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

ABSTRACT: (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.

SUBMITTER: Porter RF

PROVIDER: S-EPMC6767236 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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

Porter Richard Frederick RF Szczesniak Anna-Maria AM Toguri James Thomas JT Gebremeskel Simon S Johnston Brent B Lehmann Christian C Fingerle Jürgen J Rothenhäusler Benno B Perret Camille C Rogers-Evans Mark M Kimbara Atsushi A Nettekoven Matthias M Guba Wolfgang W Grether Uwe U Ullmer Christoph C Kelly Melanie E M MEM

Molecules (Basel, Switzerland) 20190913 18

(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</ ...[more]

PMID: 31540271

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Project description:Background and purposeCannabinoid CB2 receptors mediate immunomodulation. Here, we investigated the effects of CB2 receptor ligands on leukocyte-endothelial adhesion and inflammatory mediator release in experimental endotoxin-induced uveitis (EIU).Experimental approachEIU was induced by intraocular injection of lipopolysaccharide (LPS, 20?ng·?L(-1) ). Effects of the CB2 receptor agonist, HU308 (1.5% topical), the CB2 receptor antagonist, AM630 (2.5?mg·kg(-1) i.v.), or a combination of both compounds on leukocyte-endothelial interactions were measured hourly for 6?h in rat iridial vasculature using intravital microscopy. Anti-inflammatory actions of HU308 were compared with those of clinical treatments for uveitis - dexamethasone, prednisolone and nepafenac. Transcription factors (NF-?B, AP-1) and inflammatory mediators (cytokines, chemokines and adhesion molecules) were measured in iris and ciliary body tissue.Key resultsLeukocyte-endothelium adherence was increased in iridial microvasculature between 4-6?h after LPS. HU308 reduced this effect after LPS injection and decreased pro-inflammatory mediators: TNF-?, IL-1?, IL-6, CCL5 and CXCL2. AM630 blocked the actions of HU-308, and increased leukocyte-endothelium adhesion. HU-308 decreased levels of the transcription factors NF-?B and AP-1, while AM630 increased levels of NF-?B. Topical treatments with dexamethasone, prednisolone or nepafenac, failed to alter leukocyte adhesion or mitigate LPS-induced increases in inflammatory mediators during the 6?h of EIU.Conclusion and implicationsActivation of CB2 receptors was anti-inflammatory in a model of acute EIU and involved a reduction in NF-?B, AP-1 and inflammatory mediators. CB2 receptors may be promising drug targets for the development of novel ocular anti-inflammatory agents.Linked articlesThis article is part of a themed section on Cannabinoids 2013. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-6.

Project description:The melanocortin system plays an essential role in the regulation of immune activity. The anti-inflammatory microenvironment of the eye is dependent on the expression of the melanocortin-neuropeptide alpha-melanocyte stimulating hormone (α-MSH). In addition, the melanocortin system may have a role in retinal development and retinal cell survival under conditions of retinal degeneration. We have found that treating experimental autoimmune uveitis (EAU) with α-MSH suppresses retinal inflammation. Also, this augmentation of the melanocortin system promotes immune tolerance and protection of the retinal structure. The benefit of α-MSH-therapy appears to be dependent on different melanocortin receptors. Therefore, we treated EAU mice with α-MSH-analogs with different melanocortin-receptor targets. This approach demonstrated which melanocortin-receptors suppress inflammation, preserve retinal structure, and induce immune tolerance in uveitis. At the chronic stage of EAU the mice were injected twice 1 day apart with 50 μg of α-MSH or an α-MSH-analog. The α-MSH-analogs were a pan-agonist PL8331, PL8177 (potent MC1r-only agonist), PL5000 (a pan-agonist with no MC5r functional activity), MT-II (same as PL5000) and PG901 (MC5r agonist, but also an antagonist to MC3r, and MC4r). Clinical EAU scores were measured until resolution in the α-MSH-treated mice, when the eyes were collected for histology, and spleen cells collected for retinal-antigen-stimulated cytokine production. Significant suppression of EAU was seen with α-MSH or PL8331 treatment. This was accompanied with significant preservation of retinal structure. A similar effect was seen in EAU-mice that were treated with PL8177, except the suppression of EAU was temporary. In EAU mice treated with PL5000, MTII, or PG901, there was no suppression of EAU with a significant loss in whole retina and outer-nuclear layer thickness. There was significant suppression of IL-17 with induction of IL-10 by retinal-antigen stimulated spleen T cells from EAU mice treated with α-MSH, PL8331, PL8177, or PL5000, but not from EAU mice treated with MT-II, or PG901. Our previous studies show the melanocortin system's importance in maintaining ocular immune privilege and that α-MSH-treatment accelerates recovery and induces retinal-antigen-specific regulatory immunity in EAU. Our current results show that this activity is centered around MC1r and MC5r. In addition, the results suggest that a therapeutic potential to target MC1r and MC5r together to suppress uveitis induces regulatory immunity with potentially maintaining a normal retinal structure.

Dataset Information

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

Publications

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

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