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

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