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.

[1]  M. R. Iyer,et al.  Crystal Structure of the Human Cannabinoid Receptor CB2 , 2019, Cell.

[2]  T. Sakmar,et al.  Receptor Structures for a Caldron of Cannabinoids , 2019, Cell.

[3]  H. Bradshaw,et al.  Inflammation and CB2 signaling drive novel changes in the ocular lipidome and regulate immune cell activity in the eye. , 2018, Prostaglandins & other lipid mediators.

[4]  T. Boeckers,et al.  NF‐κB activation in astrocytes drives a stage‐specific beneficial neuroimmunological response in ALS , 2018, The EMBO journal.

[5]  H. Fessi,et al.  Ophthalmic Drug Delivery Systems for Antibiotherapy—A Review , 2018, Pharmaceutics.

[6]  B. Johnston,et al.  Cannabinoid 2 receptor is a novel anti-inflammatory target in experimental proliferative vitreoretinopathy , 2017, Neuropharmacology.

[7]  H. de Vries,et al.  Cannabinoid CB2 receptor ligand profiling reveals biased signalling and off-target activity , 2017, Nature Communications.

[8]  R. Stevens,et al.  Crystal Structure of the Human Cannabinoid Receptor CB1 , 2016, Cell.

[9]  A. Szcześniak,et al.  Seeing over the horizon – targeting the endocannabinoid system for the treatment of ocular disease , 2016, Journal of basic and clinical physiology and pharmacology.

[10]  K. Mackie,et al.  An Introduction to the Endogenous Cannabinoid System , 2016, Biological Psychiatry.

[11]  V. Di Marzo,et al.  Are cannabidiol and Δ9‐tetrahydrocannabivarin negative modulators of the endocannabinoid system? A systematic review , 2015, British journal of pharmacology.

[12]  M. Honer,et al.  Radioligand development for molecular imaging of the central nervous system with positron emission tomography. , 2014, Drug discovery today.

[13]  R. Barry,et al.  Pharmacotherapy for uveitis: current management and emerging therapy , 2014, Clinical ophthalmology.

[14]  C. Lehmann,et al.  Experimental Cannabinoid 2 Receptor-Mediated Immune Modulation in Sepsis , 2014, Mediators of inflammation.

[15]  R. Laprairie,et al.  Anti‐inflammatory effects of cannabinoid CB2 receptor activation in endotoxin‐induced uveitis , 2014, British journal of pharmacology.

[16]  우베 그레터,et al.  Pyridine-2-amides useful as cb2 agonists , 2013 .

[17]  R. Geremia,et al.  The Endocannabinoid System and Spermatogenesis , 2013, Front. Endocrinol..

[18]  P. Pacher,et al.  Selective activation of cannabinoid receptor 2 in leukocytes suppresses their engagement of the brain endothelium and protects the blood-brain barrier. , 2013, The American journal of pathology.

[19]  M. Glogauer,et al.  Filamin-A Regulates Neutrophil Uropod Retraction through RhoA during Chemotaxis , 2013, PloS one.

[20]  N. Hogg,et al.  PHAGOCYTES , GRANULOCYTES , AND MYELOPOIESIS Mast cell and macrophage chemokines CXCL 1 / CXCL 2 control the early stage of neutrophil recruitment during tissue in fl ammation , 2013 .

[21]  L. Di,et al.  Demystifying brain penetration in central nervous system drug discovery. Miniperspective. , 2013, Journal of medicinal chemistry.

[22]  R. Mechoulam,et al.  The endocannabinoid system and the brain. , 2013, Annual review of psychology.

[23]  R. Pertwee Targeting the endocannabinoid system with cannabinoid receptor agonists: pharmacological strategies and therapeutic possibilities , 2012, Philosophical Transactions of the Royal Society B: Biological Sciences.

[24]  P. Malherbe,et al.  Functional monoclonal antibody acts as a biased agonist by inducing internalization of metabotropic glutamate receptor 7 , 2012, British journal of pharmacology.

[25]  M. Glass,et al.  A new cannabinoid CB2 receptor agonist HU‐910 attenuates oxidative stress, inflammation and cell death associated with hepatic ischaemia/reperfusion injury , 2012, British journal of pharmacology.

[26]  Juan Zhou,et al.  Cannabinoid receptor 2 activation reduces intestinal leukocyte recruitment and systemic inflammatory mediator release in acute experimental sepsis , 2012, Critical Care.

[27]  S. Gupta,et al.  Endotoxin‐induced uveitis is primarily dependent on radiation‐resistant cells and on MyD88 but not TRIF , 2011, Journal of leukocyte biology.

[28]  A. Veves,et al.  Cannabidiol Attenuates Cardiac Dysfunction , Oxidative Stress , Fibrosis , and Inflammatory and Cell Death Signaling Pathways in Diabetic Cardiomyopathy , 2017 .

[29]  J. Forrester,et al.  Investigation and management of uveitis , 2010, BMJ : British Medical Journal.

