AD is the most prevalent neurodegenerative disease in the world, affecting around 40–50 million people globally, and is the main cause of dementia, a general term used to describe memory loss and cognitive impairment in individuals over 65 years old. AD is characterized by the presence of extracellular deposits of β-amyloid protein aggregates (Aβ), forming amyloid plaques, and by the presence of intracellular neurofibrillary tangles containing hyperphosphorylated tau protein (Pooler et al., 2014; Spires-Jones and Hyman, 2014; Liu et al., 2019; Long and Holtzman, 2019; Knopman et al., 2021). Although the etiology of AD is not totally understood, it is known that several genetic and environmental factors are involved with the emergence of this disease. Mutations in some genes, such as amyloid precursor protein (APP) and presenilin-1 (PSEN1) and presenilin-2 (PSEN2) proteins, related to the production of β-amyloid peptides, are directly associated to the development of familiar AD, distinguished by early onset. Therefore, mutations in these genes, that correspond to 1–5% of all cases, are considered determinant to the emergence of this disease (Selkoe, 2011; Knopman et al., 2021). On the other hand, mutations or polymorphisms in other genes that code proteins such as apolipoprotein E (ApoE) are considered risk factors to the development of late onset AD. Polymorphism in the allele 4 of ApoE (ApoE-e4), for example, appears in about 40–65% of the individuals diagnosed with AD and it is related to the unbalance in Aβ clearance, leading to its accumulation and aggregation (Long and Holtzman, 2019; Yamazaki et al., 2019). Non-genetic and environmental factors, such as exposure to toxins/viruses, head trauma, cardiovascular diseases, diabetes, and sedentary lifestyle may also be associated with a higher probability of AD development (Selkoe, 2011; Karch and Goate, 2015; Armstrong, 2019). Therefore, it is believed that alterations in Aβ metabolism and dysfunctional tau protein phosphorylation may be the main causes to the emergence of AD, leading to altered synaptic signaling, activation of glial inflammatory responses, alterations in ionic homeostasis and oxidative stress, as well as activation of intracellular pathways in response to stress. Thus, the homeostatic alterations produced by these factors lead to neuronal damage and consequent cell death, contributing to the already known cognitive and memory deficits (Sperling et al., 2011; Heneka et al., 2015; Masters et al., 2015; Ding et al., 2019; Hampel et al., 2021; Figure 1).

Despite the growing scientific advances regarding the comprehension of cellular and molecular mechanisms underlying AD physiopathology, this disease still has no cure. This way, the pharmacotherapy used in the clinic aims to reduce some of the symptoms, though not preventing the progression of the disease and the considerable decrease in life quality of both patients and caretakers.

Although there are few conflicting studies, research involving animal models and post-mortem brain tissue of diagnosed patients have already shown that elements of the ECS may be altered in this pathology (Figure 1). Some studies show, for example, that the expression of CB1 receptors in brain areas such as cortex, hippocampus and basal ganglia is decreased in AD patients (Westlake et al., 1994; Solas et al., 2013), while other studies show no difference in the expression of this receptor in cortex and hippocampus (Lee et al., 2010; Ahmad et al., 2014). On the other hand, studies appear to be more consistent regarding the increase in CB2 receptor expression in AD, which may be correlated to the microglial alteration found in this pathology (Benito et al., 2003; Solas et al., 2013; Basavarajappa et al., 2017; Hopperton et al., 2018). Evidence also shows that 2-AG levels were reduced in the plasma of AD individuals, besides its correlation to cognitive decline presented by these patients, suggesting a possible protective role of 2-AG (Altamura et al., 2015). Additionally, the levels of DAGL and MAGL, enzymes responsible by the canonical pathway of synthesis and degradation of 2-AG, respectively, were increased in the hippocampus of post-mortem AD patients in Braak VI stage, indicating that 2-AG metabolism might be altered according to the stage of the disease (Mulder et al., 2011).

The ECS modulation has been studied as an alternative to AD treatment (Mulder et al., 2011; Manuel et al., 2014). Preclinical studies performed in vitro and in vivo using synthetic drugs to modulate the ECS have already shown positive results regarding the reduction of Aβ plaque deposition and tau phosphorylation, improvement in the cognitive performance and reduction of glial activation and neuroinflammation (Ramírez et al., 2005; Chen et al., 2011, 2019, 2012; Aso et al., 2012; Murphy et al., 2012; Zhang and Chen, 2018; Zhao et al., 2020; Grieco et al., 2021). On the other hand, the use of synthetic compounds, such as THC analogs, in clinical studies promoted an improvement only regarding aggressiveness and agitation in patients, not showing effectiveness in terms of primary symptoms of AD (Passmore, 2008; Woodward et al., 2014; Herrmann et al., 2019).

