Selective Cannabinoid 2 Receptor Agonists as Potential Therapeutic Drugs for the Treatment of Endotoxin-Induced Uveitis.

(1) Background: The cannabinoid 2 receptor (CB₂R) is a promising anti-inflammatory drug target and development of selective CB₂R ligands may be useful for treating sight-threatening ocular inflammation. (2) Methods: This study examined the pharmacology of three novel chemically-diverse selective CB₂R ligands: CB₂R agonists, RO6871304, and RO6871085, as well as a CB₂R inverse agonist, RO6851228. In silico molecular modelling and in vitro cell-based receptor assays were used to verify CB₂R interactions, binding, cell signaling (ß-arrestin and cAMP) and early absorption, distribution, metabolism, excretion, and toxicology (ADMET) profiling of these receptor ligands. All ligands were evaluated for their efficacy to modulate leukocyte-neutrophil activity, in comparison to the reported CB₂R ligand, HU910, using an in vivo mouse model of endotoxin-induced uveitis (EIU) in wild-type (WT) and CB₂R^-/- mice. The actions of RO6871304 on neutrophil migration and adhesion were examined in vitro using isolated neutrophils from WT and CB₂R^-/- mice, and in vivo in WT mice with EIU using adoptive transfer of WT and CB₂R^-/- neutrophils, respectively. (3) Results: Molecular docking studies indicated that RO6871304 and RO6871085 bind to the orthosteric site of CB₂R. Binding studies and cell signaling assays for RO6871304 and RO6871085 confirmed high-affinity binding to CB₂R and selectivity for CB₂R > CB₁R, with both ligands acting as full agonists in cAMP and ß-arrestin assays (EC₅₀s in low nM range). When tested in EIU, topical application of RO6871304 and RO6871085 decreased leukocyte-endothelial adhesion and this effect was antagonized by the inverse agonist, RO6851228. The CB₂R agonist, RO6871304, decreased in vitro neutrophil migration of WT neutrophils but not neutrophils from CB₂R^-/-, and attenuated adhesion of adoptively-transferred leukocytes in EIU. (4) Conclusions: These unique ligands are potent and selective for CB₂R and have good immunomodulating actions in the eye. RO6871304 and RO6871085, as well as HU910, decreased leukocyte adhesion in EIU through inhibition of resident ocular immune cells. The data generated with these three structurally-diverse and highly-selective CB₂R agonists support selective targeting of CB₂R for treating ocular inflammatory diseases.