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Clinical Immunology (2012) 142, 57–67

REVIEW

Neuroprotective agents: CannabinoidsA.J. Sánchez, A. García-Merino⁎

Neuroimmunology Laboratory and Neurology Service, Hospital Universitario Puerta de Hierro, Majadahonda,Universidad Autónoma de Madrid, Joaquín Rodrigo 2, 28222 Majadahonda, Madrid, Spain

Received 11 October 2010; accepted with revision 3 February 2011Available online 21 March 2011

⁎ Corresponding author. Fax:+349119E-mail addresses: jgarciam.hpth@s

1521-6616/$ - see front matter © 201doi:10.1016/j.clim.2011.02.010

KEYWORDSCannabinoids;Experimentalautoimmuneencephalomyelitis;Multiple sclerosis;Immune modulation;Neuroprotection

Abstract Chronic inflammation and neurodegeneration are the main pathological traits ofmultiple sclerosis that coexist in all stages of the disease course, with complex and stillnonclarified relationships. Currently licensed medications have efficacy to control aspectsrelated to inflammation, but have been unable to modify pure progression. Experimental workhas provided robust evidence of the immunomodulatory and neuroprotective properties thatcannabinoids exert in animal models of multiple sclerosis. Through activation of the CB2receptor, cannabinoids modulate peripheral blood lymphocytes, interfere with migration acrossthe blood-brain barrier and control microglial/macrophage activation. CB1 receptors present inneural cells have a fundamental role in direct neuroprotection against several insults, mainly

excitotoxicity. In multiple sclerosis, several reports have documented the disturbance of theendocannabinoid system. Considering the actions demonstrated experimentally, cannabinoidsmight be promising agents to target the main aspects of the human disease.© 2011 Elsevier Inc. All rights reserved.

16796.alud.madrid.org, gmerino@me

1 Elsevier Inc. All rights reserv

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 582. The cannabinoid system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 583. Cannabinoids, immune modulation and neuroprotection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

3.1. Cannabinoids and the systemic immune compartment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 593.2. Cannabinoids in CNS inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

4. The role of CB1 and CB2 receptors in neuroprotection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 615. Disturbance of the endocannabinoid system in MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 626. Beyond symptomatic improvement. Towards a therapy for MS course . . . . . . . . . . . . . . . . . . . . . . . . . 62Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

ditex.es (A. García-Merino).

ed.

58 A.J. Sánchez, A. García-Merino

1. Introduction

Over the last decades, the progress in understanding basicpathogenetic mechanisms of multiple sclerosis (MS) has beenremarkable. The classical view, based largely on experimentalmodels, considered MS as an immune-mediated disorder whereinflammation against myelin was the predominant feature of theearly, relapsing, phase, and neurodegeneration the pathologicaltrait of the late, progressive, phase. However, this view has beenchallenged by the demonstration that damage of neurons and axonsmay be an initial finding, both in MS [1,2] and experimentalautoimmune encephalomyelitis (EAE) [3]. The observation that, insome cases, central nervous system (CNS) damage may occur beforethe presence of inflammatory cells in the area [4] has alsocomplicated the interpretation of the relationship between inflam-mation and neurodegeneration. In experimental models, it has beenshown that a primary axonal injury may damage secondarily themyelin sheath, in an inside-out model opposite to the classicaloutside-in model, where myelin damage precedes axonal injury [5].Recent pathological studies have demonstrated the close associationbetween inflammation and neurodegenerative changes in all stagesof MS [6], but the relationship between these two components of MSpathology is far from being clarified. Possible interpretations havebeen proposed elsewhere [7].

The view of MS as a “two-stage” disease has also been questionedby neuroimaging studies showing the coexistence of both processes[8]. Clinical trials with drugs able to control CNS inflammation havenot yielded comparative results on brain atrophy, a paradox leadingto the concept of inflammation/neurodegeneration mismatch [9].

A number of reasons may be invoked to explain the low efficacy ofanti-inflammatory drugs to prevent the progressive atrophy of theCNS: inability to access the CNS, involvement of the innate immunity,role of ill-defined specific mechanisms acting on degeneration ofneurons and axons, and, in sum, lack of significant neuroprotectiveproperties of the available medication.