Phytocannabinoids have also been used in preclinical and clinical trials. In SH-SY5Y cell lineage transfected with APP (695), the treatment with CBD in several concentrations (10^–9–10^–6 M) decreased the content of APP and Aβ42 through activation of the PPARγ receptors (Scuderi et al., 2014). In rats injected with intrahippocampal Aβ, the treatment with CBD (10 mg/kg, i.p.) for 15 days led to the decrease in neuroinflammation and increase of hippocampal neurogenesis. Interestingly, these effects were abolished after administration of GW9662 (a PPARγ antagonist; 1 mg/kg, i.p.), reinforcing the involvement of this receptor in the beneficial effects following CBD administration (Esposito et al., 2011). Later, it has been shown that the treatment with CBD 2.5 or 10 mg/kg, i.p. during 7 days promoted a dose-dependent decrease in the iNOS, GFAP, and IL-1β expressions in the hippocampus of C57BL/6J mice injected with intrahippocampal Aβ (Esposito et al., 2007; Figure 2). Additionally, PC12 cultures incubated for 36 h with Aβ1-42 and treated with CBD (10^–6–10^–4 M) also had reduction in p38 MAPK phosphorylation, as well as decrease in the activity of NF-κB transcription factor, suggesting another mechanism of action to the protective effects observed with the use of this phytocannabinoid (Esposito et al., 2006).

Moreover, the synergic role of THC and CBD in animal models of AD has also been evaluated. In 6 months old APP/PS1 mice, the neuroprotective role of the treatment with isolated THC (0.75 mg/kg, i.p.), CBD (0.75 mg/kg, i.p.), or their combination for 5 weeks was assessed. Even though animals treated with either isolated phytocannabinoids or their combination showed improvement in tests designed to assess memory and learning, such as the novel object recognition and active avoidance, only animals that received THC + CBD had a decrease in soluble Aβ42 levels and gliosis in cerebral cortex (Aso et al., 2015). On the other hand, when the same experimental design was performed with 12-months old mice, the positive results observed regarding Aβ42 levels and gliosis were lost, suggesting that the mechanisms of action mediated by these phytocannabinoids might be more effective at initial stages of AD (Aso et al., 2016; Figure 2).

Another important molecular feature found in neurodegenerative diseases is the failure in protein homeostasis mechanisms, resulting in undesirable aggregation of misfolded proteins. In this context, CBD has exerted its protective effect over several signaling cascades involved with proteostasis, consequently reducing oxidative stress in cells (Dash et al., 2021). It has been demonstrated that CBD is able to downregulate genes involved in development of AD in gingiva derived mesenchymal stem cells (GMSCs), more precisely in secretases responsible for Aβ generation and kinases involved in tau protein phosphorylation. It was also shown that CBD can upregulate the expression of several heat shock proteins (HSP) and the activity of ubiquitin systems, responsible to maintain proteostasis (Libro et al., 2017).

Regarding clinical studies using phytocannabinoids, the data are still feeble. However, studies show improvements in some important secondary symptoms related to AD. In a randomized controlled study with 11 patients diagnosed with AD or vascular dementia, the outcome of treatment with THC 1.5 mg, orally administered three times a day for 3 weeks, was evaluated. In this study, patients did not show improvements in the Neuropsychiatric Inventory (NPI) scale, therefore no amelioration of agitation, life quality aspects and performance in daily activities was reported (van den Elsen et al., 2015). Moreover, the same group showed that THC in the same dosage, twice a day, did not promote adverse effects regarding balance and gait of patients (van den Elsen et al., 2017). On the other hand, in an open-label pilot study with 11 AD patients, THC 2.5–5 mg twice a day for 4 weeks promoted improvement in the Clinical Global Impression (CGI) scale, as well as reduction in aggressiveness/agitation, apathy, irritability, and sleep disturbances, assessed by NPI. However, there was no effect regarding MMSE (Mini Mental State Examination) scores (Shelef et al., 2016).

