory pain in rodents125,127–129;

conditioned fear130, stress-induced corticosterone release47 and anxiety- or depression-like signs109,131–133 in rodents;

glutamate-mediated excitotoxicity 134–136, experimentalPD29,137 and β-amyloid-induced neurotoxicity and mem-ory retention loss in rodents32. Beneficial effects havealso been demonstrated in stress-induced suppressionof hippocampal cell proliferation138; low motor perform-ance and spasticity, respectively, in rats and mice withEAE37,139–142, but also neuroinflammation and demyeliniza-tion in Theiler’s virus-induced EAE143; thyroid epitheliomagrowth in athymic mice117; experimental colitis in mice79;hypertension in spontaneously hypertensive rats105; highintraocular pressure in rabbits144; and emesis in ferrets and

Figure 3 | Physiological roles of endocannabinoids, and potential consequences of their dysregulation during

central neuroinflammation. Following the initial phases of several neurodegenerative disorders, cannabinoid 2 (CB2

)

receptors are expressed in microglial cells, which become activated and start counteracting neuronal damage. As the

disorder progresses, the blood–brain barrier becomes partly disrupted and blood macrophages, B lymphocytes and

natural killer (NK) cells start expressing CB2 receptors. The activation of these receptors stimulates the migration of these

cells into the nervous tissue and towards the endocannabinoids produced by both microglial and neuronal cells, thereby

initiating a neuroinflammatory response and causing gliosis, exaggerated microglial activity and neuronal death62,63,67,86,87.

Thus, both CB2 agonists and antagonists might be beneficial in counteracting the inflammatory consequences of

neurodegenerative disorders depending on the disease phase.

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|

Oedema,itch or pain

↑CB2

↑CB2↑CB1 Histaminecytokines

Keratinocytedamage

CGRP,substance P,NGF

Small vesselplasmaextravasation

Sensoryneuronexcitation

Neuropathy,inflammation

Neuron

Macrophage

Mast cell

↑TRPV1 ↑CB1

Least shrews107,145. As in the case of URB-597, OMDM-2and AM404 do not exhibit drug-reinforcing effects innormal rodents, and hence are not likely to inducedependence110,132,146. However, the concomitant effectof uptake inhibitors on endocannabinoid levels, or theintermediacy of CB

1 or CB

2 receptors in their effects,

were investigated in only a few of the above studies(TABLES 1,2). This leaves open the possibility that some ofthese compounds might also act via mechanisms that aredifferent from those hypothesized. Furthermore, as mostof the inhibitors used in these studies inhibit FAAH athigh concentrations, even in those cases in which theirenhancing effect on tissue endocannabinoid levels was

demonstated32,79,105,107,117,145, it is not always clear whetherthis effect is due to inhibition of the cellular uptake ofendocannabinoids alone or also of their catabolism.

Potential disadvantages of indirect agonists. MostFAAH inhibitors (AA-5-HT being perhaps the onlyexception) have been designed from the chemical modi-fication of known classes of serine hydrolase inhibitors,and therefore are likely to have off-target effects. Rapidand accurate proteomic approaches have been devel-oped to identify possible off-targets for FAAH andendocannabinoid reuptake inhibitors. They are basedon activity-based protein profiling, which uses active

site-directed chemical probes and allows the screeningof inhibitors against multiple enzymes in parallel. Thesemethods showed that URB-597 and other FAAH inhibi-tors (OL-135, BMS-1), as well as the inhibitor of theputative endocannabinoid transporter LY2183240, butnot SA-47 and PF-750 (TABLE 1), act on other carboxyl-esterases100,147–149. However, even when extremely selec-tive, FAAH inhibitors are likely to prolong the lifespanalso of non-endocannabinoid substrates of this promis-cuous enzyme, including fatty acid ethanolamides suchas oleoylethanolamide and palmitoylethanolamide,which act on non-cannabinoid receptors (BOX 1). Tocomplicate things further, the increase of anandamide

levels induced by FAAH inhibitors might result in theactivation of non-cannabinoid receptor targets for thiscompound150 and in particular of the TRPV1 recep-tor151. This has been demonstrated so far in animalmodels of PD152,153; in the periaqueductal grey, follow-ing direct injection of the inhibitor in this area, withsubsequent influence on descending antinociception85;and in mice being tested for anxiety-like behaviours154.As TRPV1 stimulation triggers intracellular events thatare opposite to those produced by CB

1 receptor activa-

tion, selective FAAH inhibitors might cause effects thatare less efficacious than expected, as recently observedwith URB-597 in models of anxiety-like behaviour53 and

Figure 4 | Physiological roles of endocannabinoids and potential consequences of their dysregulation during

peripheral neuroinflammation. At the onset of neuropathic conditions or immune reactions against external agents,

cannabinoid 1 (CB1) receptors and transient receptor potential, vanilloid subtype 1 receptor (TRPV1) channels are

present in sensory neurons of the dorsal root ganglia, and CB2 receptors in eosinophils, mast cells and monocytes87.

