3.1. CB1R and CB2R Ligands
Cannabinoids exert neuroprotective effects acting at multiple molecular sites that are in all key cellular elements for the control of neuronal survival (e.g., neurons, astrocytes, resting and reactive microglia, oligodendrocytes) and also in key brain structures (e.g., BBB) [
84]. These effects are due to activation of CB1R and CB2R.
CB1R is widely expressed within the CNS (cortical neurons and interneurons, oligodendrocytes, astrocytes) and also in several leukocytes infiltrating the brain [
85]. Initially CB2R has been restricted exclusively to immune cells (macrophages, mast cells, B and T lymphocytes) and immune organs (spleen, thymus, lymph nodes) [
86]. However, some evidence showed the expression of CB2R in microglia of the CNS [
87], and more recently, it has been also reported to be expressed in brainstem neurons and astrocytes upon cellular activation by an insult or inflammation [
36,
88,
89].
The multiplicity of action of cannabinoids allows to reduce the excitotoxicity by acting through neuronal CB1R, as well as the toxic influence of reactive microgliosis by acting through microglial CB2R, or enhancing the trophic and metabolic support to neurons by acting through astroglial CB1R or CB2R. In particular, the activation of CB1R provides neuroprotection regulating glutamate homeostasis [
90]. In fact, it is well-known that glutamate is a key mediator in neuronal and oligodendrocyte damage in MS [
91], and CB1R agonists exert direct neuroprotective effects by limiting glutamate release and the excitotoxic damage characteristic of several neurodegenerative disorders [
92]. Furthermore, the protective effects of CB2R activation in microglial cells upon inflammatory-induced CNS damage have been demonstrated in preclinical models of multiple sclerosis [
92]. Microglia may be, in two activated states: M1 and M2. The classical M1 state is characterized by release of pro-inflammatory factors, i.e., interleukins (IL-1beta, IL-18, and IL-6), prostanoids and inducible nitric oxide synthase (NOS2)-derived NO. On the other hand, the neuroprotective M2 state, known as “alternative activation” is associated with the release of anti-inflammatory factors, such as IL-10, IL-4, and NGF [
93]. Microglia has a functional endocannabinoid signaling system, composed of cannabinoid receptors and the complete machinery for the synthesis and degradation of endocannabinoids. The expression of cannabinoid receptors, mainly CB2R, and the production of endocannabinoids have been related to the activation profile of these cells [
94].
In preclinical studies, the beneficial effects of cannabinoids have been reported in different animal models of MS including experimental autoimmune encephalomyelitis (EAE), chronic relapsing experimental allergic encephalomyelitis (CREAE), and Theiler’s murine encephalomyelitis virus induced demyelinating disease (TMEV-IDD) [
95]. More specifically, autoimmune encephalomyelitis is a demyelinating autoimmune disease of the CNS that is characterized by mononuclear cell infiltration and mainly induced by auto-reactive CD4
+ T cells. EAE is a useful animal model of MS since many of the pathologies observed in the CNS of mice with EAE show strong similarity to those found in the CNS of MS patients [
96]. CREAE animal model also presents relapsing-remitting paralytic episodes and tremor and spasticity of limb muscles during post-relapse remission strongly similar to MS [
97]. Finally, TMEV-IDD is an immune-mediated demyelinating disease dependent on persistent virus infection of the macrophages, microglia and astrocytes within the CNS [
98] and the inflammation and demyelination observed in TMEV-IDD are similar to those described in MS patients [
99].
One of the first studies of cannabinoids in MS model is reported by Lyman et al. in 1989 [
100], who demonstrated the effects of daily administration of ∆
9-THC, a partial CB1R agonist with limited effects on CB2R, on EAE progression in rats. Indeed, the development of EAE was ameliorated, indicating the counteracting effect of ∆
9-THC in the disease [
100]. Subsequently, the effects of the phytocannabinoid ∆
8-THC (
Table 2) on EAE were studied by Wirguin et al. [
101]. ∆
8-THC is an analogue of ∆
9-THC which binds CB1Rs with high affinity and it is much more stable and less psychotropic than ∆
9-THC. In this study, ∆
8-THC significantly ameliorated the clinical manifestations of EAE. Among all the possible mechanisms of action postulated, one involved the inhibition of the prostanoid production by action of an active metabolite of ∆
8-THC. This active metabolite would explain the necessity of the oral, rather intraperitoneal administration due to the first-pass metabolism in the liver [
101].