Another pilot study conducted in a nursing home in Switzerland showed that the administration of CBM, with known THC:CBD concentrations (1:2 ratio) decreased the use of other psychotropic drugs used in the treatment of dementia in women with AD, besides improving behavioral and psychological symptoms of dementia. The CBM was administered with food to facilitate the intake, and the minimum dosage used was 7 mg of THC and 14 mg of CBD, while the maximum was 13 mg of THC and 26 mg of CBD per day, during the course of 2 months. It was reported improvement in neuropsychiatric and motor scores and in aggressiveness. The feedback offered by nurses and relatives was positive, when taking into consideration the noticeable reduction in aggressive behaviors, such as screaming and irritability, and motor issues, such as muscle stiffness, that collectively disturbed the life quality of other patients, staff and caregivers (Broers et al., 2019; Figure 2).

Recently, an extensive systematic review evaluating the use of phytocannabinoids, especially THC, in the treatment of AD, showed that this compound is effective in improving some secondary symptoms of this pathology, but not in aspects regarding memory deficits and cognition (Bosnjak Kuharic et al., 2021). Although in preclinical studies CBD has shown promising effects in controlling the characteristic neuroinflammation associated with AD models and behavioral improvements, there are still no clinical studies evaluating positive effects of CBD in the control of AD symptoms in patients.

In summary, the need for more studies using phytocannabinoids in different therapeutic dosages and treatment regimens is notorious, in order to better understand the effect of these compounds in AD, as well as to establish standardized therapies with phytocannabinoids.

PD is considered the second most prevalent neurodegenerative disease, only behind Alzheimer’s disease. It is estimated that about 1–2% of the population between 65 and 70 years old will present the diagnosis and, with the increase of life expectancy, it is believed that about 9 million people might present the disease in the next 10 years (Wirdefeldt et al., 2011; Tysnes and Storstein, 2017). One of the main PD symptoms is the motor disturbance characterized by bradykinesia, that consists in the presence of stiffness, tremors and slowing down of voluntary movements (Larsen et al., 1994). As the disease develops, other symptoms can appear, such as: cognitive alterations, depression, and anxiety (Lees and Smith, 1983; Schrag and Taddei, 2017). Additionally to the motor, cognitive and psychiatric damages, sensorial, autonomic, and sleep disturbances can develop in parkinsonian patients, most of the time manifesting years before the appearance of motor alterations (Chaudhuri K. R. et al., 2006; Sauerbier et al., 2017). These symptoms result from a series of molecular alterations that occur gradually during the disease.

At the cellular level, PD consists in the progressive death of dopaminergic neurons found in substantia nigra pars compacta and consequent loss of projections to the striatum, which is responsible for most of the motor deficit observed in patients (Dauer and Przedborski, 2003; Olanow et al., 2009). This neurodegenerative process results in the depletion of dopamine, promoting the dysfunction of important pathways related to the control of voluntary movements, involving the basal ganglia, cerebral cortex, thalamus, and brainstem (Alexander and Crutcher, 1990; Dauer and Przedborski, 2003; Olanow et al., 2009). The degeneration of dopaminergic neurons results from a series of dysfunctions intertwined, promoting a disbalance in cellular homeostasis and production of trophic factors. Among the processes involved are alterations in proteostasis, oxidative stress, mitochondrial damage, inflammation, and apoptosis (Olanow et al., 2009; Winklhofer and Haass, 2010; Stojkovska et al., 2015; Guo et al., 2018; Figure 1). The etiology of PD has not been clarified yet. However, it is suggested that its development might be related to environmental factors (Olanow et al., 2009; Ascherio and Schwarzschild, 2016), such as exposure to toxins and lifestyle, and genetic factors, such as mutations in genes like PARK1/4, PARK2, and PINK1 (Winklhofer and Haass, 2010; Ali et al., 2011; Riederer et al., 2019). Another important physiological feature in PD is the presence of Lewy bodies in substantia nigra, structures consisting of cytoplasmic inclusions mainly composed by the accumulation of α-synuclein aggregates, closely related to multiple neurodegenerative processes (Hurtig et al., 2000; Riederer et al., 2019).

