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

(1) Background: The cannabinoid 2 receptor (CB ₂ R) is a promising anti-inflammatory drug target and development of selective CB ₂ R ligands may be useful for treating sight-threatening ocular inflammation. (2) Methods: This study examined the pharmacology of three novel chemically-diverse selective CB ₂ R ligands: CB ₂ R agonists, RO6871304, and RO6871085, as well as a CB ₂ R inverse agonist, RO6851228. In silico molecular modelling and in vitro cell-based receptor assays were used to verify CB ₂ R 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 CB ₂ R ligand, HU910, using an in vivo mouse model of endotoxin-induced uveitis (EIU) in wild-type (WT) and CB ₂ R ^-/- mice. The actions of RO6871304 on neutrophil migration and adhesion were examined in vitro using isolated neutrophils from WT and CB ₂ R ^-/- mice, and in vivo in WT mice with EIU using adoptive transfer of WT and CB ₂ R ^-/- neutrophils, respectively. (3) Results: Molecular docking studies indicated that RO6871304 and RO6871085 bind to the orthosteric site of CB ₂ R. Binding studies and cell signaling assays for RO6871304 and RO6871085 confirmed high-affinity binding to CB ₂ R and selectivity for CB ₂ R > CB ₁ R, with both ligands acting as full agonists in cAMP and ß-arrestin assays (EC ₅₀ s 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 CB ₂ R agonist, RO6871304, decreased in vitro neutrophil migration of WT neutrophils but not neutrophils from CB ₂ R ^-/- , and attenuated adhesion of adoptively-transferred leukocytes in EIU. (4) Conclusions: These unique ligands are potent and selective for CB ₂ R 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 CB ₂ R agonists support selective targeting of CB ₂ R for treating ocular inflammatory diseases.