Melanie E. M. Kelly

Cannabinoid receptor 1 (CB1) activation has been reported to reduce transient receptor potential cation channel subfamily V member 1 (TRPV1)-induced inflammatory responses and is anti-nociceptive and anti-inflammatory in corneal injury. We examined whether allosteric ligands, can modulate CB1 signaling to reduce pain and inflammation in corneal hyperalgesia. Corneal hyperalgesia was generated by chemical cauterization of cornea in wildtype and CB2 knockout (CB2^−/−) mice. The novel racemic CB1 allosteric ligand GAT211 and its enantiomers GAT228 and GAT229 were examined alone or in combination with the orthosteric CB1 agonist Δ⁸-tetrahydrocannabinol (Δ⁸-THC). Pain responses were assessed following capsaicin (1 µM) stimulation of injured corneas at 6 h post-cauterization. Corneal neutrophil infiltration was also analyzed. GAT228, but not GAT229 or GAT211, reduced pain scores in response to capsaicin stimulation. Combination treatments of 0.5% GAT229 or 1% GAT211 with subthreshold Δ⁸-THC (0.4%) significantly reduced pain scores following capsaicin stimulation. The anti-nociceptive effects of both GAT229 and GAT228 were blocked with CB1 antagonist AM251, but remained unaffected in CB2^−/− mice. Two percent GAT228, or the combination of 0.2% Δ⁸-THC with 0.5% GAT229 also significantly reduced corneal inflammation. CB1 allosteric ligands could offer a novel approach for treating corneal pain and inflammation.

Background and Purpose: Corneal injury can result in dysfunction of corneal nociceptive signaling and corneal sensitization. Activation of the endocannabinoid system has been reported to be analgesic and anti-inflammatory. The purpose of this research was to investigate the antinociceptive and anti-inflammatory effects of cannabinoids with reported actions at cannabinoid 1 (CB₁R) and cannabinoid 2 (CB₂R) receptors and/or noncannabinoid receptors in an experimental model of corneal hyperalgesia. Methods: Corneal hyperalgesia (increased pain response) was generated using chemical cauterization of the corneal epithelium in wild-type (WT) and CB₂R knockout (CB₂R^−/−) mice. Cauterized eyes were treated topically with the phytocannabinoids Δ⁸-tetrahydrocannabinol (Δ⁸THC) or cannabidiol (CBD), or the CBD derivative HU-308, in the presence or absence of the CB₁R antagonist AM251 (2.0 mg/kg i.p.), or the 5-HT_1A receptor antagonist WAY100635 (1 mg/kg i.p.). Behavioral pain responses to a topical capsaicin challenge at 6 h postinjury were quantified from video recordings. Mice were euthanized at 6 and 12 h postcorneal injury for immunohistochemical analysis to quantify corneal neutrophil infiltration. Results: Corneal cauterization resulted in hyperalgesia to capsaicin at 6 h postinjury compared to sham control eyes. Neutrophil infiltration, indicative of inflammation, was apparent at 6 and 12 h postinjury in WT mice. Application of Δ⁸THC, CBD, and HU-308 reduced the pain score and neutrophil infiltration in WT mice. The antinociceptive and anti-inflammatory actions of Δ⁸THC, but not CBD, were blocked by the CB₁R antagonist AM251, but were still apparent, for both cannabinoids, in CB₂R^−/− mice. However, the antinociceptive and anti-inflammatory actions of HU-308 were absent in the CB₂R^−/− mice. The antinociceptive and anti-inflammatory effects of CBD were blocked by the 5-HT_1A antagonist WAY100635. Conclusion: Topical cannabinoids reduce corneal hyperalgesia and inflammation. The antinociceptive and anti-inflammatory effects of Δ⁸THC are mediated primarily via CB₁R, whereas that of the cannabinoids CBD and HU-308, involve activation of 5-HT_1A receptors and CB₂Rs, respectively. Cannabinoids could be a novel clinical therapy for corneal pain and inflammation resulting from ocular surface injury.

The endocannabinoid system (ECS) has emerged as an important regulator of both physiological and pathological processes. Notably, this endogenous system plays a key role in the modulation of pain and inflammation in a number of tissues. The components of the ECS, including endocannabinoids, their cognate enzymes and cannabinoid receptors, are localized in the eye, and evidence indicates that ECS modulation plays a role in ocular disease states. Of these diseases, ocular inflammation presents a significant medical problem, given that current clinical treatments can be ineffective or are associated with intolerable side-effects. Furthermore, a prominent comorbidity of ocular inflammation is pain, including neuropathic pain, for which therapeutic options remain limited. Recent evidence supports the use of drugs targeting the ECS for the treatment of ocular inflammation and pain in animal models; however, the potential for therapeutic use of cannabinoid drugs in the eye has not been thoroughly investigated at this time. This review will highlight evidence from experimental studies identifying components of the ocular ECS and discuss the functional role of the ECS during different ocular inflammatory disease states, including uveitis and corneal keratitis. Candidate ECS targeted therapies will be discussed, drawing on experimental results obtained from both ocular and non-ocular tissue(s), together with their potential application for the treatment of ocular inflammation and pain.

The observation that marijuana reduces intraocular pressure was made by Hepler and Frank in the 1970s. Since then, there has been a significant body of work investigating cannabinoids for their potential use as therapeutics. To date, no endocannabinoid system (ECS)-modulating drug has been approved for clinical use in the eye; however, recent advances in our understanding of the ECS, as well as new pharmacological tools, has renewed interest in the development of ocular ECS-based therapeutics. This review summarizes the current state-of-affairs for the use of ECS-modulating drugs for the treatment of glaucoma and ocular inflammatory and ischemic disease.

