The molecular pharmacology of phytocannabinoids

Phytocannabinoids may be defined as C21 or C22 compounds predominantly found in Cannabis sativa. Over 100 of these compounds have been identified and 89(-)-tetrahydrocannabinol (THC) is the principal psychoactive compound. THC and Cannabidiol (CBD), which make up the most abundant constituent of some cannabis chemotypes, are the most studied phytocannabinoid. The biological activities of other phytocannabinoids are less clear. We do not fully understand how they interact with protein targets, which means their therapeutic potential may not be fully explored. We also do not have enough evidence to refute or accept the hypothesis of entourage effect. Entourage effect means that inactive constituents of Cannabis augment the biological effect of active constituents. Without understanding the biological effect of each phytocannabinoid, it will be difficult explaining observations that have been reported in preclinical models like rats and mice.

This thesis investigates the molecular activities of phytocannabinoids namely CBN, CBG, CBC, CBDV, CBGA, THCA, CBDA, CBDVA at CB₁, CB₂ and TRPAl channels. We also investigated the structure-activity relationship of synthetic analogues of cannabichromene (CBC). This was carried out by measuring cellular hyperpolarization through membrane potential assay in FlexStation 3. We also used calcium dye assay, bioluminescence resonance energy transfer (BRET) assay and AlphaLISA assay to measure increase in intracellular calcium, inhibition of cAMP formation and stimulation of ERK phosphorylation respectively.

We found that CBC is a CB₂ receptor agonist and does not activate CB₁ receptors. CBC activated CB₂ receptors and transmitted downstream signals through multiple pathways. It induced cellular hyperpolarization (by opening of G-protein inwardly rectifying potassium channels), phosphorylation of ERK, and inhibited forskolin-induced cAMP stimulation. We provided evidence that CBC activity was mediated downstream through Gi-protein. CBC was also capable of internalising surface CB₂ and receptors in a mechanism that is independent of G-protein receptor kinase (GRK)

We also discovered that the biological activity of CBC at CB₂ receptors was due to one enantiomer. CBC enantiomers are herein referred to as 'slow-CBC' and 'fast-CBC' pending the resolution of their circular dichroism (i.e. assigning (-) and ( +) to the appropriate enantiomer). Slow-CBC stimulated CB₂ receptors with similar efficacy to the racemate, while fast-CBC was relatively inactive. The structure-activity relationship of CBC showed that the complete truncation of the pentyl side chain will lead to loss of activity. Extensive expansion of the aromatic core will also lead to loss of activity. However, 5-fluoro substitution on the pentyl chain retained activity similar to the parent compound.

We also found that cannabigerol (CBG) does not activate CB₁ or CB₂ receptors, unlike cannabinol (CBN), which activates CB₁ receptors and behaves as a CB₂ receptor antagonist. Our data also shows that while cannabidivarinic acid (CBDA), tetrahydrocannabinolic acid (THCA) and cannabigerolic acid (CBGA) caused cellular hyperpolarization, this effect was not mediated through CB₁ or CB₂ receptors. 

Our findings are important because it has identified CBC as a CB₂ agonist and provided evidence that it can be further exploited for the treatment of diseases such as neuropathic pain without concern for a psychoactive effect. Overall, this work has provided information about the molecular activities of some phytocannabinoids at CB₁, CB₂ and TRP Al channels, which is relevant in the global research towards the opimal exploitation of Cannabis and its constituents for recreational and medicinal purposes.