Early activation of CB1 receptors counteracts the release of inflammatory and algesic peptides such as, nerve growth

factor (NGF), substance P and calcitonin gene-related peptide (CGRP). These would normally orchestrate the

inflammatory response together with inflammatory cytokines, whose release from immune cells is inhibited by CB2

receptors. However, in a later stage, CB2 and TRPV1 receptors are strongly upregulated, and 2-arachidonoylglycerol

(2-AG) and anandamide might start enhancing the migration of eosinophils, mast cells and macrophages and

stimulating substance P and CGRP release, via these two receptors, respectively. This leads to neurogenic

inflammation, plasma extravasation and keratinocyte damage, and eventually to oedema, itch and pain, which can

be counteracted by TRPV1 and CB2 antagonists86,151. By contrast, CB

1 agonists, endocannabinoid hydrolysis inhibitors

and, in some cases, CB1 antagonists (possibly via indirect desensitization of TRPV1 receptors) might be effective in

both the initial and late phase of these conditions.

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Table 1 | Endocannabinoid indirect agonists: inhibitors of FAAH and MAGL

Compound (FAAH/MAGL inhibitor) Potency invitro*

Off-targets‡ Effective doses§ Potential indications tested inanimal models

O

H2N O

O

HN

URB-597 (FAAH)

• IC50

of109–113 nMby ABPP100,148

• IC50

of 3–5 nM

by enzymaticassaywith pre-incubation101

• Carboxylesteraseinhibition100,148,208

in the liverwith IC

50 of

210–1,620 nM100 • TRPA1 channels55

• 0.15–0.3 mgper kg101

• 1–50 mgper kg by oral

administration102

• Reduces neuropathic pain aftersystemic and oral administration102

• Inflammation104 • Blood pressure in spontaneously

hypertensive rats105• Glaucoma106

• Emesis107-108

• Locomotor impairment in a PD29

• Anxiety- and depression-likesigns in animal models52,97

O

HN

OH

NH

CH3

AA-5-HT (FAAH)

• IC50

of1–10µM byenzymaticassay,with no pre-incubation98,208

• Antagonisticactivity on TRPV1with IC

50 of

37–40 nM63

• 0.3–5 mg per kg • Colitis82 • Colon carcinogenesis79 • Neuropathic pain63 • Hyperactivity in

hyperdopaminergia119

O

O

N

NOL-135 (FAAH)

• IC50

of 2.1 nMby ABPP148

• Carboxylesteraseinhibition100,207

• 1.7–9.0 mgper kg148,207

• Neuropathic pain209

N

HN O

N O

F

BMS-1 (FAAH)

• IC50

of 200 nMby ABPP148

• IC50

of 2 nMby enzymaticassay with pre-

incubation210

• Carboxylesteraseinhibition 100

• 20 mg per kg210 • Neuropathic pain210

N NNH

O

OHN

O

SA-47 (FAAH)

• IC50

of5–1,000 nMby enzymaticassaywith pre-incubation99

• None so far100 • 2 mg per kg • Analgesia99

N

HN

O

N

PF-750 (FAAH)

• IC50

of16.2–595 nMby enzymaticassay anddepending on

pre-incubationtime207

• None so far207 • No study so far • No study so far

HN O

O

URB-602 (MAGL)

• IC50

of 28µMby enzymaticassay45

• FAAH211 • 5–10 mgper kg157

• Inflammation157

*Potency in vitro, expressed here as the concentration necessary to exert half-maximal inhibition of anandamide or 2-arachidonoylglycerol hydrolysis (IC50

).Values vary according to the assay conditions, the animal species and the type of tissue or cells used to prepare the enzyme. ‡All types of off-targets identifiedso far are listed, even though the inhibitor might interact with them at concentrations higher than those required to inhibit fatty acid amide hydrolase (FAAH) ormonoacylglycerol (MAGL). §Systemic (generally intraperitoneal) doses are shown unless otherwise stated. ABPP, activity-based protein profiling; PD, Parkinson’sdisease; TRPA1, transient receptor potential, ankyrin-like type 1; TRPV1, transient receptor potential, vanilloid receptor subtype 1.

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PD153. Instead, in the case of supraspinal antinociceptionand emesis, the capability of URB-597 to also indirectlyactivate TRPV1 receptors seems to afford higher effi-cacy 85,107. AA-5-HT was recently found to also antagonizeTRPV1 receptors, a property that might explain the highefficacy of this compound against neuropathic pain and

anxiety 63,154, and thus prompt the development of furthertherapeutically useful dual FAAH/TRPV1 blockers.High concentrations of URB-597, instead, were foundto activate TRPA1 channels155, which are also involvedin pain transduction, and this property might affect theanalgesic efficacy of high doses of this compound.

Table 2 | Endocannabinoid indirect agonists: inhibitors of endocannabinoid cellular uptake