In the last years, a vast literature on cannabinoids has providedrobust evidence on their abilities as neuroprotective agents underdifferent pathological situations, first demonstrated in 1994 inexperimental brain ischemia [10]. In animal models of MS, thebeneficial role of cannabinoids has been shown for more than20 years [11–19]. Their mechanisms of protection are complex andinvolve receptors in the brain, but also in immune cells, and thesemechanisms will be analyzed below. As they combine neuroprotec-tion and immune modulation, cannabinoids have the potential tobecome useful agents to therapeutically deal with the two basicaspects of MS.

2. The cannabinoid system

Two subtypes of receptors mediate most cannabinoid actions,namely the CB1 receptor [20], located in the CNS and in peripheraltissues, and the CB2 receptor [21], found in the periphery, mainly incells of the immune system. Human CB1 and CB2 receptors share only44% overall amino acid identity with a 68% in the transmembranedomains. Although CB1 receptors are expressed by certain nonneur-onal cells, such as immune cells, and some tissues, CB1 receptors arelocalized predominantly at the terminals of central and peripheralneurons, where they mediate regulation of neurotransmitter releaseand psychoactivity.

Cannabinoid CB2 receptors are expressed mainly by immune cellsaccording to a rank order of B cellsNnatural killer cells≥polymor-phonuclear neutrophils≥CD8+ cellsNmonocytes NCD4+ cells[22,23], but recently they have been detected in the CNS [24],neurons [25], and neural stem cells [26].

Some actions of certain cannabinoid ligands may not be mediatedjust by the CB1/CB2 receptors. Such non-CB1/non-CB2 effects havebeen observed through other receptors like the serotonin type 3

receptor, α7-nicotinic acetylcholine receptor [27] and the transientreceptor potential vanilloid type 1 receptor (TRPV1) [28], anonselective cationic channel. Cannabinoid effects independent ofany kind of known receptor have also been reported, what suggests adirect diffusion through the lipid bilayer of cell membranes or thepresence of other unidentified cannabinoid receptor subtypes [29].In fact, GPR55 and GRP119 have recently attracted much attentionas another member of the cannabinoid receptor family [30,31].

Receptor ligands can be classified in two main groups: exoge-nous, divided into naturals (plant-derived) and synthetics(designed); and endogenous, also known as endocannabinoids(Fig. 1). The Cannabis sativa plant contains more than 60phytocannabinoids, a group of chemical compounds not found inother plants, including, amongst others, Δ8-THC, cannabinol,cannabidiol and Δ9-THC, the main active component and the primarypsychoactive cannabinoid. Δ9-THC behaves as a partial agonist ofboth CB1 and CB2 receptors [32]. The discovery of cannabinoidreceptors promoted the development of new CB1 and CB2 selectiveagonists and antagonists. The most commonly used antagonists/inverse agonists for CB1 are SR141716A (rimonabant), AM251 andAM281; and SR144528 and AM630 for CB2. On the other hand, thenumber of compounds that possess agonist properties is very high,some of them exhibit nonselective affinity for CB1/CB2 receptors orare more selective for CB2 receptors (for review see [33]).

The cannabinoid receptors can be also activated by endogenouscannabinoids. Cannabinoid receptors, their endogenous ligands andenzymes that catalyze their biosynthesis, transport and degradationconstitute the endocannabinoid system (ECS) (Fig. 1). Endocanna-binoids are a family of polyunsaturated fatty acid derivatives thatfunction as lipid signaling molecules by acting as endogenous ligandsat CB1 and CB2 receptor. To date, N-arachidonoylethanolamine(anandamide, AEA) [34] and 2-arachidonoylglycerol (2-AG) [35] arethe most thoroughly studied endocannabinoids, each of whichbehaves as a partial agonist at both CB1 and CB2 receptors. N-arachidonoyl-dopamine (NADA) [36] and 2-arachidonyl glycerylether (noladin ether) [37] are partial agonists selective for CB1; O-arachidonoyl ethanolamine (virodhamine, OAE) selectively activatesCB2 receptors and antagonizes CB1 receptors [38].