To date there is no pharmacological treatment to prevent the progression of PD, with the control of the main symptoms being the protocol used. The main pharmacological treatment used in PD patients is levodopa, administered with peripheral dopa decarboxylase inhibitors, such as benserazide or carbidopa, due to its rapid peripheral metabolization. The synergic effect between these drugs allows a greater availability of levodopa to the brain, besides reducing the adverse effects related to the increase in peripheral dopamine. After a certain period of treatment, other pharmacological interventions may be necessary to maintain the dopaminergic levels and act over the other non-motor symptoms (Connolly and Lang, 2014). Because of the progressive neurodegeneration, the fluctuations in the response with levodopa treatment, referred to as “on-off,” become more frequent. There are “on” periods, when the drug exerts its effect over motor control, and “off” periods, when the clinical effects cease and motor symptoms reappear (Poewe et al., 2017). Consequently, patients that are chronically users of levodopa may develop dyskinesia—a collateral effect known as levodopa-induced dyskinesia (LID)—in the “on” period. The mechanisms to the development of LID are complex and, besides the strong dopaminergic component, there is also the involvement of glutamatergic, serotoninergic and endocannabinoid systems, neuroinflammatory mechanisms, among others (Zheng and Zhang, 2021). Several scientific studies have brought even more understanding over the mechanisms underlying PD, allowing the development of new strategies to treat it. In this way, several antioxidants, anti-inflammatory, and nutraceuticals have been indicated (Hang et al., 2016; Sarkar et al., 2016). Besides that, current adjuvant pharmacotherapies try to attenuate the disease’s symptoms without developing LID, such as anticholinergic drugs, dopaminergic agonists, and amantadine (Connolly and Lang, 2014).

Scientific evidence obtained in animal models of PD and patients show that there are alterations in components of the ECS in PD pathology. Using the experimental model of 6-hydroxydopamine (6-OHDA) injection into the substantia nigra of rats, it was observed an increasing in AEA levels in the striatum of lesioned animals, while observing a decreasing in the activity of the AEA membrane transporter, as well as the endocannabinoid degradation enzyme FAAH (Figure 1). It was also observed that the inhibition of this enzyme promoted a decrease in the glutamatergic activity in the striatum of animals injected with 6-OHDA. Other studies showed in PD models that the pharmacological increase in the endocannabinoid levels and/or direct activation of cannabinoid receptors was capable of reducing the death of dopaminergic neurons, recovering the dopamine levels and the activity of the enzyme tyrosine hydroxylase, as well as improving motor deficits and neuroinflammatory parameters (García-Arencibia et al., 2007; Price et al., 2009; Fernández-Suárez et al., 2014; Mounsey et al., 2015). These data suggest that the modulation of this system’s activity might be an interesting strategy to regulate the excessive excitability in the circuits connected to the basal ganglia in PD (Gubellini et al., 2002). To reinforce the relationship between the ECS alterations and PD, the analysis of the cerebrospinal fluid of patients showed an increase of almost twofold in the AEA levels (Pisani et al., 2005). Besides, a decrease in the expression of CB1 mRNA was detected in the striatum and globus pallidus external of post-mortem samples of individuals with PD (Hurley et al., 2003; Figure 1).

Regarding the physiological control of voluntary movements, the modulatory effects of the ECS are well established in the literature, mainly through the regulation of neurotransmission in structures of basal ganglia, such as striatum, globus pallidus external and internal, and substantia nigra. Moreover, the activation of cannabinoid receptors in subpopulations of GABAergic and glutamatergic neurons located in these regions also modulate the dopaminergic signaling, specially through interaction between CB1 receptors and dopaminergic receptors D1 and D2 (Benarroch, 2007; Bassi et al., 2017).