Compound Potency invitro*

Off-targets‡ Effective doses§ Potential indicationstested in animalmodels

NH

OOH

AM-404

IC50

of1–8 µM120-121

• TRPV1 receptor activation121,161 • Fatty acid amide hydrolase158 • CB

1 receptors120

5–10 mg per kg • Neuropathic pain127-129

• Anxiety132

• Hypolocomotion inParkinson’s disease137

• Glaucoma144

NH

OOH

VDM-11

IC50

of5–10µM121,158

• Maybe fatty acid amidehydrolase120,158

5–10 mg per kg • Thyroid carcinoma117

• Colitis82

• β-amyloid-inducedneurotoxicity32

NH

O

OH

OH

OMDM-1/OMDM-2

IC50

of2.6–5.0µM122,158

• None so far122,158 5–10 mg per kg • Multiple sclerosisprogress43

• Spasticity in multiplesclerosis141

NH

O

O

UCM-707

IC50

of0.8–30µM123,158

• Fatty acid amide hydrolase158 • CB

2 receptors123

5–10 mg per kg • Hyperlocomotion inHuntington’s disease142

• Spasticity in multiplesclerosis142

HN

O

HO

AM1172

IC50

of2.5–24µM124,158

• CB1 and CB

2 receptors124 Not tested yet • Not tested yet

N

N

N

N

O

N

LY-2183240

IC50

of0.27 nM125 orIC

50 of 15 nM126

• Carboxylesterase inhibition100

• Fatty acid amide hydrolase andmonoacylglycerol lipase149

• Diacylglycerol lipase126

3–30 mg per kg125 • Analgesia125

Cl OH

R = or

OH

N

O

R

R

O-3246 O-3262

IC50

of1.4µM140 orIC

50 of

2.8µM140

• Not tested 1–30 mg per kg140 • Spasticity in multiplesclerosis140

*Potency in vitro, expressed here as the concentration necessary to exert half-maximal inhibition of anandamide uptake (IC50

). Values vary according to the assayconditions and the type of cells used to study the cellular uptake of radiolabelled anandamide. ‡Only off-targets for which the inhibitor interacts at concentrationscomparable with those required to inhibit the cellular uptake of anandamide are listed here. §Systemic (generally intraperitoneal) doses are shown unless otherwisestated. CB

1/2, cannabinoid receptor 1/2; TRPV1, transient receptor potential vanilloid receptor subtype 1.

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As the MAGLs, unlike FAAH, only recognize 2-AGas a substrate, and this compound, unlike anandamide,activates cannabinoid receptors selectively over othertargets, inhibitors of these enzymes should produce amore specific indirect activation of these receptors.Although, to date, no specific and potent MAGL inhibi-tor suitable for use in vivo has been developed, the benefi-cial effects observed with the relatively weak URB-602(REF. 48) against inflammatory pain156,157 raise optimisticexpectations also in this direction.

Although they inhibit the inactivation of endocannabi-noids selectively versus other FAAH substrates158, uptakeinhibitors also have off-target effects. All such compoundsdescribed so far in the literature, except for OMDM-1 andOMDM-2 (REF. 122) and O-3246 and O-3262 (REF. 140),also inhibit FAAH at concentrations that, depending onthe assay conditions, might be similar to those necessaryto inhibit anandamide reuptake158,159. AM404, which isalso a product of the in vivo metabolism of paracetamol160,potently activates TRPV1 receptors121,161. Non-endocan-nabinoid-related in vitro effects of VDM-11 and AM404

were also reported162,163. Furthermore, evidence existsdemonstrating that uptake inhibitors also block endocan-nabinoid release from cells164,165. Therefore, the effect ofthese compounds, particularly if they are administeredbefore the disease-induced biosynthesis and release ofprotective endocannabinoids, might be to reduce endo-cannabinoid signalling rather than enhancing it. Thetiming of administration was, although for differentreasons, a crucial issue in a study of VDM-11. This com-pound, in order to be effective against β-amyloid-inducedneurotoxicity and loss of memory retention, had to beadministered within 3 days from the insult, whereas, ifadministered later, it worsened the effect ofβ-amyloid32.

One further possible problem with indirect agonistsis represented by the high degree of redundancy of path-ways and enzymes through which endocannabinoids areinactivated4,5 (BOX 1; FIG. 1). However, because of theirnature as local mediators, it is predicted that endocan-nabinoids are biosynthesized and degraded via cell-specific pathways, and that different enzymes come intoplay only in different tissues and organs, thus making itdifficult for alternative pathways to compensate for thosethat have been inhibited. This might explain why FAAHand uptake inhibitors have proved to be effective in somany animal models of diseases.

Therapeutic use of inverse agonists/antagonists

CB1 receptor antagonists. Several CB1 receptor antago-nists have been developed to be used therapeutically andare already being tested in several clinical trials (TABLE 3).The first such compound, rimonabant, successfullycompleted four Phase III trials166–169, and is currently onthe market in the European Union and in several othercountries. Rimonabant is used as an aid to diet and exer-cise for body-weight reduction in obese patients (bodymass index >30), or to reduce associated risks (such asdyslipidaemia and type 2 diabetes) in overweight patients(body mass index >27). It also improves the odds of suc-cess in individuals who are willing to quit smoking170,and is currently being tested in no less than 15 Phase IIIb

and IV clinical trials (as determined from searches inClinicalTrials.gov database) aimed at directly assessing itsefficacy against atherogenic dyslipidaemia, cardiometa-bolic risk factors, cardiovascular events and type 2 dia-betes. These ongoing trials are based on recent evidenceshowing that CB

1 stimulation enhances lipogenesis and

inhibits glucose and fatty-acid oxidation by acting at thelevel of adipocytes, hepatocytes, endocrine pancreas andskeletal muscle21,171,172. Moreover, an overactive endocan-nabinoid system in the visceral adipose tissue, liver andpancreas might contribute to glucose intolerance anddyslipidaemia in a direct way and hence independentlyfrom its effects on food intake and weight gain21,171,172.Accordingly, in subsets of treated and untreated obesepatients that lost the same amount of weight, rimonabantalways reduced the plasma levels of glycated haemoglobin(a marker of type 2 diabetes), and increased those of adi-ponectin (an insulin-sensitizing adipokine) significantlymore than placebo167,173. Subsequently, rimonabant andother CB

1 antagonists were found to stimulate lipid oxi-

dation, glucose clearance and energy expenditure, and

hence to ameliorate the metabolic consequences of obesityin lean and obese rodents, also independently from theireffects on food intake174–176.