Further research deepened the study on the role of synthetic and endogenous cannabinoids in CREAE animal model. Baker et al. showed evidence that cannabinoid CBRs agonists, in particular R(+)-WIN 55212 (
Table 2), ∆
9-THC, methanandamide (
Table 2) and JWH-133 (
Table 2) quantitatively ameliorated both tremor and spasticity in CREAE mice [
102]. In order to address these effects to the modulation of the ECS and with the aim to understand which of the CBRs was the most involved, animals were pre-treated with CBRs selective antagonists. The results suggested a role of CB1R to control tremor and an implication of both CBRs in the development of spasticity [
102]. In the same work was reported that the endocannabinoid palmitoylethanolamide (PEA) (
Table 2) caused a transient inhibition of spasticity [
102]. However, more recently it was reported that co-administration of PEA with CBD in EAE was not as active as treatment with each compound alone, indicating that these non-psychoactive cannabinoids could have antagonistic interactions in EAE [
103].
New findings indicated that cannabinoids could also target the development of progressive forms of MS, using the TMEV-IDD model of disease [
104]. In particular, in TMEV-infected mice, WIN-55212-2 (
Table 2), arachidonyl-2-chloroethylamide (ACEA), a selective CB1R agonist (
Table 2), and JWH-015, a weak selective CB2R agonist (
Table 2), was demonstrated to improve motor function on established neurological symptomatology, to promote the remyelination, and to reduce microglial activation and the number of CD4+ infiltrated T cells [
105]. Recent studies focused on the development and study of CB2R selective agonists as the best therapeutic approach thanks to their lack of central side effects usually associated with a CB1R modulation. Recent research aimed at the synthesis of CB2R selective ligands bearing different chemical scaffolds to find new agents for the treatment of MS. First of all, a resorcinol derivative, O-1966 (
Table 2), was tested in the chronic EAE model. This compound significantly improved motor function in the chronic EAE model, at concentration of 1 mg kg
−1. Moreover, O-1966 reduced rolling and adhesion of endogenous leukocytes [
106].
1,4-dihydro-6-methylindeno[1,2-c]pyrazole derivative, Gp-1a (
Table 2), with a four log higher affinity for CB2R than for CB1R, was demonstrated to be able to reduce clinical scores and ameliorate the recovery in EAE mice presenting a long term reduction in demyelination and axonal loss. Two different mechanisms were established since it was able to affect Th1/Th17 differentiation in peripheral immune organs and pathogenic T cell accumulation in the CNS and reduce the expression of chemokine and adhesion molecules in the CNS [
107].
Furthermore, in 2015, Fu et al. [
108] showed that intrathecal administration of JWH-133 (
Table 2), a selective CB2R agonist, in EAE mice, dose-dependently reduced both mechanical and cold hypersensitivity without any signs of ataxia or sedation. The co-administration of JWH-133 with a selective CB2R antagonist dose-dependently attenuated the inhibitory effects of JWH-133. This data suggested that the selective targeting of spinal CB2R reduced signs of neuropathic pain in EAE mice without any side effects [
108].
In the same year, Han et al. synthesized new quinoline-2,4(1H,3H)-dione derivatives as CB2R agonists. Among all the synthesized derivatives, compound 21 (
Table 2) was the one shown to significantly reduce the clinical scores and symptoms of the mice EAE model, as shown by the remarkably decreased leukocyte infiltration in the spinal cord and demyelination in white matter [
109].
The following year, chromenopyrazole was identified as a promising scaffold to obtain CBRs ligands [
110]. Structural modifications have been studied in order to achieve CB2R selectivity and the structural changes led to the synthesis of chromenoisoxazole derivative PM-226 (
Table 2) that was shown to be fully CB2R selective with a high affinity constant. PM-226 was found to dampen neuroinflammation in the TMEV mouse model by reducing microglial activation to levels close to those quantified in the control group [
110]. This decrease in the microglia activation lead, as already reported, to a reduction of inflammatory events and an improvement of the neurological status of treated animals [
111].
