I have investigated the pharmacological properties of cannabichromene (CBC), cannabigerol (CBG) and Δ⁸-tetrahydrocannabivarin (Δ⁸-THCV).  A concentrations in the micromolar range, CBG binds to the cannabinoid CB₁ and CB₂ receptors and acts as a CB₁ receptor antagonist.  In the functional [³⁵S]GTPγS binding assay performed with mouse brain membranes, CBG also behaves as a potent partial agonist at concentrations less than 100 nM. CBG is an α₂-adrenoceptor agonist, and behaves as a competitive 5-HT_1A antagonist at concentrations ranging from 300 nM to 10 μM. Further experiments were conducted to explore interactions between established 5-HT_1A ligands and the CB₁ receptor.  In mouse brain membranes, although not in hCB₁ transfected cells, the 5-HT_1A receptor agonist, 8-OH-DPAT, has the ability both to displace [³H]CP55940 from specific binding sites and to modulate the rate of [³H]CP55940 dissociation from these sites.  These results may reflect the presence of CB₁-5-HT_1A dimers in the brain. Δ⁹-THCV has been reported to be a potent CB₂ receptor antagonist with a <i>K</i>_B value lower than its binding affinity for CB₂.  Experiments were performed in the cAMP assay to establish if Δ⁸-THCV did in fact target the CB₂ receptor.  Antagonism of Δ⁸-THCV was attempted by using a selection of compounds but unfortunately these either behaved as inverse agonists in the assay or as partial agonists.  Δ⁸-THCV induced agonism was <i>Pertussis </i>toxin sensitive.  In untransfected CHO cells, Δ⁸-THCV did not stimulate or inhibit forskolin induced stimulation of cAMP, hence the effects of Δ⁸-THCV are most likely CB₂ dependent. As CBG was found to behave as a potent α₂-adrenoceptor agonist and 5-HT_1A receptor antagonist, it may be useful clinically, for example for the treatment of depression.  Interactions between the 5-HT_1A and CB₁ receptors need to be explored more fully, not least because it might be possible to exploit such interactions in the clinic.

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The effects of cannabinoid agonists on spontaneous neuronal network activity were characterized in murine spinal cord and auditory cortical cultures with multichannel extracellular recording using photoetched electrode arrays. Different cultures responded reproducibly with global decreases of spiking and bursting to anandamide and methanandamide, but each agonist showed unique minor effects on network activity. The two tissues responded in a tissue-specific manner. Spontaneous activity in spinal tissue was terminated by 1 μM anandamide and 6.1 μM methanandamide. Cortical activity ceased at 3.5 μM and 2.8 μM respectively. Irreversible cessation of activity was observed beyond 8 μM for both tissues and test substances. Palmitoylethanolamide, demonstrated that CB2 receptors were not present or not responsive. However, the data strongly suggested the presence of CB1 receptors.

Cannabinoid receptors play an important role in regulating bone mass and bone turnover. Studies in our laboratories have shown that young mice lacking type 1 cannabinoid receptor (CNR1-/-) had increased bone mass and were resistant to ovariectomy-induced bone loss. Other workers have reported that type 2 cannabinoid receptor knockout mice (CNR2-/-) develop age-related osteoporosis. The aim of this PhD thesis was to further investigate the role of CNR2 in bone metabolism in vitro and in vivo, using genetic and pharmacological approaches. This study showed that CNR2-/- mice had normal bone mass and bone turnover at 3 months of age, but following ovariectomy, CNR2-/- mice were partially protected from bone loss, because of a mild defect in osteoclast formation and bone resorption. In keeping with this, studies in vitro showed that RANKL-stimulated bone marrow cultures from CNR2-/- mice had fewer osteoclasts than cultures from wild type littermates. The CNR2-selective antagonist/inverse agonist AM630, inhibited osteoclast formation in wild type bone marrow cultures in vitro and prevented ovariectomy-induced bone loss in wild type mice in vivo. In contrast, osteoclast cultures from CNR2-/- mice were resistant to the inhibitory effects of AM630 at low concentrations and CNR2-/- ovariectomised mice did not respond to its protective effects at low doses, consistent with a CNR2- mediated effect. These results indicate that CNR2 regulates bone loss under conditions of increased bone turnover, such as ovariectomy, by affecting osteoclast differentiation and function. CNR2-deficient mice developed accelerated age-related osteoporosis and by 12 months of age they had a significant reduction in osteoblast numbers and bone formation, whereas osteoclast numbers remained comparable to wild type littermates. In agreement with this, osteoblasts derived from bone marrow of CNR2-/- mice had reduced PTHstimulated alkaline phosphatase activity and ability to form bone nodules, when compared with wild type cultures. The CNR2-selective agonist, HU308, stimulated bone nodule formation in wild type calvarial osteoblast cultures in vitro and reversed ovariectomy-induced bone loss in wild type mice in vivo. HU308 had blunted effects on bone nodule formation in cultures from CNR2-/- mice and no significant effects on ovariectomy-induced bone loss in CNR2-/- mice, indicating a CNR2-mediated effect. These studies demonstrate that CNR2 protects against age-related bone loss by mainly enhancing osteoblast differentiation and bone formation. In conclusion, type 2 cannabinoid receptors protect from bone loss by maintaining bone remodelling at balance. In addition, type 2 cannabinoid receptor agonists show evidence of anabolic activity, whereas antagonists/inverse agonists show evidence of antiosteoclastic activity in vitro and in vivo.