Nowadays, some preclinical and clinical studies try to unravel the mechanisms underlying the benefits that cannabinoids and CBM may have on the symptoms of PD. In this context, the neuroprotective effects of THC and CBD, as well as the possible action pathways of these molecules, have already been observed in toxicity models in vitro. In the cell lineage SH-SY5Y of human neuroblastoma incubated with MPP + toxin, the treatment with THC 10 μM increased the viability of these cells. Besides, THC also decreased cell death in response to other toxins, such as paraquat (inductor of oxidative stress) and lactacystin (inhibitor of the proteasome-ubiquitin system). Interestingly, these neuroprotector effects were not mediated by the activation of CB1 receptors, but PPARγ and its consequent involvement in regulating of redox homeostasis (Carroll et al., 2012). Additionally, the same group showed that the effects promoted by THC were related to prevention in mitochondrial mass reduction through increasing in PPARγ coactivator 1α (PGC-1α) expression, involved with metabolism regulation and mitochondrial biogenesis, as well as preventing reduction of the mitochondrial transcription factor A levels (TFAM), a protein involved in replication of mtDNA. This evidence highlights the protective role of THC through modulation of mitochondrial homeostasis (Zeissler et al., 2016). Cultures treated with CBD 10 μM for 48 h showed increased cell viability through activation of CB2 and TRPV1 receptors (Gugliandolo et al., 2020). Additionally, in another study using in vitro toxicity with MPP+, using PC12 cells, it was demonstrated that the treatment with CBD 1 μM for 24 h increased cell viability and differentiation, as well as a higher expression of GAP-43 protein, associated to neurite growth, and proteins associated to synaptic vesicles, such as synaptophysin and synapsin I (Santos et al., 2015; Figure 2).

The use of phytocannabinoids has also shown positive results in murine models of PD. With the 6-OHDA model, a consecutive treatment with isolated CBD (3 mg/kg/day, intraperitoneally) or THC (3 mg/kg/day, intraperitoneally) in rats showed a recovery in dopamine and DOPAC levels and tyrosine hydroxylase activity, both in substantia nigra and striatum (Lastres-Becker et al., 2005; Figure 2). Two years later, using the same isolated CBD treatment, the same group showed that the neuroprotective effects were only efficient if the treatment was initiated concurrently with the lesion. In this study, it was still shown that CBD promoted increase in the expression of mRNA of the enzyme Cu,Zn-Superoxide Dismutase (SOD1), suggesting that this compound might act over antioxidant pathways (García-Arencibia et al., 2007).

Other Cannabis components, besides CBD and THC, were also used as therapeutic strategies in experimental models of PD. β-caryophyllene, a terpene with cannabimimetic properties for acting as a CB2 agonist, showed antioxidant and anti-inflammatory effects, thus reducing the death of dopaminergic neurons in substantia nigra and striatum in a PD model induced by rotenone (Ojha et al., 2016). Besides, THCV, a minor phytocannabinoid, has also shown neuroprotective effects in a model induced by 6-OHDA (García et al., 2011). An important problem to be considered in the current treatment of PD concerns the development of LID. This way, studies testing the effects of phytocannabinoids in this aspect are extremely relevant in the clinic. In animal models of LID, for example, the use of CBD and THCV showed positive results, decreasing stereotyped movements as well as proinflammatory markers (Figure 2). Regarding CBD, these effects were mainly attributed to their action over PPARγ and TRPV1 receptors (Dos-Santos-Pereira et al., 2016; Sonego et al., 2018; Espadas et al., 2020).

Clinical trials using phytocannabinoids with parkinsonian patients, besides presenting variable effects, point to the promising effects in the use of these phytocannabinoids and CBM in the disease’s symptoms. An open pilot study aimed to evaluate the efficacy of isolated CBD in patients diagnosed with PD presenting secondary psychotic symptoms. The use of crescent doses of isolated CBD (150–400 mg/day) for 4 weeks showed a progressive improvement in the psychosis symptoms related to the disease, but not motor parameters (Zuardi et al., 2009; Figure 2). Similarly, the same group showed, in a double-blind exploratory study, that the treatment with isolated CBD in capsules (300 mg/day) during 6 weeks induced improvement in daily activities and emotional wellness of patients when compared to those treated with placebo. However, changes related to motor behavior were not reported (Chagas et al., 2014).

In a crossed, randomized, double-blind, placebo-controlled study, Carroll et al. (2004) assessed the potential effect of a CBM (Cannabis extract containing a THC:CBD proportion of about 2:1, each capsule containing 2.5 mg of THC and 1.25 mg of CBD) in PD patients that presented LID. Although the authors had observed that oral treatment was well tolerated, the patients did not show relief in parkinsonian symptoms. Besides, despite the double-blind design, 71% of the patients correctly identified their respective treatment group. Additionally, it was not observed any effect of the use of CBM over the LID symptoms, as evaluated by the UPDRS questionnaire (Unified Parkinson’s Disease Rating Scale) and by Rush dyskinesia evaluation scale. There was also no improvement in any measures of secondary results, such as other motor parameters of UPDRS score, PDQ-39, pain or sleep quality (Carroll et al., 2004).