In 2017, Ying Shi et al. reported the identification of new potent and selective indole based CB2R agonists [
112]. Compound 57 (
Table 2) was selected as a representative analogue to be studied in a mouse EAE model of MS. This compound was significantly shown to alleviate the clinical symptoms and to protect the murine central nervous system from immune damage in EAE mouse model. Further histological examination of spinal cords demonstrated significant reduction in leukocyte infiltration and the extent of demyelination [
112]. This study supporting again the efficacy of selective CB2R agonists in animal models of MS.
Very recently, Navarrete et al. [
113] provided evidence that VCE-004.8 (
Table 2), an aminoquinone derivative of cannabidiol (CBD), is a promising small molecule to modulate relevant MS targets, being a dual PPARγ and CB2 agonist with potent anti-inflammatory activity. VCE-004.8 showed immunomodulatory activity in EAE and TMEV mice models, inhibiting several inflammatory chemokines, chemokines receptors, and cytokines that play a key role in the pathogenesis of MS. In addition, VCE-004.8 inhibited the expression of adhesion molecules such as VCAM and ICAM-1. Also remarkable is the finding that VCE-004.8 strongly induced the expression of the hypoxia-inducible factor (HIF), which can have a beneficial role in MS by modulating the immune response and favoring neuroprotection and axonal regeneration.
Regarding phytocannabinoids, excluding CBD and ∆
9-THC, which are discussed in a specific section of this review, the sesquiterpene β-caryophyllene (BCP) (
Table 2) is worth mentioning. It is a CB2R selective agonist already reported in literature for its anti-inflammatory and analgesic effects in mouse models of inflammatory and neuropathic pain [
114]. In a very recent work, BCP was shown to attenuate disease progression by reducing mechanical hyperalgesia, inflammation, and pain in a mouse EAE model [
115]. In order to prove that BCP effects were due to its actions on CB2Rs, BCP was co-administered with a selective CB2R antagonist, which reversed the BCP effects. Altogether, this data suggested that BCP, binding to CB2R, blocks the development and progression of clinical and neurological signs of EAE [
115].
3.2. Inhibitors of Metabolic Enzymes of ECs
Alternative strategies to modulate ECS are focused on blocking the enzymes that degrade the two main endocannabinoid compounds (2-AG and AEA). This is an interesting therapeutic approach, as enhancing ECs levels is expected to preserve the beneficial effects derived from the direct activation of CBRs but limiting potential side effects mostly associated to direct CB1R agonists. It has been widely demonstrated that, in MS patients, there is a significant alteration of the metabolic enzymes mainly of FAAH and of MAGL [
116,
117].
The effects linked to alterations of the metabolism of endocannabinoids are not completely clear, also because of the different experimental models used and the variations in the recruitment of patients. Different studies in TMEV-IDD mice have assessed that the inhibition of AEA degradation by FAAH determines an improvement of the motor symptoms, with a reduction of inflammatory response and the downregulation of macrophage and of microglial function [
118,
119]. Webb et al. have demonstrated, in tests carried out on an EAE mouse model, that chronic and long-term inhibition of FAAH, via genetic ablation, produces clinical remission and ameliorates long-term results [
120]. Other researchers have shown that high levels of 2-AG can make improvements in the acute phase of MS. Indeed, 2-AG is able to inhibit spasticity when administered at dose of 10 mg kg
−1 and determines a delayed onset in acute and chronic EAE models when given at a concentration of 100 mg kg
−1 [
121].
The focus of the researchers has been mainly on the MAGL inhibitors, as 2-AG is the main endocannabinoid present in the brain, and it is a full agonist of CB1R and CB2R. However, Scholosburg et al. [
122] have shown that increased levels of 2-AG in the brain, due to the chronic MAGL inhibition, provokes a functional antagonism of the cerebral endocannabinoid system [
122]. This was evidenced with tolerance to the analgesic effects of acute enzymatic inhibition, cross-tolerance to CB1R agonists, reduction of expression and function of the CB1R, and interruptions in endocannabinoid-dependent synaptic plasticity.