Endocannabinoids are produced and released in an activity-dependent manner. AEA synthesis from N-arachidonoyl phosphati-dylethanolamine (NArPE) includes enzymes such as phospholipaseA2, phospholipase C and N-acyl-phosphatidylethanolamine phospho-lipase D (NAPE-PLD). The major degradative enzyme for AEA is fattyacid amide hydrolase (FAAH) [39]. 2-Arachinodate-containingdiacylglycerols are the main precursors of 2-AG and are mediatedby the activities of phospholipase C (PLC) and diacylglycerol lipase(DAGL). Several enzymes can hydrolyze 2-AG, this reaction appearsto be primarily catalyzed by monoacylglycerol lipase (MAGL) [40]. Inthis context, the use of potent and selective inhibitors ofendocannabinoid re-uptake and enzymatic hydrolysis might enhanceendocannabinoid actions both in the CNS and the peripheral tissues(for review see [41]).

Both CB1 and CB2 receptor are single polypeptides with seventransmembrane α-helices, a glycosylated amino-terminus and anintracellular carboxyl-terminus. They are G-protein-coupled recep-tors (GPCR) to Gi or Go protein and thereby can stimulate themitogen-activated protein kinase pathway [42] and inhibit adenylylcyclase activity, thus attenuating the conversion of ATP to cyclicAMP [43]. Another major action of cannabinoids is the direct Gprotein-mediated modulation of ion channels, positively to A-typeand inwardly rectifying potassium channels, and negatively to N-type and P/Q-type calcium channels, modulating the releases ofneurotransmitters like the inhibition of glutamate, noradrenalineand acetylcholine [44].

Cannabinoid receptor stimulation involves the activation of thephosphatidylinositol 3-kinase and Akt (PI3K–Akt) pathway andincreases the lipid second messenger ceramide, linked to a pro-survival and a pro-apoptotic effect, respectively [45,46]. All these

Figure 1 Principal components of the cannabinoid system. The cannabinoid system includes: (I) cannabinoid CB1 and CB2 receptors,(II) exogenous (natural and synthetic) and endogenous agonists, of which anandamide and 2-arachidonoylglycerol are the mostcharacterized endocannabinoids, and (III) enzymes responsible for the production, transport, and degradation of these ligands.Anandamide and 2-AG are synthesized in an inducible manner from lipid precursors, such as N-arachidonoyl-phosphatidylethanol-amine (NArPE) and diacylglycerols (DAGs), respectively. Once synthesized, endocannabinoids are released into the extracellular spaceby a putative endocannabinoid transporter. The degradation requires re-uptake by cells, through membrane associated bindingproteins or by a transmembrane carrier protein and inactivation by enzymatic hydrolysis of the amide and ester bonds. The keyenzymes responsible for these reactions are the fatty acid amidehydrolase (FAAH) and monoacylgly-cerol lipase (MAGL), foranandamide and 2-AG, respectively.

59Neuroprotective agents: Cannabinoids

interactions are implicated in regulating many physiological func-tions in a wide range of systems.

3. Cannabinoids, immune modulationand neuroprotection

Cannabinoids exert a very wide spectrum of actions on cells of theadaptive and innate branches of the immune system, both in theperiphery and the CNS. In most cases, those actions depend on thepresence of the CB2 receptor, and will reduce the inflammatoryinsult against neuroaxonal structures. Inside the CNS, other welldefined actions will protect neurons against injury, and they aremediated basically through CB1 receptor activation. Nevertheless, itis not always possible to clearly differentiate anti-inflammatoryfrom neuroprotective actions. In the following sections we willreview the cannabinoid-induced effects in the periphery, and thoseof the CNS in experimental models of MS and in MS, when available(Fig. 2).

3.1. Cannabinoids and the systemic immunecompartment

The effects of cannabinoids on immunity have been studied for morethan 30 years [47]. Even though many of them are clearly defined, auniform view on the immune effects of these compounds is not yetavailable, as many results have been contradictory. Reasons toexplain discrepancies are multiple. Some results are derived fromanimal and in vitro studies, and they depend on type and dose of thecannabinoid employed [48] and type of cell where the cannabinoid isacting. Immune modulatory effects of cannabinoids are extensiveand reported mechanisms include inhibition of pro-inflammatory

cytokine production, antigen presentation, T cell proliferation, andeffector cell function [49]. Other actions like activation of anti-inflammatory cytokines, induction of apoptosis and modulation ofregulatory cells are clearly established [50] (Fig. 2).

