Inflammation is a multifactorial response towards noxious stimuli, however appropriate regulation and resolution of inflammation is crucial for the prevention of chronic inflammatory diseases such as atherosclerosis. The endocannabinoid (eCB) system is an endogenous immunomodulatory system which consists of a series of lipophilic ligands that signal via two G-protein-coupled receptors. Cannabinoid receptor 1 (CB1R) is mainly expressed in the central nervous system and its activation has psychoactive effects. Cannabinoid receptor 2 (CB2R) is mainly expressed on leukocytes and receptor activation has anti-inflammatory actions in mouse models of atherosclerosis and chronic inflammatory pain. It is considered that CB2R activation is involved in modulation of the recruitment of inflammatory cells, especially monocytes/macrophages; however the exact mechanism of action has not been fully elucidated. We hypothesised that activation of CB2R modulates monocyte/ macrophage recruitment and signalling, thus providing a homeostatic mechanism to limit macrophage activation in inflammatory responses. The high lipophilicity of cannabinoid ligands and their lack of selectivity for CB2R over CB1R limits CB2R drug development. In collaboration with Dr Angela Russell, we used virtual screening and a CB2R cAMP assay that we validated to discover a novel CB2R agonist, 3-((2’-Cyanobenzyl)thio)-5H-[1,2,4]triazino[5,6-b]indole, (DIAS2). In collaboration with Dr Russell’s group who did chemical synthesis, we extended this novel scaffold to include over 80 compounds. Using the same hCB2R cAMP screening assay we demonstrated that 16 compounds with the same scaffold are at active at CB2R in the nanomolar range. At least 3 compounds, including DIAS2, were found to be ≥ 300-fold selective for CB2R over CB1R in cAMP assays and radioligand binding studies. In the inflammatory model of zymosan-induced peritonitis, DIAS2 dose-dependently inhibited inflammatory monocyte recruitment by 50% at highest dose of 5 mg/kg with no effect on neutrophils. In further zymosan-induced peritonitis experiments 5 mg/kg of DIAS2 and a structurally-similar CB2R agonist from the same family of triazino-indoles inhibited monocyte recruitment while a different CB2R agonist (JWH-133) at 5 mg/kg did not inhibit monocyte recruitment. Analysis of peritoneal exudates showed that the inhibition of monocyte recruitment was not associated with changes in the levels of JE, MIP-1α and nitric oxide but was associated with increased levels of the chemokine KC. Using in vitro cell biology approaches, we demonstrated that 10μΜ dose of both DIAS2 and JWH-133 reduced forskolin-induced cAMP production in primary murine macrophages. Also 2.5 to 10 μΜ οf JWH-133 and HU-308 dose-dependently induced primary murine macrophage chemotaxis which could be blocked a CB2R antagonist (SR 144528, 1 μΜ) while DIAS2 at doses up to 10 μΜ was not a chemoattractant. Accordingly HU-308 and JWH-133 were at least 3-fold more efficacious than DIAS2 at recruiting β-arrestin to the murine CB2R. Moreover in studies with primary murine macrophages 10 μΜ dose of JWH-133 and HU-308 induced ERK1/2 and Akt phosphorylation within 30 minutes, while 2-AG (an endogenous eCB ligand) and DIAS2 at 10 μΜ had no such effect. In summary, we have discovered a novel family CB2R agonists and demonstrated that some devoid of chemotactic active CB2R agonists can reduce monocyte recruitment in vivo while other chemoattractant CB2R agonists have no in vivo anti-inflammatory effect. We propose that non-chemotactic CB2R agonists represent a new class of anti-inflammatory drugs with a novel mode of action.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:692844 |
| Date | January 2012 |
| Creators | Christou, Ivy |
| Contributors | Greaves, D. R. |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://ora.ox.ac.uk/objects/uuid:fd7a7b39-7225-4cdf-b01d-2d5ccb9cd0da |
Page generated in 0.0021 seconds