Other CB1 antagonists that are being tested against

obesity and related co-morbidities include tarana-bant (developed by Merck 177), for which the resultsof a 12-week weight-loss Phase II trial have just beenpublished178, and which is soon due to complete aPhase III trial. Taranabant is also being tested forsmoking cessation and for weight maintenance afterweight loss (TABLE 3). Additional CB

1 antagonists in the

pipeline include CP-945,598 (otenabant) and BMS-646256, developed by Pfizer and Bristol–Myers Squibb,respectively, as anti-obesity drugs; and surinabant andAVE1625, also developed by Sanofi–Aventis176,179.BMS-646256 and AVE1625 are in clinical trials forthe treatment of nicotine-dependence and/or obesity;AVE1625 is also being tested in patients with mild tomoderate AD, as well as against cognitive impairmentin schizophrenia (TABLE 3). Indeed, as outlined above,and apart from AD32 and schizophrenia180,181, thereare several other pathological conditions in whichelevated endocannabinoid levels acting at CB

1 receptors

might contribute to disorder symptoms and progress.Accordingly, thus far limited to animal models, variousCB

1 antagonists were found to ameliorate locomotion,

alone or together with -DOPA, and/or to improve

-DOPA-induced dyskinesia, in rodent and primatePD models25,27,30,31,182; improve memory retention inβ-amyloid-treated mice33; reduce both alcohol andnicotine self-administration183,184 and reinstatement ofheroin185 and cocaine186 self-administration in rats. Inaddition, CB

1 antagonists also produce antidepressant-

like effects in rodents187,188; inhibit inflammatory painand inflammation189; prevent bone loss in a mousemodel of osteoporosis76; retard progression of liverfibrosis in mice73; reduce hypotension and cardiopathyin cirrhosis190–192; and inhibit breast cancer cell growthin vivo80. Therefore, preclinical bases exist now to testCB

1 receptor antagonists in clinical trials targeting at

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Table 3 | CB1 receptor antagonists/inverse agonists in clinical trials*

Compound Affinity orpotencyin vitro

‡

Effective dosesin animals§

Potential indications tested inanimal models

Most important clinicaltrial results

N NH

O

N N

ClCl

Cl

Rimonabant

K i of 2 nM

in bindingassays179

0.3–10 mg per kg • Reduces body weight,dyslipidaemia, hyperglycaemiaand steatohepatitis, preservesrenal function70,174–176

• Nicotine and alchol abuse,relapse of heroin and cocaineabuse183–186

• Hypotension, cardiopathies,encephalopathy and liver fibrosisin cirrhosis73,190,192,213

• Parkinson’s disease25,27,30–31

• Alzheimer’s disease33

• Osteoporosis76

• Paradoxical beneficial effects ininflammatory and neuropathicpain, anxiety and depression187–189

• Five Phase III trialscompleted; at 20 mg perday it reduces body weightand cardiovascular riskfactors in overweight/obese patients with orwithout dyslipidaemiaand with or without type 2diabetes166–169

• Increases the oddsof quitting cigarettesmoking170

• Adverse events leading todiscontinuation includetransient diarrhoea,dizzines, nausea, signs ofdepression and anxiety

N NH

O

N N

ClCl

Br

Surinabant

K iof 0.56 nM

in binding

assays

179

3.8 mg per kg • Reduces ethanol or sucroseconsumption in mice and rats,

and food intake in fasted andnon-deprived rats179

• Has an anti-alcohol profilein selectively bred Sardinianalcohol-preferring rats184

• Phase II, smoking cessation(NCT00432575)||

• Phase II, obesity

178

(NCT00239174)||

• Results not yet disclosed• Adverse events not yet

disclosed

AVE-1625

Cl

N

N Cl

S O

C

F

F

IC50

of 25nM in afunctionalassay176

3–30 mg perkg by oraladministration176

• Induces lipolysis from fat tissueand glycogenolysis from the liverwhen acutely administered toWistar rats176

• Three ongoing Phase IItrials, for the treatmentof obesity withatherogenic dyslipidaemia(NCT00345410)||,Alzheimer’s disease(NCT00380302)|| andcognitive impairmenent(NCT00439634)||

• Results not yet disclosed• Adverse events not yet

disclosed

Taranabant

NNH

Cl

O

O

N

F

F

F

K i of 0.13 nM

in bindingassays177

0.3–1 mg perkg177

• Produces significant weightloss in rats with diet-inducedobesity177

• Phase III trial ongoingfor the reduction ofbody weight in obesepatients (NCT00131391)||,and smoking cessation(NCT00109135)|| • Phase II trial in obesityreduced dose-dependentlybody weight• Adverse events leadingto discontinuation includegastrointestinal andpsychiatric effects178

N

N

N N

N

Cl

Cl

NH

NH2

O

Otenabant

Not yetdisclosed

Not yet disclosed – • One Phase II studycompleted, in comparisonwith sibutramine( NCT00134199)||

• Reduces body weight andcardiovascular risk factorsin overweight/obesepatients

• Adverse events not yetdisclosed

*A Phase II clinical trial with the CB1 antagonist/inverse agonist BMS-646256 has not been completed yet (NCT00388609). ‡Affinity, usually expressed here as

affinity constant (K i). Values vary according to the assay conditions and the type of cells used to study and the animal species. The K

i for cannabinoid receptor 2

(CB2) is not shown, and is usually at least 50-fold higher than the K

ivalues shown here. ‡Systemic (generally intraperitoneal) doses are shown unless otherwise

stated. ||Identifying code of trial from ClincalTrials.gov database (http://clinicaltrials.gov/ct2/home ).

