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Introduction: Preclinical studies suggest that cannabinoid receptor type 2 (CB2R) activation has a therapeutic effect in animal models on chronic inflammation and vascular permeability, which are key pathological features of diabetic retinopathy (DR). A novel CB2R agonist, triazolopyrimidine RG7774, was generated through lead optimization of a high-throughput screening hit. The aim of this study was to characterize the pharmacology, absorption, distribution, metabolism, elimination, and toxicity (ADMET) profile of RG7774, and to explore its potential for managing the key pathological features associated with retinal disease in rodents., Methods: The in vitro pharmacology of RG7774 was investigated for CB2R binding and receptor activation using recombinant human and mouse CB2R expression in Chinese hamster ovary cells, and endogenous CB2R expression in human Jurkat cells, and rat and mouse spleen cells. The ADMET profile was evaluated and the effects of RG7774 on retinal permeability, leukocyte adhesion, and choroidal neovascularization (CNV) were investigated in rodent models of retinal disease. Pharmacokinetic (PK) parameters and the exposure-response relationship were characterized in healthy animals and in animals with laser-induced CNV., Results: RG7774 was found to be a potent (EC 50 : 2.8 nM and K i : 51.3 nM), selective, and full CB2R agonist with no signs of cannabinoid receptor type 1 (CB1R) binding or activation. The ligand showed a favorable ADMET profile and exhibited systemic and ocular exposure after oral delivery. Functional potency in vitro translated from recombinant to endogenous expression systems. In vivo , orally administered RG7774 reduced retinal permeability and leukocyte adhesion in rodents with lipopolysaccharide (LPS)-induced uveitis and streptozotocin (STZ)-induced DR, and reduced lesion areas in rats with laser-induced CNV with an ED 50 of 0.32 mg/kg. Anatomically, RG7774 reduced the migration of retinal microglia to retinal lesions., Discussion: RG7774 is a novel, highly selective, and orally bioavailable CB2R agonist, with an acceptable systemic and ocular PK profile, and beneficial effects on retinal vascular permeability, leukocyte adhesion, and ocular inflammation in rodent animal models. Results support the development of RG7774 as a potential treatment for retinal diseases with similar pathophysiologies as addressed by the animal models., Competing Interests: UG, RF, SG, CK, AO, EZ, SU, MN, RM, CB, WG, AA, FR, AP, CP, AC, SF, CU: Employee: F. Hoffmann-La Roche Ltd. AK: Employee: Chugai Pharmaceuticals Co. Ltd. JT, NC, MR-E, SR, J-MA, JF: Employee: F. Hoffmann-La Roche Ltd., at the time of writing. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Grether, Foxton, Gruener, Korn, Kimbara, Osterwald, Zirwes, Uhles, Thoele, Colé, Rogers-Evans, Röver, Nettekoven, Martin, Adam, Fingerle, Bissantz, Guba, Alker, Szczesniak, Porter, Toguri, Revelant, Poirier, Perret, Winther, Caruso, Fezza, Maccarrone, Kelly, Fauser and Ullmer.)

Introduction: The cannabinoid receptor (CBR) subtypes 1 (CB 1 R) and 2 (CB 2 R) are key components of the endocannabinoid system (ECS), playing a central role in the control of peripheral pain, inflammation and the immune response, with further roles in the endocrine regulation of food intake and energy balance. So far, few medicines targeting these receptors have reached the clinic, suggesting that a better understanding of the receptor signalling properties of existing tool compounds and clinical candidates may open the door to the development of more effective and safer treatments. Both CB 1 R and CB 2 R are Gα i protein-coupled receptors but detecting Gα i protein signalling activity reliably and reproducibly is challenging. This is due to the inherent variability in live cell-based assays and restrictions around the use of radioactive [ 35 S]-GTPγS, a favoured technology for developing higher-throughput membrane-based Gα i protein activity assays. Methods: Here, we describe the development of a membrane-based Gα i signalling system, produced from membrane preparations of HEK293TR cells, stably overexpressing CB 1 R or CB 2 R, and components of the Gα i -CASE biosensor. This BRET-based system allows direct detection of Gα i signalling in both cells and membranes by monitoring bioluminescence resonance energy transfer (BRET) between the α and the βγ subunits. Cells and membranes were subject to increasing concentrations of reference cannabinoid compounds, with 10 μM furimazine added to generate RET signals, which were detected on a PHERAstar FSX plate reader, then processed using MARS software and analysed in GraphPad PRISM 9.2. Results: In membranes expressing the Gi-CASE biosensor, the cannabinoid ligands profiled were found to show agonist and inverse agonist activity. Agonist activity elicited a decrease in the BRET signal, indicative of receptor activation and G protein dissociation. Inverse agonist activity caused an increase in BRET signal, indicative of receptor inactivation, and the accumulation of inactive G protein. Our membrane-based Gi-CASE NanoBRET system successfully characterised the potency (pEC 50 ) and efficacy (E max ) of CBR agonists and inverse agonists in a 384-well screening format. Values obtained were in-line with whole-cell Gi-CASE assays and consistent with literature values obtained in the GTPγS screening format. Discussion: This novel, membrane-based Gα i protein activation assay is applicable to other Gα i -coupled GPCRs, including orphan receptors, allowing real-time higher-throughput measurements of receptor activation., Competing Interests: Authors WG, AR, and UG was/were employed by F Hoffmann-La Roche Ltd. Authors DV and DS was/were employed by Z7 Biotech Limited. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Scott-Dennis, Rafani, Yi, Perera, Harwood, Guba, Rufer, Grether, Veprintsev and Sykes.)

The distribution and roles of the cannabinoid CB 2 receptor in the CNS are still a matter of debate. Recent data suggest that, in addition to its presence in microglial cells, the CB 2 receptor may be also expressed at low levels, yet biologically relevant, in other cell types such as neurons. It is accepted that the expression of CB 2 receptors in the CNS is low under physiological conditions and is significantly elevated in chronic neuroinflammatory states associated with neurodegenerative diseases such as Alzheimer's disease. By using a novel mouse model (CB 2 EGFP/f/f ), we studied the distribution of cannabinoid CB 2 receptors in the 5xFAD mouse model of Alzheimer's disease (by generating 5xFAD/CB 2 EGFP/f/f mice) and explored the roles of CB 2 receptors in microglial function. We used a novel selective and brain penetrant CB 2 receptor agonist (RO6866945) as well as mice lacking the CB 2 receptor (5xFAD/CB 2 -/- ) for these studies. We found that CB 2 receptors are expressed in dystrophic neurite-associated microglia and that their modulation modifies the number and activity of microglial cells as well as the metabolism of the insoluble form of the amyloid peptide. These results support microglial CB 2 receptors as potential targets for the development of amyloid-modulating therapies., Competing Interests: Authors CK, CR and UG were employed by the company F. Hoffmann-La Roche Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ruiz de Martín Esteban, Benito-Cuesta, Terradillos, Martínez-Relimpio, Arnanz, Ruiz-Pérez, Korn, Raposo, Sarott, Westphal, Elezgarai, Carreira, Hillard, Grether, Grandes, Grande and Romero.)