This way, it is observed that clinical studies involving PD and CBM or isolated phytocannabinoids are also very incipient and with few conclusive data. Nevertheless, the use of CBM is promising, especially when considering all previous evidence involving basic science with synthetic drugs or phytocannabinoids. Moreover, a relevant factor regarding the variety of results obtained in clinical research points to the variability in establishing and standardizing the time of treatment, the dosage of CBM or isolated components, and administration routes.

MS is a chronic autoimmune and progressive neurological disorder that affects more than 3 million people worldwide and is considered the most prevalent neurodegenerative disease in young adults (Reich et al., 2018). Disturbances in the neurovascular unit allow the infiltration of auto-reactive T cells into the CNS, which is facilitated by the activity of endothelial adhesion molecules (ICAM-1 and VCAM-1) and matrix metalloproteinases (MMP-2 and MMP-9), therefore promoting the degradation of myelin sheath and multifocal demyelinating lesions, mostly in the white matter, due to several immune-mediated mechanisms already postulated, such as cytokine release, cytotoxic attack of CD8⁺ T cells and macrophage-induced digestion of surface myelin antigens (Podbielska et al., 2021; Figure 1).

Even though the precise cause of MS may not be fully elucidated, several risk factors that could lead to disease onset have already been identified. Among environmental triggers for MS, infection by Epstein-Barr virus, vitamin D deficiency, obesity, tobacco consumption and exposure to toxins are highlighted, while genome-wide association studies identified more than 200 gene variants that could lead to elevated risk of MS development, such as the human leukocyte antigen DRB1*1501 haplotype (Reich et al., 2018).

The clinical progression of MS is classified according to the stages of progression and/or improvement of MS severity. Most patients experience a relapsing course of MS progression (relapsing-remitting multiple sclerosis, RRMS), which could become progressive in later stages, showing signs of steadily worsening neurological damage (secondary progressive multiple sclerosis, SPMS). In other cases, which accounts for the minority of patients, the disease gets progressive since the onset, with no relapsing stages (primary progressive multiple sclerosis, PPMS) (Absinta et al., 2020). Despite the different etiologies of MS, clinical manifestations may include optic neuritis, myelitis, motor and vestibular impairment, paroxysms, Uhthoff’s phenomenon, fatigue, and cognitive disorders (Frohman E. M., 2003).

In the pathogenesis of MS, glutamate-mediated excitotoxicity may greatly contribute to disease progression due to induced neuronal death, axonal loss and demyelination (Macrez et al., 2016). Not only neurons are responsible for increased release of glutamate in synapses, but also other cell types may contribute to the elevated level of this neurotransmitter. In the context of infiltration of peripheral immune cells into the CNS, monocytes, macrophages and dendritic cells, besides microglia, are able to release glutamate through the cysteine/glutamate antiporter Xc^– activity (Pacheco et al., 2006; Pampliega et al., 2011; Evonuk et al., 2015). The excessive glutamate may also induce AMPA-mediated excitotoxic cell death of oligodendrocytes, NMDA-mediated damage to myelin integrity, and overexpression of sodium-channel subunits Na_v1.2 and Na_v1.6 the white matter, resulting in Na⁺ accumulation in axons (Stys et al., 1993; Domercq et al., 2005; Young et al., 2008; Micu et al., 2016).

Furthermore, glial cells can exert a significant effect on neuroinflammatory-induced changes in CNS, which leads to aggravation of MS. Following injure in the neurovascular unit, T-cells infiltrate the CNS and release proinflammatory cytokines, such as TNF-α, IL-17, and IFN-γ, which in turn induce the activation of astrocytes and microglia (Rothhammer and Quintana, 2015). Excessive microglial activation is correlated to elevated release of reactive oxygen and nitrogen species (ROS and RNS, respectively) in MS and is particularly harmful to oligodendrocytes precursor cells (OPC) due to their low level of antioxidant enzymes and antiapoptotic proteins (Butts et al., 2008; Gray et al., 2008; Fischer et al., 2012; Figure 1).

The activation of CB1 and CB2 receptors is essential to control pre-synaptic release of glutamate in excitatory synapses and to modulate the release of proinflammatory cytokines produced by glial cells, respectively, contributing to mitigate excitotoxicity and neuroinflammation (Ligresti et al., 2016). In the pathological context of MS, imbalance of ECS components and cannabinoid-mediated signaling pathways may account for the perpetration of abnormal insults to homeostasis and consequent MS symptomatology (Rossi et al., 2010).