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least some of these disorders. However, although thesecompounds exhibit an overall relatively safe profileof side effects, some concern is raised by the fact thattheir chronic use in obese patients is associated withincreased risk of developing signs of depression173,178.

CB2 receptor antagonists. Several CB

2 antagonists/

inverse agonists have also been developed (TABLE 4). Thefollowing compounds in particular received attentionby virtue of their anti-inflammatory effects: JTE-907(REF. 193) SR144528 (REF. 194), and Sch.336, Sch.036 and

Sch.414319 (REFS 67,86). Iwamura et al .193 showed thatoral administration of JTE-907 and SR144528 inhibitcarrageenin-induced paw oedema in mice. The samegroup also reported that orally administered JTE-907and SR144528 significantly inhibit dinitrofluoroben-zene-induced ear swelling195. SR144528 blocked 12-O-tetradecanoylphorbol-13-acetate-induced ear swelling,while decreasing leukotriene B4 production and neu-trophil infiltration into the ear196. Ear swelling was alsosuppressed by administration of SR144528 in oxazolone-induced contact dermatitis58. This CB

2 antagonist also

suppressed pro-inflammatory cytokine mRNA expres-sion and attenuated eosinophil recruitment into the

treated ear, which suggests that CB2 antagonists have

high potential against allergic states such as contactdermatitis. When the effects of Sch.414319 were inves-tigated on different types of inflammatory disorders, thecompound reduced bone damage in antigen-inducedmonoarticular arthritis and counteracted the clinicalsigns of EAE in the Lewis rat strain. The authors sug-gested that these effects can all result from the controlof inflammatory cell migration, possibly involving-plastin phosphorylation86. Despite these promisingpreclinical results, however, clinical trials of JTE-907,

SR144528 and Sch.336 or Sch.414319 in allergic contactdermatitis or autoimmune disorders do not seem to bein the pipeline.

Potential disadvantages of cannabinoid receptor

antagonists. Similar to indirect agonists, antagonistsshould exert pharmacological effects preferentially inthose tissues and organs where the receptor they targetis tonically activated by locally acting endogenous ago-nists. However, all CB

1 and CB

2 receptor antagonists

tested so far in preclinical and clinical experimentationbehave — in in vitro assays of functional activity — notas neutral antagonists but as inverse agonists. That is,

Table 4 | CB2 receptor antagonists/inverse agonists in preclinical studies

Compound Affinity orpotencyin vitro*

Effective dosesin animals‡

Potential indications testedin animal models

SR-144528

Cl

N

N

NH

O

H

K i of 0.6 nM

in bindingassays194

0.35 mgper kg; 10 mgper kg by oraladministration

• Blocks the 12-O-tetradecanoyl-phorbol-13-acetate-induced earswelling196

• Attenuates the recruitment ofeosinophils and ear swelling inchronic contact dermatitis inducedby repeated challenge withoxazolone58

• Inhibits leukocyte recruitment ina murine model of delayed-typehypersensitivity67

O HN

O

O

HN

OO

O

JTE-907

K i of

0.38–1.55 nMin bindingassays193

0.1–10 mgper kg by oraladministration

• Inhibits carrageenin-inducedmouse paw oedema193

• Inhibits dinitrofluorobenzene-induced ear swelling195

• Alleviates dermatitis symptomsin a mouse model of contactdermatitis58

O S

O

O

SO

O

O

HN S

O

O

Sch.336

K i of 0.3 nM

(human)67,863–10 mg perkg by oraladministration

• This compound and its congenersare potent modulators of immunecell mobility in vivo and of bonedamage in antigen-inducedmono-articular arthritis andin experimental autoimmuneencephalomyelitis67,86

*Affinity, usually expressed here as affinity constant (K i). Values vary according to the assay conditions and the type of cells used to

study and the animal species. The K i for cannabinoid receptor 1 (CB

1) is not shown, and is usually at least 50-fold higher than the K

i

values shown here.

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they appear to destabilize a receptor conformationthat preferentially couples to the G-protein also in theabsence of endogenous agonists. This might raise thepossibility that these compounds exert effects (oppositeto those of agonists) also on organs where endocannabi-noid levels are not aberrantly elevated, with subsequentloss of specificity. Yet, this does not often seem to bethe case, as cannabinoid receptor antagonists have beenshown so far to be significantly more efficacious in vivo under physiological or pathological conditions in whichlocal endocannabinoid levels are elevated (see above).However, it is almost impossible to determine whetheror not inverse agonism occurs in vivo. The finding of apharmacological profile similar to those of cannabinoidreceptor antagonists also with inhibitors of endocan-nabinoid biosynthesis might be used in the future asindirect evidence that tonic activity of receptors occursbecause of endogenous ligands and not of pre-couplingto G-proteins. Unfortunately, the only endocannabinoidbiosynthesis inhibitors developed so far, although selec-tive, are not suitable for systemic in vivo use197.