[30]  Xiaofeng Hu,et al.  Expression of TLR4-MyD88 and NF-κB in the Iris during Endotoxin-Induced Uveitis , 2010, Mediators of inflammation.

[31]  C. Constantinescu,et al.  Cannabinoids and the immune system: an overview. , 2010, Immunobiology.

[32]  C. Kilkenny,et al.  Guidelines for reporting experiments involving animals: the ARRIVE guidelines , 2010, British journal of pharmacology.

[33]  I. Cuthill,et al.  Animal Research: Reporting In Vivo Experiments: The ARRIVE Guidelines , 2010, British journal of pharmacology.

[34]  Ming Yan,et al.  The axonal repellent, Slit2, inhibits directional migration of circulating neutrophils , 2009, Journal of leukocyte biology.

[35]  Hong Zhang,et al.  Activation of the cannabinoid 2 receptor (CB2) protects against experimental colitis , 2009, Inflammatory bowel diseases.

[36]  N. Stella,et al.  The therapeutic potential of novel cannabinoid receptors. , 2009, Pharmacology & therapeutics.

[37]  K. Ramana,et al.  Prevention of endotoxin-induced uveitis in rats by benfotiamine, a lipophilic analogue of vitamin B1. , 2009, Investigative ophthalmology & visual science.

[38]  J. Kolls,et al.  A novel IL‐17‐dependent mechanism of cross protection: Respiratory infection with mycoplasma protects against a secondary listeria infection , 2009, European journal of immunology.

[39]  W. Yeh,et al.  LPS/TLR4 signal transduction pathway. , 2008, Cytokine.

[40]  D. McHugh,et al.  Inhibition of Human Neutrophil Chemotaxis by Endogenous Cannabinoids and Phytocannabinoids: Evidence for a Site Distinct from CB1 and CB2 , 2008, Molecular Pharmacology.

[41]  S. Friedman,et al.  Regression of Fibrosis after Chronic Stimulation of Cannabinoid CB2 Receptor in Cirrhotic Rats , 2008, Journal of Pharmacology and Experimental Therapeutics.

[42]  Michael D. Connolly,et al.  Use of Ly6G‐specific monoclonal antibody to deplete neutrophils in mice , 2008, Journal of leukocyte biology.

[43]  J. Forrester,et al.  Anti‐inflammatory property of the cannabinoid receptor‐2‐selective agonist JWH‐133 in a rodent model of autoimmune uveoretinitis , 2007, Journal of leukocyte biology.

[44]  P. Pacher,et al.  The Endocannabinoid System as an Emerging Target of Pharmacotherapy , 2006, Pharmacological Reviews.

[45]  D. Broadway,et al.  Corticosteroid-induced glaucoma: a review of the literature , 2006, Eye.

[46]  R. Nitsch,et al.  The Endocannabinoid Anandamide Protects Neurons during CNS Inflammation by Induction of MKP-1 in Microglial Cells , 2006, Neuron.

[47]  E. Ponomarev,et al.  Modulation of the cannabinoid CB2 receptor in microglial cells in response to inflammatory stimuli , 2005, Journal of neurochemistry.

[48]  M. L. de Ceballos,et al.  Prevention of Alzheimer's Disease Pathology by Cannabinoids: Neuroprotection Mediated by Blockade of Microglial Activation , 2005, The Journal of Neuroscience.

[49]  W. Campbell,et al.  Cultured rat microglial cells synthesize the endocannabinoid 2-arachidonylglycerol, which increases proliferation via a CB2 receptor-dependent mechanism. , 2004, Molecular pharmacology.

[50]  Alberto Rábano,et al.  Cannabinoid CB2 Receptors and Fatty Acid Amide Hydrolase Are Selectively Overexpressed in Neuritic Plaque-Associated Glia in Alzheimer's Disease Brains , 2003, The Journal of Neuroscience.

[51]  J. Rosenbaum,et al.  Digital video-imaging of leukocyte migration in the iris: intravital microscopy in a physiological model during the onset of endotoxin-induced uveitis. , 2000, Journal of immunological methods.

[52]  R. Mechoulam,et al.  HU-308: a specific agonist for CB(2), a peripheral cannabinoid receptor. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[53]  K Gubernator,et al.  Physicochemical high throughput screening: parallel artificial membrane permeation assay in the description of passive absorption processes. , 1998, Journal of medicinal chemistry.

[54]  S. Galiègue,et al.  Expression of central and peripheral cannabinoid receptors in human immune tissues and leukocyte subpopulations. , 1995, European journal of biochemistry.

[55]  S. Munro,et al.  Molecular characterization of a peripheral receptor for cannabinoids , 1993, Nature.

[56]  W. Guba,et al.  Novel Triazolopyrimidine‐Derived Cannabinoid Receptor 2 Agonists as Potential Treatment for Inflammatory Kidney Diseases , 2016, ChemMedChem.

[57]  V. Di Marzo Targeting the endocannabinoid system: to enhance or reduce? , 2008 .

[58]  A. Ivashchenko TO PYRIDINE DERIVATIVES , 1986 .