In animal model of experimental autoimmune encephalomyelitis (EAE), AEA, and 2-AG levels have been found downregulated in striatum, midbrain, brainstem, hippocampus, and cerebral cortex, which could be reverted by administration of AM404, an inhibitor of FAAH enzyme (Cabranes et al., 2005). Moreover, lower levels of endocannabinoids were also reported in cerebrospinal fluid of MS patients, highly correlated to disease subtype and relapse stage (Di Filippo et al., 2008). On the other hand, increased levels of AEA have been reported in cerebrospinal fluid, inflammatory lesions, lymphocytes and plasma of MS patients, despite lack of change in 2-AG levels (Eljaschewitsch et al., 2006; Centonze et al., 2007; Jean-Gilles et al., 2009; Figure 1).

Besides altered levels of endocannabinoids, the expression of FAAH has been reported as upregulated in MS plaques obtained from brain tissue of human patients, which may seem controversial when compared to AEA concentration in other tissues and/or fluids (Benito et al., 2007; Figure 1). Usually, delivery route, tissue origin, cannabinoid pharmacokinetics and pharmacodynamics, outcome measurement and other bias are accounted as responsible for diverging observations.

Cannabinoids have been used as a tool to better elucidate in vitro and in vivo the potential role of ECS in reducing excitotoxicity and neuroinflammation. It has been reported that cannabinoid receptor agonists exert neuroprotective effects in mixed cortical cultures exposed to AMPA and NMDA, therefore reducing neuronal death (Docagne et al., 2007; Loría et al., 2010). Additionally, the CB1-mediated signaling is essential for tremor and spasticity control in animal model of chronic relapsing experimental autoimmune encephalomyelitis (CREAE) and EAE in CB1 knockout mice (Baker et al., 2000; Pryce and Baker, 2007). Besides, the dual activation of CB1 and CB2 by WIN 55,212-2, a cannabinoid agonist, improves the clinical score shown by animals, reduces inflammation and restores tolerance to self-myelin antigen (Arevalo-Martin et al., 2012), while WOBE437, an inhibitor of eCB reuptake, reduces MS severity and infiltration of immune cells into the CNS (Reynoso-Moreno et al., 2021).

Considering the use of phytocannabinoids in animal models of MS, it has been described that CBD is able to attenuate infiltration of T cells into the spinal cord, the brain and also reduce microglial reactivity and motor deficit, in A2 adenosine receptor-dependent mechanism (Kozela et al., 2011; Mecha et al., 2013). Further to migration and morphological adaptation of immune cells, the release of proinflammatory cytokines is reduced in EAE animals treated with CBD (Rahimi et al., 2015; Elliott et al., 2018; Figure 2).

Another formulation of CBM can be used in the form of THC and CBD combination, due to pharmacological interaction and targets of both compounds in physiological systems. Even though isolated CBD and THC were effective in the clinical score reported for EAE mice, only the combination CBD/THC and isolated THC was able to induce long-term effectiveness in a CB1-dependent manner, which led to decrease in the disease progression (Moreno-Martet et al., 2015). Furthermore, a 1:1 combination of CBD/THC decreases the expression of Tumor Necrosis Factor-α (TNF-α) and increases Brain-Derived Neurotrophic Factor (BDNF) release (Zhou et al., 2019; Figure 2).

Considering that Cannabis extracts contain a large number of secondary metabolites from Cannabis sativa, the combined potential of these molecules could be important to induce a potentiated combined effect, called entourage effect. As an example, it has been shown that the terpene β-caryophyllene has the property of inhibiting T cell activity, along with reduced proinflammatory cytokine release, thus attenuating MS severity, inflammatory stress and spinal cord demyelination (Alberti et al., 2017).