Most studies show that cannabinoids induce a Th2 shift [51]. IFN-γ and TNF-α production has been repeatedly found to be diminishedby cannabinoids [52–56], although other experiments have reportedthe opposite effect on TNF-α [57,58]. In MS, the first study with oralΔ9-THC showed a modest increase of TNF-α in LPS-stimulated wholeblood [59]. Effects on IL-4, IL-10 and IL-12 are complex; in general,IL-4 and IL-10 levels were increased [51,55], although the oppositewas reported for IL-10 [53,60]. IL-12 secretion has been founddecreased [51] or increased [60].

Other studies describe immunomodulatory effects in prolifera-tion, activation, apoptosis, and induction of T-regulatory cells[49,61–64]. More recently, current research on cannabinoids andTh17 cells, a cell subpopulation involved in the pathogenesis of MS[65], has found that these compounds induce a suppressive effect[48,66].

Macrophages are particularly sensitive to cannabinoids, as theymay alter nitric oxide production [67], impair killing of cells [68],suppress phagocytic activity [69] and decrease in vitro migration[70]. The production of TNF-α, IL-10 or IL-12 cytokines by macro-phages/microglia is inhibited through the CB2 receptors [71,72].Antigen processing and presentation by macrophages/microglia to Tcells is critical for generating pathogenic cells in EAE [73];cannabinoids interfere with antigen processing via CB2 [74].

On B cells, low dose cannabinoids may increase proliferation [75]and inhibit response to LPS [76]. Serum levels of immunoglobulinshave been analyzed in marijuana smokers with divergent results.However, in vitro, they suppress the humoral response by directinhibition of adenylate cyclase [77] and indirectly through T cells ormacrophages required for B cell activation [78,79].

60 A.J. Sánchez, A. García-Merino

Cannabinoid-induced apoptosis, described in several cell types [50],might contribute to down-regulation of T cell activity and, therebywould terminate the inflammatory process that precedes the clinical

signs of EAE [80]. Dendritic cells are highly sensitive to cannabinoid-induced apoptosis [81]. They may inhibit Th1 activation by targetingessential dendritic cell functions [82]. It has been demonstrated that

61Neuroprotective agents: Cannabinoids

WIN55, 212-2 blocks a passive form of EAE by inducing apoptosis inencephalitogenic cells through partial activation of the CB2 receptor[19].

The damage of the blood-brain barrier (BBB) and migration ofinflammatory cell across the BBB into the CNS represent key eventsin the immunopathogenesis of EAE/MS [83]. In an EAE context, theleukocyte/endothelial interaction can be attenuated with theadministration of a different cannabinoid agonist by a CB2receptor-dependent mechanism [84], and even by a CB1 receptorantagonist [85]. Other authors have also demonstrated, by in vivoand in vitro studies, that WIN55, 212-2 suppressed the expression ofcell adhesion molecules ICAM-1 and VCAM-1, in brain endothelium[15,86] (Fig. 2).

3.2. Cannabinoids in CNS inflammation

Chronic activation of microglia plays a major role in disorderscharacterized by nervous tissue inflammation, such as EAE [87]. CB1is expressed constitutively in microglial cells; CB2 expression is notpresent in resting cells and is related to the cell activation state[88]. Microglia secrete mediators that regulate phenotype of Thelper cells and inflammation inside the CNS, including cytokinessuch as TNF-α, IL-1, and IL-12 [89]. Microglial cells are activated bybrain injury and may amplify damage by releasing toxic cytokinesand mediators. The ECS is highly up-regulated under thosecircumstances. Cannabinoids may help the survival of neurons andaxons by interfering with microglia. In EAE, WIN55, 212-2 was able toreduce the levels of TNF-α, IL-1β, IL-6 and IFN-γ [16]. In the Theiler'smurine encephalomyelitis virus model of demyelination, pharmaco-logical increase of tissue levels of AEA, down-regulated microglialactivation, TNF-α, IL-6 and IL-1β [90], IL-12, and nitric oxidesynthase-2 [91]. AEA, produced in high levels in MS brains,particularly in active lesions, acts on CB1 and CB2 receptors ofactivated microglial cells and protects neurons by inducing mitogen-activated protein kinase phosphatase-1 [92]. Several experimentshave shown the key importance of CB2 in the inhibition of pro-inflammatory cytokines by cannabinoids [93,94].