Another potential limitation of cannabinoid recep-tor antagonists targeting a certain disorder to whosesymptoms or progress dysregulated endocannabinoidsmight contribute, is that they might interfere also withother concomitant disorders in which endocannabinoidsmight instead be playing a protective effect. Indeed, theuse of CB

2 receptor antagonists against allergic contact

dermatitis or multiple sclerosis might be limited bythe fact that endocannabinoids do not only contributeto inflammation with chemotaxic effects, but can alsoinhibit inflammatory cytokine release198, to the pointthat both CB

2/CB

1 agonists produce beneficial effects

in the same model of allergic dermatitis in which CB2

antagonists do58,62. Likewise, while being used againstobesity and metabolic disorders, CB

1 antagonists might

interfere with endocannabinoid-mediated adaptation tonew stressful conditions, thus explaining the anxiogenicand pro-depressant effects observed in obese patientstreated with these compounds166–169,178. However, onedoes not expect cannabinoid receptor antagonists tobe capable per se of inducing adverse events, but onlyto interfere with some of the protective effects of endo-cannabinoids that might arise when these compoundsare produced de novo during a new acute pathologicalcondition. It is also clear that specific cannabinoid recep-tor antagonists produce fewer adverse events than thoseexpected from the many protective actions postulated for

endocannabinoids. This is because, in the pathologicalconditions that they target, CB

1 or CB

2 sometimes play

opposing functions and hence blockade of only one of

these receptors does not prevent the beneficial effects ofthe other. Finally, in view of the high specificity of endo-cannabinoid protective role, the tissue-specific distribu-tion/accumulation/targeting of non-receptor-saturatingdoses of these drugs might prevent them from acting inthe wrong place. In this respect, it is interesting to notethat doses of taranabant that are clinically efficacious atreducing body weight in humans only occupy 30–40%of human brain CB

1 receptors178.

Concluding remarksPerhaps no other signalling system discovered duringthe past 15 years is raising as many expectations for thedevelopment of new therapeutic drugs, encompass-ing such a variety of pathological conditions, targetingso many different organs and tissues, and using such awide range of potential strategies for treatment, as theendocannabinoid system. The articles published overthe past 4 years have shown that both direct or indirectagonists and antagonists of cannabinoid receptors canproduce beneficial effects, sometimes even in the same

condition, in agreement with the pleiotropic homeo-static function of this system and with its unfortunatetendency to become dysregulated. While this might lookattractive for drug developers, it can also be a drawbackwhen the time comes to go from preclinical to clinicalstudies. Nevertheless, we know from the experience ofdronabinol, nabilone, tetrahydrocannabinol/cannabidiol,rimonabant and of several other compounds currentlyin the clinical pipeline, that good drugs can be made byeither increasing or decreasing the tone of the endocan-nabinoid system, while keeping at bay most side effects.

Therefore, perhaps, while we wait to understand moreabout the physiological function of this system, it is just amatter of trying what is the best endocannabinoid-baseddrug for a certain condition. One, however, needs to becautious as the possibility exists that, when someoneis being treated with a selective enhancer or blocker ofendocannabinoid action for long periods of time, a co-morbidity develops that is worsened or counteracted bysuch action, respectively, and this might cause problems.However, experience has shown that collateral events ofthese drugs can be controlled, in both clinical trials andin the medical practice, by using the appropriate dosage,selecting the right patient and making the most of clinicalsurveillance. In conclusion, for those who are engaged indeveloping new therapeutics by targeting the endocan-nabinoid system, this task can be described by Giuseppe

Verdi’s definition (in “La Traviata”) of love as “Croce eDelizia”: a series of painstaking, and sometimes frustrating,efforts alternating with immense gratifications.

1. Russo, E. & Guy, G. W. A tale of two cannabinoids:

the therapeutic rationale for combining

tetrahydrocannabinol and cannabidiol.

Med. Hypotheses 66, 234–246 (2006).

2. Mechoulam, R. Discovery of endocannabinoids and

some random thoughts on their possible roles in

neuroprotection and aggression. Prostaglandins

Leukot. Essent. Fatty Acids 66, 93–99 (2002).

3. Pertwee, R. G. Cannabinoid pharmacology: the first 66

years. Br. J. Pharmacol. 147 (Suppl. 1), 163–171

(2006).

4. Di Marzo, V. & Petrosino, S. Endocannabinoids and

the regulation of their levels in health and disease.

Curr. Opin. Lipidol . 18, 129–140 (2007).

5. Alexander, S. P. & Kendall, D. A. The complications

of promiscuity: endocannabinoid action and

metabolism. Br. J. Pharmacol. 152, 602–623

(2007).6. Di Marzo, V., Bifulco, M. & De Petrocellis, L.

The endocannabinoid system and its therapeutic

exploitation. Nature Rev. Drug Discov. 3,

771–784 (2004).

7. Piomelli, D. The endocannabinoid system: a drug

discovery perspective. Curr. Opin. Investig. Drugs.

6, 672–679 (2005).

8. Hohmann, A. G. & Suplita, R. L. 2nd. Endo-

cannabinoid mechanisms of pain modulation. AAPS J .

8, e693–e708 (2006).

