• 2-ag
• anandamide
• cannabinoid receptor
• CB1/CB2 dual agonist
• CNS-disorders
• endocannabinoid reuptake
• endocannabinoid system
• multiple sclerosis
• neuroinflammation
• PAM
• polypharmacology
• positive allosteric modulator

The endocannabinoid system is a widespread neuromodulatory system with wide-range of actions. It comprises cannabinoid receptors, endocannabinoids (endogenous cannabinoids) and the enzymes responsible for the synthesis and degradation of the endocannabinoids.
The two main receptors of this system are G-protein coupled receptors (GPCRs): cannabinoid receptor type-1 (CB1) and type-2 (CB2). CB1 receptor is mainly expressed in the brain, while CB2 receptor in the periphery, especially in the immune system.
Endocannabinoids are endogenous ligands for CB1 and CB2 receptors and are produced “on demand” from membrane lipid precursors by multiple biosynthetic pathways. The main endocannabinoids are 2-arachidonylglycerol (2-AG), a full agonist for both CB1 and CB2 receptors and N-arachidonyl-ethanolamine (anandamide, AEA) which act as partial agonist for both CB1 and CB2. The biological activities of these lipid mediators are terminated upon cellular re-uptake and subsequent metabolism. The main metabolic enzymes are fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) that are involved in AEA and 2-AG metabolism, respectively.
The endocannabinoid system plays a role in many biological conditions as immune response, neuroprotection, regulation of appetite, metabolism, pain, endocrine and vascular response.
Many evidences suggest the involvement of the endocannabinoid system in the modulation of neuroinflammatory disorders such as Alzheimer’s Disease (AD), Multiple Sclerosis (MS), Huntington and Parkinson Disease (Chiurchiù, 2018) .
In these conditions, the immune response damages component of the nervous system.
A common feature of neuroinflammatory disorders is the hyperactivation of microglia that lead to secretion of many reactive species, include cytokines, chemokines, glutamate, prostanoids and free radicals that cause oxidative stress.
Multiple sclerosis is characterized by demyelination of the axons in the central nervous system (CNS). It gradually leads to progressive neurodegeneration that entail neuronal dysfunction and a wide spectrum of neurological symptoms. Characteristic symptoms of MS are spasticity, sensory alterations, weakness, painful spasms, bladder dysfunction, tremor, ataxia, optic neuritis, fatigue, and dysphagia.
Available treatments for multiple sclerosis are able to control some symptoms and to improve disease progression of certain forms of the disease.
The endocannabinoid system provides an attractive target for managing microglial-derived neuroinflammation and the modulation of the endocannabinoids could be beneficial for specific symptoms and for the slowdown of disease progression.
Several evidences show that targeting CB1 receptor can provide neuroprotection by regulating glutamate homeostasis and limiting excitotoxic damage (De Lago, 2012), moreover the neuroprotective action of CB2 activation have been shown in preclinical models of MS (Croxford et al., 2008).
The aim of this project is to assess the effect of novel multi-target approaches to target the endocannabinoid system. We evaluated three different pharmacological approaches:
• CB1/CB2 dual agonist; we tested the compound FM6b, synthetized in the laboratory of Prof. Manera and still unpublished. We assessed the effect of the activation of both cannabinoid receptors in the active experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. We also estimated the effect of FM6b in a full tetrad test, in order to evaluate the impact of CB1 direct activation.
• First prototype of selective endocannabinoid reuptake inhibitor, WOBE437 (Chicca et al., 2017; Reynoso-Moreno et al., 2018). WOBE437 was designed, synthetized and biologically profiled in the laboratory of Prof. J. Gertsch at the Institute of Biochemistry and Molecular Medicine of the University of Bern (Switzerland), where I performed the experimental part of my master thesis. This molecule is able to increase the endocannabinoid system through the modulation of the endogenous AEA and 2-AG. We evaluated the in vivo effect of the inhibition of the endocannabinoid transporter in EAE mouse model.
• Combination of WOBE437 and the first synthetic positive allosteric modulator of CB2 receptor, EC21a (Gado et al., 2018). Since in preliminary experiments with WOBE437 there was a significant CB2 mediated pharmacological effect, we wanted to evaluate the combination of the increase of endocannabinoid levels and the allosteric modulation of CB2 receptor in the same animal model of multiple sclerosis.
After the evaluation of the effect of these molecules in vivo, we collected the organs and we quantified the endocannabinoid levels in plasma, brain, cerebellum, spleen and lumbar spinal cord, using LC-MS.
As last, we evaluated the functionality of cannabinoid receptors, performing GTPS assay in brain samples, in order to assess the possible desensitization of the receptors upon chronic treatment.