Accumulation of excessive amounts of glutamate leading toexcitoxicity is a common trait of several neurological disorders, andresults from the overstimulation of the ionotropic glutamatereceptors, provoking the entry of calcium inside the cells andaltering the sodium/potassium balance. In addition to neurons,oligodendrocytes are quite vulnerable to prolonged activation ofglutamate receptors, and this may contribute to tissue damage in MS[95]. A number of observations indicate a pathophysiologic role ofglutamate excitotoxicity both in EAE [96,97], and in CSF [98,99],brain tissue [100,101] and MRI spectroscopy [102] from MS patients.The ability of cannabinoids to control glutamate release throughactivation of CB1 [103], located at presynaptic terminals [104] hasrevealed the crucial role of this receptor for excitotoxicity control inneuroinflammation [105] (Fig. 2). Brain-derived neurotrophic factoris a key mediator in protection against excitotoxicity through CB1[106]. In addition to the activation of CB1 in neurons, other reportshave demonstrated that the neuroprotective effect of cannabinoids

Figure 2 Multiple sclerosis immunopathogenesis and possiblecannabinoids may modulate the immune system in the periphery byeffector function, and by promoting release of anti-inflammatory cytreduce the ability of white cells to cross the BBB and enter the CNleukocytes across the BBB. At the CNS level, cannabinoids may inhibantigen presentation, thus diminishing pro-inflammatory cytokines,cannabinoid receptor can also increase the apoptotic elimination ofagainst excitoxicity and oxidative stress, promote oligodendrocyteprecursor cells with implication in neurogenesis and repair. Abs, antCNS, central nervous system; IFN-γ, interferonγ; IL, interleukin; NO, nTNF-α, tumor necrosis factor α; Treg, regulatory T cell; VCAM, vascu

depends as well on CB2 activation in astrocytes [107]. HU-211, acannabinoid with no affinity for either CB1 or CB2, suppresses EAE byblocking the NMDA receptor [108]. Enhancement of the endocanna-binoid tone by means of UCM707, an inhibitor of endocannabinoiduptake, protected against AMPA-induced excitotoxicity by activat-ing CB1 and CB2 receptors and the peroxisome proliferator-activatedreceptor-gamma (PPAR-γ) [109]. On the contrary, a reduction ofendocannabinoid levels, as found in EAE and related to the IFN-γrelease by primed T cells infiltrating the CNS, is associated withmore tissue damage [110].

As a consequence of excitotoxicity, toxic levels of calciumaccumulate intracellularly and can generate a cascade of eventsleading to cell death. Cannabinoids may counteract this phenomenonby acting on voltage-sensitive calcium channels, an action mediatedpartly through CB1 receptors and by direct pathways [105,111].

Reactive oxygen and nitrogen species increase in excess duringCNS inflammation creating oxidative stress that can damage lipids,proteins, nucleic acids and mitochondria, eventually resulting in celldeath [112]. In MS, altered antioxidant defense systems mayincrease susceptibility to oxidative stress [113]. Cannabinoidscould afford protection against oxidative stress in MS, consideringthat, in addition to their ability to reduce nitric oxide production onmacrophage/microglial cells [91] and astrocytes [46], they havestrong antioxidant properties in vitro [114,115].

In chronic MS lesions, remyelination failure is a contributingfactor to axonal loss due to the lack of trophic support byoligodendrocytes [116]. In this context, cannabinoids have shownremyelinating properties in viral-induced models of demyelination,in close association with attenuation of the inflammatory response[15]. Furthermore, they enhance survival of oligodendrocyteprogenitors through the modulation of phosphatidylinositol-3 ki-nase/Akt signaling [46].

4. The role of CB1 and CB2 receptorsin neuroprotection

Compelling evidence shows that CB1 receptors in the CNS play afundamental role in neuroprotection [117]. Besides their involve-ment in controlling excitotoxicity and inflammation, the generationof knockout mice deficient in CB1 receptor by Baker's group hasrevealed specific roles of this receptor in neuroinflammation: micedeficient in CB1 were more susceptible to EAE, had more neurode-generation and worse recovery, compared to wild type mice [105],and had increased caspase 3 activation [118]. Further experimentsdemonstrated that the presence of CB1 in neurons, but not in T cells,was an essential requisite for cannabinoid-mediated neuroprotectionin EAE [119], and that this effect was independent on the inflam-matory control, as low doses of cannabinoids may protect againstnerve loss, despite being unable to suppress relapses [120].