9. Jhaveri, M. D., Richardson, D. & Chapman, V.

Endocannabinoid metabolism and uptake:

novel targets for neuropathic and inflammatory

pain. Br. J. Pharmacol. 152, 624–632

(2007).

R EVI EWS



15/19

10. Lambert, D. M. Allergic contact dermatitis and

the endocannabinoid system: from mechanisms

to skin care. ChemMedChem 2, 1701–1702 (2007).

11. Bisogno, T. & Di Marzo, V. Short- and long-term

plasticity of the endocannabinoid system in

neuropsychiatric and neurological disorders.

Pharmacol. Res. 56, 428–442 (2007).

12. Cota, D. CB1 receptors: emerging evidence for central

and peripheral mechanisms that regulate energy

balance, metabolism, and cardiovascular health.

Diabetes Metab. Res. Rev. 23, 507–517 (2006).

13. Matias, I. & Di Marzo, V. Endocannabinoids and thecontrol of energy balance. Trends Endocrinol. Metab.

18, 27–37 (2007).

14. Ashton, J. C. & Smith, P. F. Cannabinoids and

cardiovascular disease: the outlook for clinical

treatments. Curr. Vasc. Pharmacol. 5, 175–185

(2007).

15. Bifulco, M., Laezza, C., Gazzerro, P. & Pentimalli, F.

Endocannabinoids as emerging suppressors of

angiogenesis and tumor invasion. Oncol. Rep. 17,

813–816 (2007).

16. Storr, M. A. & Sharkey, K. A. The endocannabinoid

system and gut–brain signalling. Curr. Opin.

Pharmacol. 7, 575–582 (2007).

17. Mallat, A., Teixeira-Clerc, F., Deveaux, V. &

Lotersztajn, S. Cannabinoid receptors as new targets

of antifibrosing strategies during chronic liver

diseases. Expert Opin. Ther. Targets 11, 403–409

(2007).18. Pacher, P., Batkai, S.. & Kunos, G. The endocannabinoid

system as an emerging target of pharmacotherapy.

Pharmacol. Rev. 58, 389–462 (2006).

19. Kirkham, T. C., Williams, C. M., Fezza, F. &

Di Marzo, V. Endocannabinoid levels in rat limbic

forebrain and hypothalamus in relation to fasting,

feeding and satiation: stimulation of eating by

2-arachidonoyl glycerol. Br. J. Pharmacol. 136,

550–557 (2002).

20. Hanus, L. et al . Short-term fasting and prolonged

semistarvation have opposite effects on 2-AG levels in

mouse brain. Brain Res. 983, 144–151 (2003).

21. Matias, I. et al. Regulation, function, and

dysregulation of endocannabinoids in models of

adipose and β-pancreatic cells and in obesity andhyperglycemia. J . Clin. Endocrinol. Metab. 91,

3171–3180 (2006).

22. Jamshidi, N. & Taylor, D. A. Anandamide

administration into the ventromedial hypothalamus

stimulates appetite in rats. Br. J. Pharmacol. 134,

1151–1154 (2001).

23. Di Marzo, V. et al. Leptin-regulated endocannabinoids

are involved in maintaining food intake. Nature 410,822–825 (2001).

24. Monteleone, P. et al . Blood levels of the

endocannabinoid anandamide are increased in

anorexia nervosa and in binge-eating disorder, but not

in bulimia nervosa. Neuropsychopharmacology 30,

1216–1221 (2005).

25. Di Marzo, V., Hill, M. P., Bisogno, T., Crossman, A. R.

& Brotchie, J. M. Enhanced levels of endogenous

cannabinoids in the globus pallidus are associated

with a reduction in movement in an animal model of

Parkinson’s disease. FASEB J. 14, 1432–1438

(2000).

26. Gubellini P et al. Experimental parkinsonism alters

endocannabinoid degradation: implications for

striatal glutamatergic transmission. J. Neurosci. 22,

6900–6907 (2002).

27. van der Stelt, M. et al . A role for endocannabinoids in

the generation of parkinsonism and levodopa-

induced dyskinesia in MPTP-lesioned non-human

primate models of Parkinson’s disease. FASEB J. 19,1140–1142 (2005).

28. Ferrer, B., Asbrock, N., Kathuria, S., Piomelli, D. &

Giuffrida, A. Effects of levodopa on endocannabinoid

levels in rat basal ganglia: implications for the

treatment of levodopa-induced dyskinesias. Eur. J.

Neurosci. 18, 1607–1614 (2003).

29. Kreitzer, A. C. & Malenka, R. C. Endocannabinoid-

mediated rescue of striatal LTD and motor deficits in

Parkinson’s disease models. Nature 445, 643–647

(2007).

30. Fernandez-Espejo, E. et al. Cannabinoid CB1

antagonists possess antiparkinsonian efficacy only in

rats with very severe nigral lesion in experimental

parkinsonism. Neurobiol. Dis. 18, 591–601 (2005).

31. Gonzalez S. et al . Effects of rimonabant, a selective

cannabinoid CB1 receptor antagonist, in a rat model

of Parkinson’s disease. Brain Res. 1073–1074,

209–219 (2006).

32. van der Stelt, M. et al. Endocannabinoids and

β-amyloid-induced neurotoxicity in vivo: effect ofpharmacological elevation of endocannabinoid levels.

Cell. Mol. Life Sci. 63, 1410–1424 (2006).

