Characterisation of Ligand Selectivity, Receptor Trafficking and Novel Cell Models for the Cannabinoid CB2 Receptor

Abstract

A participation of the cannabinoid system in many pathological processes has been defined. More recently the importance of the Cannabinoid Receptor 2 (CB2) in inflammatory reactions of the central and peripheral nervous systems has drawn interest. In vivo evidence suggests targeting the CB2 receptor therapeutically could aid in prevention of inflammatory mediated neurodegeneration and pain management. Thorough pharmacological characterisation of the CB2 receptor is necessary for our understanding of the specific role this target plays in both the periphery and the central nervous system. Therefore, this thesis is concerned with the investigation of the activation, regulation and expression pattern of human CB2 receptors. Activation of the CB2 receptor was investigated through the search for potent, CB2 selective agonists. A series of compounds, based on the previously reported HU-308 structure, were screened for binding and activity at human CB2 receptors through radioligand binding displacement and cAMP assays. Only one of these compounds, HU-910, exhibited a higher efficacy than HU-308 and no compounds matched the potency of the lead drug although several CB2 selective partial agonists were identified. These compounds may prove useful for future in vivo assessment of the therapeutic potential of CB2 agonism. Receptor desensitisation by internalisation is a problem encountered with chronic drug treatment both in vivo and in vitro. However, the agonist induced regulation of the CB2 receptor has been poorly explored in the literature, prompting characterisation of the fundamental properties of the CB2 receptor trafficking with direct evidence of agonist induced internalisation and receptor recycling demonstrated here for the first time. Association of the trafficking Rabs 5 and 11 but not 4 were implicated in this process. Rab5 appears to be involved in the sequestering of CB2 receptors from the cell surface and Rab11 in their re-delivery after agonist induced internalisation. This is the first comprehensive investigation of CB2 receptor trafficking. Finally, a model of the expression of these receptors was pursued using human derived Ntera2/D1 (NT2) cells. NT2 cells were differentiated into neurons and astrocytes and evaluated for their potential to act as a model of human cannabinoid receptor functioning in the central nervous system. In particular, we wished to determine if these cells could provide a model system for the investigation of CB2 function on astrocytes. While functional expression of CB1 receptors by neuronal cells was detected, the presence of either cannabinoid receptor could only be detected at the mRNA level in astrocytes. This agrees with recent in vivo expression profiles of these receptors in the normal human brain. The ability of NT2 derived cells to model neuronal cell types was validated here but their astrocytic properties were questioned and an immature phenotype is suggested due to their expression of several markers of neural precursors. This thesis has therefore characterised fundamental properties of the CB2 receptor in order to advance the search for CB2 selective ligands, understand the trafficking of the receptor and develop appropriate human cell models with the intention of informing future research and the development of CB2 targeted therapeutics.