Recently, the relevance of CB2 receptors in neurodegenerativedisorders has been the subject of several reviews [15,93,121,122]. Inmodels of MS, CB2 is the target for immune modulation of peripheralblood lymphocytes: the shift towards a Th2 response, the apoptosis

therapeutic cannabinoid targets. Exogenous and endogenousinhibiting pro-inflammatory cytokines, T-cell proliferation andokines, apoptosis and regulatory T-cells. Furthermore, they mayS by inhibition of molecules implicated in the extravasation ofit microglia and astrocyte activation, macrophage function andtoxic mediators and cell damage. The inhibition/activation ofinflammatory cells. Cannabinoids may afford direct protection

progenitor survival, and stimulate proliferation of neural stem/ibodies; APC, antigen-presenting cells; BBB, blood brain barrier;itric oxide; ODG, oligodendrocyte; ROS, reactive oxygen species;lar cellular adhesion molecule; VLA-4, very late antigen 4.

62 A.J. Sánchez, A. García-Merino

induction of encephalitogenic cells [19], the interference withleukocyte rolling and adhesion to endothelium [84] are factors thatdiminish significantly the cell infiltration in the nervous tissue, thusreducing the inflammatory damage to myelin, glia and neurons. CB2 isexpressed in microglia and astrocytes and regulates microglialneurotoxicity [93]. In addition to those effects, all related to inflam-matory control, one more action of CB2 refers to the replenishment ofat least one subpopulation of microglial cells from bone-marrow pro-genitors, demonstrated in EAE [123]; this process is under the negativecontrol of CB2 [124]. CB2 is also associated with neurogenesis; it ishighly expressed in neural progenitors and they proliferateboth in vitroand in vivo by selective-CB2 agonists [125]. The article by Maresz et al.[119] with transgenic mice deficient in CB2 has provided information toclarify CB2 impact on EAE: CB2 deficient miceweremore susceptible todisease induction, had a protracted course, and that was dependent onthe expression or absence of CB2 in encephalitogenic T cells, not in CNStissue, the opposite role of CB1 expression in neuroprotection. Thesuggestion of this work is that T-cell CB2 receptors are activated in thebrain in the presence of high concentrations of 2-AG, and that thisactivation is able to suppress T-cell function [126].

Several actions of cannabinoid-induced neuroprotection are clearlyindependent on both CB1 and CB2 receptors. Induction of T-cell apop-tosis is partly receptor independent [19], the antioxidant properties ofphenol-containing cannabinoids, such as Δ9-THC and cannabidiol, arenotmediated through these receptors [114,115], HU-211 alleviates EAEby blocking directly theNMDA receptor, theAEA-induced antagonismofthe TWIK-related acid-sensitive potassium channel (TASK 1) is asignificant contribution to the therapeutic benefit in EAE [127], certaincannabinoids, both synthetic and endogenous, such as AEA, are PPARligands and some of their neuroprotective properties seem to dependon PPAR-α [128] and PPAR-γ activation [109].

5. Disturbance of the endocannabinoid systemin MS

Indirect evidence of the involvement of the ECS in MS has beenprovided by randomized controlled clinical trials using cannabinoidsfor symptomatic therapy. Several trials have analyzed the effects oforal and sublingually applied cannabinoids on spasticity and pain[129–136] with variable results. However, direct information on theinvolvement of the ECS in MS patients is limited. In CNS samples fromMS patients, increased levels of AEA were reported [92]. Benito et al.[137] identified CB1 receptors in cortical neurons, oligodendrocytes,and also oligodendrocyte precursor cells, whereas CB2 receptorswere present in T-lymphocytes, astrocytes, and perivascular andreactive microglia in MS plaques; the expression of the FAAH enzymewas also up-regulated in lesions. In another study, AEA, but not 2-AG, was increased in the CSF of relapsing patients, and was also highin peripheral lymphocytes of MS patients [138]. Other authors foundthat AEA was higher in plasma from RRMS, SPMS and PPMS comparedto controls [139]. We have also found differential expression ofcannabinoid receptors and endocannabinoids in cell populationsduring the course of MS (unpublished data).