Considering the diverse nature of MS symptoms, treatments currently available are not sufficient to mitigate the progression of this disorder and may lead to side effects, tolerance and toxicity. Thus, the search for new therapeutic approaches have intensified and phytocannabinoids have been demonstrated as important allies for MS treatment (Figure 2). In a parallel group, double-blind, randomized, placebo-controlled study, 160 MS patients were treated with oromucosal spray of CBM (2.7 mg THC and 2.5 mg CBD) during 6 weeks, with a mean dose of 2.5–120 mg of each daily. The Visual Analogue Scale (VAS) score was used to assess the primary outcome and results showed reduction of mean 74.36–48.89 in patients treated with Cannabis extract, significantly relevant when compared to placebo group (Wade et al., 2004). Later, the same group published a study to assess the safety and efficacy of this CBM, which included the evaluation of 137 MS patients refractory to standard drugs and described lack of efficacy in 42.3% of patients. For those who experienced improvement in clinical conditions, the amelioration of symptoms remained stable even after the acute phase and only mild to moderate adverse effects were reported (Wade et al., 2006).

The evaluation of CBM efficacy on spasticity of 189 patients in a double-blind study over 6 weeks, which showed positive change from baseline in the severity of spasticity measured by the 0–10 numerical rating scale (NRS), besides the Ashworth Scale and Motricity Index in muscles affected by spasticity, analyzed by intention-to-treat (ITT) (Collin C. et al., 2007). The group has performed a similar ITT analysis after NRS in a larger number of patients, refractory to anti-spasticity therapies, describing the efficiency of CBM in reducing treatment-resistant spasticity in treated subjects within the first 4 weeks of treatment (Collin et al., 2010).

In 2011, a multicenter study was conducted during 19 weeks to assess efficacy and safety of CBM in two phases. In phase A, which was single-blind and preliminary, subject responsiveness to CBM treatment has been evaluated for 4 weeks, and only those that showed at least 20% reduction in spasticity scores were allowed to proceed into phase B, a double-blind, randomized, parallel-group and placebo-controlled design, during 12 weeks. ITT analysis corroborated previous findings supporting CBM use for treating spasticity, besides positive outcome in Spasm Frequency Score, Sleep Disturbance NRS Patient, Carer and Clinician Global Impression of Change (Novotna et al., 2011). Posterior post hoc analysis of these data showed that results reported could be observed regardless of anti-spasticity pre-treatment history, accounting for consistent relief of symptoms and good tolerability (Haupts et al., 2016).

The phase 3 trial named MUSEC (MUltiple Sclerosis and Extract of Cannabis) consisted of a double-blind, randomized, placebo-controlled, multicenter study with 279 stable MS patients, initiated by a screening period, followed by a 2-week dose titration phase, then a 10-week maintenance period. Differently from the studies previously reported, capsules containing THC as the main cannabinoid were administered to patients twice daily. Results demonstrated relief of muscle stiffness, body pain, spasms and sleep quality after 12 weeks in approximately 29.4% of patients (Zajicek J. P. et al., 2012).

An observational, prospective, multicenter, and non-interventional study conducted in Germany, named MOVE 2, evaluated the response of patients with moderate to severe MS spasticity to CBM treatment. About 74.6% of patients showed relief in spasticity after 1 month, which was stable after 3 months in patients who could undergo follow-up analysis, in accordance with previous studies. Furthermore, improvements in sleep disorders and quality of life have also been reported, besides being well tolerated, once the most common adverse effects reported were dizziness, drowsiness, fatigue, nausea, and dry mouth (Flachenecker et al., 2014). To expand discoveries from the German experience, MOVE 2 design was applied to other European countries. Thus, results from Italy describe a similar phenomenon previously observed, once there at least 49% of patients showed stable improvement of NRS for spasticity after 3 months of treatment, whose main adverse events were dizziness and confusion, reported by 13% of patients in the 3-month follow-up period (Trojano and Vila, 2015).

More recently, the Belgian experience has also been reported in a retrospective study with 238 patients from 8 centers. Improvement in the MS spasticity NRS was significantly lower from 4 to 12 weeks when compared to baseline, which accounted for 73% of patients with improvement ≥ 20% in NRS. Additionally, the self-rated health-related life quality score (EuroQoL Visual Analog Scale) during treatment expressed a decreased disease burden perceived by patients, once 33% of them reported apparent amelioration of this aspect by 4 weeks and stable at 12 weeks (D’hooghe et al., 2021).

In summary, these data suggest that CBM composed by equal concentration of THC and CBD provides an efficient, well-tolerated and safe option to treat spasticity due to MS progression, besides being effective for amelioration of co-morbidities associated to quality of life of patients, such as sleep disorders, even though the progression of disease at neurodegenerative level is not completely assessed.