33. Mazzola, C., Micale, V. & Drago, F. Amnesia induced

by β-amyloid fragments is counteracted bycannabinoid CB

1 receptor blockade. Eur. J.

Pharmacol. 477, 219–225 (2003).

34. Esposito, G. et al . Opposing control of cannabinoid

receptor stimulation on amyloid-β-induced reactivegliosis: in vitro and in vivo evidence. J. Pharmacol.

Exp. Ther. 322, 1144–1152 (2007).35. Ramirez, B. G., Blazquez, C., Gomez del Pulgar, T.,

Guzman, M. & de Ceballos, M. L. Prevention of

Alzheimer’s disease pathology by cannabinoids:

neuroprotection mediated by blockade of microglial

activation. J. Neurosci. 25, 1904–1913 (2005).

36. Benito, C. et al . Cannabinoid CB2 receptors and fatty

acid amide hydrolase are selectively overexpressed in

neuritic plaque-associated glia in Alzheimer’s disease

brains. J. Neurosci. 23, 11136–11141 (2003).

37. Cabranes, A. et al. Decreased endocannabinoid levels

in the brain and beneficial effects of agents activating

cannabinoid and/or vanilloid receptors in a rat model

of multiple sclerosis. Neurobiol. Dis. 20, 207–217

(2005).

38. Witting, A. et al. Experimental autoimmune

encephalomyelitis disrupts endocannabinoid-

mediated neuroprotection. Proc. Natl Acad. Sci. USA

103, 6362–6367 (2006).39. Centonze, D. et al., The endocannabinoid system is

dysregulated in multiple sclerosis and in

experimental autoimmune encephalomyelitis. Brain

130, 2543–2553 (2007).

40. Maresz, K. et al. Direct suppression of CNS

autoimmune inflammation via the cannabinoid

receptor CB1 on neurons and CB

2 on autoreactive

T cells. Nature Med. 13, 492–497 (2007).

The first study that really clarifies the distinct

beneficial roles of the two cannabinoid receptor

types in autoimmune neuroinflammation.41. Croxford, J. L. et al. Cannabinoid-mediated

neuroprotection, not immunosuppression, may be

more relevant to multiple sclerosis. J. Neuroimmunol .

193, 120–129 (2007).

42. Bilsland, L. G. et al. Increasing cannabinoid levels by

pharmacological and genetic manipulation delay

disease progression in SOD1 mice. FASEB J. 20,

1003–1005 (2006).

43. Kim, K., Moore, D. H., Makriyannis, A. & Abood, M. E.

AM1241, a cannabinoid CB2 receptor selective

compound, delays disease progression in a mouse

model of amyotrophic lateral sclerosis. Eur. J. Pharmacol. 542, 100–105 (2006).

44. Shoemaker, J. L., Seely, K. A., Reed, R. L., Crow, J. P.

& Prather, P. L. The CB2 cannabinoid agonist

AM-1241 prolongs survival in a transgenic mouse

model of amyotrophic lateral sclerosis when initiated

at symptom onset. J. Neurochem. 101, 87–98

(2007).

45. Marsicano, G. et al. The endogenous cannabinoid

system controls extinction of aversive memories.

Nature 418, 530–534 (2002).

Perhaps the first example of the site- and time-

specific activation of the endocannabinoid system

following a stressor and with a protective function

in the adaptation to new environmental conditions.

46. Kamprath, K. et al. Cannabinoid CB1 receptor

mediates fear extinction via habituation-like

processes. J. Neurosci. 26, 6677–6686 (2006).

47. Patel, S., Roelke, C. T., Rademacher, D.J., Cullinan, W. E.

& Hillard, C. J. Endocannabinoid signaling negatively

modulates stress-induced activation of thehypothalamic–pituitary–adrenal axis. Endocrinology

145, 5431–5438 (2004).

An important study showing how the

endocannabinoids and CB1 receptors can

be involved in the control of stress.

48. Hohmann, A. G. et al. An endocannabinoid

mechanism for stress-induced analgesia. Nature 435,

1108–1112 (2005).

Another elegant example of the site- and

time-specific activation of the endocannabinoid

system following a stressor, and of the therapeutic

exploitation of specific inhibitors of

endocannabinoid degradation.49. Hill, M. N. et al . Involvement of the endocannabinoid

system in the ability of long-term tricyclic

antidepressant treatment to suppress stress-induced

activation of the hypothalamic-pituitary-adrenal axis.

Neuropsychopharmacology 31, 2591–2599 (2006).

50. Moreira, F. A., Kaiser, N., Monory, K. & Lutz, B.

Reduced anxiety-like behaviour induced by genetic

and pharmacological inhibition of the

endocannabinoid-degrading enzyme fatty acid amide

hydrolase (FAAH) is mediated by CB1 receptors.

Neuropharmacology 54, 141–150 (2007).51. Rubino, T. et al. Role in anxiety behavior of the

endocannabinoid system in the prefrontal cortex.

Cereb Cortex 5 Oct 2007 (doi:10.1093/cercor/

bhm161).

52. Gobbi, G. et al. Antidepressant-like activity and

modulation of brain monoaminergic transmission byblockade of anandamide hydrolysis. Proc. Natl Acad.

Sci. USA 102, 18620–18625 (2005).

53. Naidu, P. S. et al . Evaluation of fatty acid amide

hydrolase inhibition in murine models of emotionality.

Psychopharmacology (Berl.) 192, 61–70 (2007).

54. De

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