6. Beyond symptomatic improvement. Towardsa therapy for MS course

Despite the long tradition of marijuana use among patients, and thenumber of controlled clinical trials carried out so far, an impact onclinical course has not been demonstrated. There is convincingevidence of improvement on symptoms, mainly spasticity, howeverwe still do not know if this has an effect on relapses and disabilityaccumulation. A trial designed to specifically assess the impact ofcannabinoids on disability in 500 progressive MS patients (cannabi-noid use in progressive inflammatory brain disease—CUPID-trial) will

allow knowing, for the first time, whether or not these compoundsmodify disease evolution.

Whereas the currently licensed medications for MS have provenimmunomodulatory or immunosuppressive properties, significantefficacy on relapse-free progression has not been demonstrated. Incontrast, cannabinoids have an impressive array of beneficial effectsin models of MS, and appear as ideal agents for the human disease;they offer immunomodulatory/anti-inflammatory actions, mainlythrough CB2 receptors, and neuroprotective effects mediated to alarge extent by CB1. In spite of that, the cannabinoid-basedmedicine for MS is little developed.

A number of aspects must be taken into consideration fordeveloping a suitable cannabinoid for treating the MS course: safety,tolerability, type of compound, mechanism of action, route ofadministration, and dosage. Safety and tolerability are fundamentalconsiderations. There is some concern with regard to the possibilityof neurotoxicity in patients, as shown experimentally [140,141]; inhumans, it has been much discussed whether chronic exposure tocannabis for recreational use can have a negative impact oncognition, although literature is controversial on this regard. Clinicaltrials with cannabinoids in MS have addressed their influence oncognition, but limitations in design do not allow drawing firmconclusions about long-term adverse effects (for review see [142]).

For good tolerance, ideal cannabinoids for chronic use in MSshould avoid CB1 stimulation because of the unwanted psychoactiveeffects, a serious limiting factor of Δ9-THC based treatments. On thecontrary, drugs targeting CB2 would be free of psychic intoleranceand might favor modulation of autoreactive cells in the peripheryand neuroprotection against acute and chronic inflammatorydamage. For clinical testing, a highly selective agonist for CB2,devoid of CB1 affinity would be required. Although the existingcompounds have different levels of selectivity and are not suitableyet for human use, development of better selective-CB2 agonists is avery promising avenue [93].

Alternative approaches to cannabinoid receptor stimulationinclude the increase of endocannabinoid tone by agents able tointerfere with the degrading enzyme FAAH or with the uptake ofendocannabinoids [109,143]. This therapeutic design offers severaladvantages: it avoids unwanted effects related to the globalstimulation of receptors at the CNS and increases levels ofendocannabinoids at sites were they are being synthesized [90].

The only existing cannabinoid for human use, devoid of CB1 or CB2agonist activity, but possessing significant anti-inflammatory andneuroprotective properties, is cannabidiol [114], a component ofSativex® used in a 1:1 ratio with THC, to diminish unwanted effectsof THC. Sativex®, has been currently approved in some countries forspasticity. Selective CB2 agonists or drugs increasing the endocan-nabinoid tone, or both, appear as more interesting drugs to controlthe two main components of MS. There is still some way to go withthe pharmacological development of cannabinoids, to define thebest drug, the best route of administration, or the best dose.

Future trials on cannabinoids for MS might be assayed as singledrugs or combined with existing medications for either progressiveor relapsing MS patients, with particular emphasis on disabilityprogression. A major challenge for MS therapy is to improve pureprogression; cannabinoids could be new options for trials currentlydesigned to such a purpose.

Conflict of interest

The authors declare no conflict of interest.

Acknowledgments

Sánchez AJ is supported by Fondo de Investigaciones Sanitarias,Spanish Ministerio de Ciencia e Innovación; CA07/410. This work was

63Neuroprotective agents: Cannabinoids

supported by grants from the Fondo de Investigación Sanitaria; 04/1214 and PS09/020004, Agencia Laín Entralgo, Comunidad deMadrid; NDG07 and Fundación de Investigación Médica MutuaMadrileña, 2006 and 2008.

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