In a recent work, the utility of irreversible MAGL inhibitor, JZL184, (
Table 3), in the treatment of MS has been demonstrated [
123]. Indeed, the chronic administration of JZL184 reduced the neurological consequences of disease progression in EAE mice. This result is linked to reduce of myelin loss and of inflammation of spinal cord white matter [
123]. Furthermore, it was demonstrated that the repeated administration of JZL184 at a dose of 8 mg kg
−1 did not provoke changes in CB1 receptor expression in the hippocampus. Moreover, there was not tolerance to the anxiolytic and analgesic effects of the MAGL inhibitor [
123].
With the aim to increase the potency of JZL184, Brindisi et al. described the synthesis of new β-lactam-based inhibitors reporting compound 4a (
Table 3) as very potent hMAGL inhibitor, with high selectivity toward FAAH, other serine hydrolases, and CBRs [
124]. This compound in EAE mice showed analgesic effects that were clearly dependent on the increased levels of 2-AG and the subsequent indirect modulation of CBRs. Moreover it exerted a surprising beneficial effect on the progression of the disease, thanks to the CB1R and CB2R mediated action, confirming the hypothesis of a close intersection between the endocannabinoid system and MS. Histological evaluation of myelin showed that, when the EAE mice were treated with compound 4a, myelin-density staining was comparable to that of control animals [
124].
As the irreversible MAGL inhibition causes pharmacological tolerance and receptor desensitization, many researchers have focused on development of reversible inhibitors. An interesting example is given by the compound 21 (
Table 3) synthesized by Hernández-Torres et al. [
125]. This compound showed a submicromolar IC
50 value for MAGL inhibition and very good selectivity against FAAH, ABDH6, and ABHD12 enzymes, the CB1R and CB2R cannabinoid receptors [
125]. In AEA mouse, it was demonstrated its ability to significantly increase the levels of 2-AG in spinal cord, improving clinical symptoms and decreasing tissue damage in the spinal cords. Importantly, catalepsy or other motor impairments that are observed after the administration of irreversible MAGL inhibitors, didn’t occurred.
As reported above, the prolonged inhibition of MAGL enzymes, although effective, provokes negative effects that compromise its biological activity. These effects do not occur by FAAH inhibition. Indeed, it was demonstrated that the prolonged inhibition of FAAH produced no tolerance or no changes in the expression or function of the CB1 receptor [
122].
Pryce et al. [
126] studied the efficacy of FAAH inhibitors to control the spasticity in Biozzi ABH mice. They demonstrated that potent FAAH inhibitors such as CAY10402 (
Table 3) and CAY10400 (
Table 3) inhibited spasticity but did not induce any hypothermia, typical of cannabimimetic effects. However, CAY10400 and CAY10402 have poor pharmacokinetics, and therefore, their development as therapeutic drugs is unlikely [
126].
A valid alternative is given by compound URB597 (
Table 3), which is a potent FAAH inhibitor irreversible, with improved pharmacokinetic profile [
126]. Repeated administration of URB597 in a few days compared to the vehicle demonstrated that this compound induced spasticity alleviation immediately after administration, but spasticity was similarly inhibited after four daily doses. However, the study emphasized a benefit because the level of spasticity at baseline after four administrations was lower than baseline before treatment. Moreover, the use of this inhibitor was not associated with CB1R tolerance.
Another way to modulate the endocannabinoid system is to act on AEA reuptake, and many selective inhibitors of cellular reuptake of AEA, but inactive against other enzyme involved in the degradation of this endocannabinoid, have been developed. Ligresti et al. reported very interesting inhibitors of endocannabinoid reuptake [
127]. In particular, the most potent compound was O-3246 (
Table 3), which has a very high potency as an inhibitor of AEA cellular uptake and a negligible activity as an FAAH inhibitor, CB1R and CB2R ligand, and TRPV1 agonist. This compound was shown to inhibit spasticity in CREAE mice, confirming the potential utility of selective AEA uptake inhibitors as anti-spasticity drugs in MS [
127].
Furthermore, it has been found that UCM707 (
Table 3), a potent and selective inhibitor of the AEA reuptake [
119], was able to improve the motor function in a TMEV-IDD mouse model, and at the histological level, it reduced microglial activation, diminished major histocompatibility complex class II antigen expression, and decreased cellular infiltrates in the spinal cord [
119]. These results confirm the role played by the ECS at the level of immunomodulation, and they are in agreement with experiments that describe how the blockade of microglial activation represses the development of the EAE model of MS.
