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.