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Although there is a renewed interest in the therapeutic potential of cannabinoids, pharmacological and physiological characterisation of these promising compounds is currently not well documented in the respiratory system. The aim of this study is to increase our understanding of possible roles of cannabinoids in the airways. Apart from CB1 and CB2 receptor-mediated actions, cannabinoid compounds can also target TRPV1 receptors, ion channels or the orphan GPR55. In isolated guinea-pig bronchi, WIN55212-2 probably exerted its inhibitory effect on sensory nerves through CB2-like receptors. VIR did not act prejunctionally but its excitatory action was mediated through TRPV1 receptors. Δ9-THC activated sensory nerves presumably involving CB1 receptors. It was speculated that GPR55 might be activated by VIR and antagonized by CBD. CBD revealed multiple mechanisms of actions: it antagonized effects mediated by TRPV1 and NK2 receptors, modulated mast cell function and showed anti-allergic activity in an in vitro model of bronchial asthma. In a human bronchial epithelial cell line the functional expression of CB1 receptors could not be confirmed. Cannabinoids examined in this study were ineffective to induce signal transduction which would be linked to ion channel activity or to intracellular Ca2+ changes. Only VIR might trigger a CB1 receptor-independent signalling pathway in these cells. In conclusion, the findings presented in this thesis reflect the diversity of cannabinoid pharmacology in the airways. They show for the first time that CBD has the ability to reduce antigen-induced bronchoconstriction, indicating relevance in bronchial asthma.

We report on the design, synthesis, and structure activity relationship studies of novel Org 27569 analogues as potential allosteric modulators of the CB1 receptors. We also investigated by computer modelling the possible location of the allosteric site on CB1 and the binding confirmation of the allosteric ligands. Docking of the synthesised molecules is also performed and the results are compared to the results of the biological bioassays. The synthesis of non-fused indole analogues of Org 27569 is described. These analogues were systematically varied to study the importance of key functional groups for CB1 allosteric activity. It was found that the two NH groups of the indole derivatives are required for activity. Activity is also significantly improved for analogues possessing a hydroxymethyl group or a hydrophobic chain at position 3 of the indole moiety. SAR analysis also shows that the presence of a dialkylamino group at the <i>para-</i>position on the aromatic side chain further improves the activity. Conformationally restricted analogues (fused indoles) of Org 27569 were prepared to determine the possible binding conformation of Org 27569.<i> </i>An analogue having the two NH groups directed in the same direction exhibited a moderate ability to enhance CP55,940 affinity and gave significant decrease in [³⁵S]GTPγS binding at 1μM, indicating the possible binding conformation for the Organon derivatives. Molecular modelling studies allowed locating a possible binding pocket for the CB1 allosteric ligands. The study described here should help the design of ligands of the CB1 allosteric site that possess higher biological activities and specificities. The results should pave the way for the discovery of the anti-obesity drugs of the future.