Background and Purpose: Cannabinoid 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 Approach: EIU was induced by intraocular injection of lipopolysaccharide (LPS, 20 ng·μL⁻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‐κB, AP‐1) and inflammatory mediators (cytokines, chemokines and adhesion molecules) were measured in iris and ciliary body tissue.Key Results: Leukocyte‐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‐α, IL‐1β, 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‐κB and AP‐1, while AM630 increased levels of NF‐κB. 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 Implications: Activation of CB₂ receptors was anti‐inflammatory in a model of acute EIU and involved a reduction in NF‐κB, AP‐1 and inflammatory mediators. CB₂ receptors may be promising drug targets for the development of novel ocular anti‐inflammatory agents.Linked Articles: This 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

Introduction: Cannabinoid receptor 2 (CB2R) expression is upregulated during sepsis. However, there are conflicting results regarding the effects of CB2R modulation in the hyperinflammatory phase of the disease. The aim of this study was therefore to investigate the effects of CB2R manipulation on leukocyte activation within the intestinal microcirculation in two acute experimental sepsis models. Methods: In the endotoxemia model we studied four groups of Lewis rats: controls, lipopolysaccharide (LPS), LPS + CB2R agonist HU308 (2.5 mg/kg), and LPS + CB2R antagonist AM630 (2.5 mg/kg). In the colon ascendens stent peritonitis (CASP)-induced sepsis model we also studied four groups: sham group, CASP and CASP + CB2R agonist (HU308, 2.5 or 10 mg/kg). Intravital microscopy was performed 2 hours following LPS/placebo administration or 16 hours following CASP/sham surgery to quantify intestinal leukocyte recruitment. Additionally, hemodynamic monitoring, histological examinations and measurements of inflammatory mediators were performed. Results: HU308 administration significantly reduced intestinal leukocyte adhesion in both acute sepsis models. The systemic levels of inflammatory mediators were significantly reduced by 10 mg/kg HU308 treatment in CASP animals. Conclusion: CB2R activation reduces leukocyte activation and systemic release of inflammatory mediators in acute experimental sepsis. Drugs targeting the CB2R pathway may have therapeutic potential in sepsis.

The objective of our study was to examine the pharmacology of the intraocular pressure (IOP)-lowering actions of the behaviorally inactive cannabinoids, abnormal cannabidiol (abn-CBD), and a cannabigerol analog, cannabigerol-dimethyl heptyl (CBG-DMH), in comparison to that of the nonselective cannabinoid 1 receptor (CB₁R) and CB₂R agonist, WIN55,212-2, in Brown Norway rats. The IOP was measured noninvasively using a hand-held tonometer in nonanesthetized animals. The IOP measurements were taken every 15 min for a period of 2 h after drug administration. All drugs were administered via intraperitoneal (i.p.) injections, and abn-CBD and CBG-DMH were also given topically. Both abn-CBD and CBG-DMH reduced IOP when administrated i.p. at doses of ≥2.5 mg/kg or topically at concentrations of 1%–2%. The IOP-lowering effects of abn-CBD and CBG-DMH were reduced by i.p. administration of O-1918 (2.5 mg/kg), a selective antagonist of the abn-CBD-sensitive cannabinoid-related receptor (CBx), but were unaffected by the CB₁R antagonist, AM251 (2.5 mg/kg), or the CB₂R antagonist, AM630 (2.5 mg/kg). In contrast, the IOP-lowering action of WIN55,212-2 was completely blocked by the CB₁R-selective antagonist, AM251, and was unaffected by the CBx receptor antagonist, O-1918. However, similar to the nonpsychotropic cannabinoids, the ocular hypotensive actions of WIN55,212-2 were also insensitive to block by the CB₂R antagonist, AM630. Consistent with this, the selective CB₂R agonist, HU-308 (2 mg/kg) failed to reduce IOP in Brown Norway rats. Concurrent application of a dose of WIN55,212-2 that was subthreshold to reduce IOP (0.25 mg/kg), together with a topical dose of either abn-CBD (0.5%) or CBG-DMH (0.25%), respectively, potentiated the ocular hypotensive effect of either compound applied alone. This study demonstrates that the atypical cannabinoid, abn-CBD, and the cannabigerol analog, CBG-DMH, decrease IOP in the normotensive Brown Norway rat eye independent of CB₁R or CB₂R activation, via activation of CBx receptors. The enhanced decrease in IOP seen after coapplication of the CB₁R agonist, WIN55,212-2, together with either abn-CBD or CBG-DMH, respectively, further suggests that the ocular pharmacodynamics of abn-CBD and CBG-DMH are mediated by receptor targets distinct from CB₁R. These results indicate that both CBG-DMH and abn-CBD have the potential for further investigation as novel ocular hypotensive cannabinoids devoid of CB₁R-mediated side-effects.

This review examines the function of calcium-activated chloride currents (ICl(Ca)) in the retina with an emphasis on their physiological role in photoreceptors. Although found in a variety of neurons and glial cells of the retina, ICl(Ca) has been most prominently studied in cones, where it activates in response to depolarization-evoked Ca2+ influx. The slow and complex gating kinetics of the chloride current have been considered to reflect the changing submembrane concentration of intracellular calcium. It is likely that the role of ICl(Ca) is to stabilize the membrane potential of cones during synaptic activity and presynaptic Ca channel modulation. Several candidates in the molecular identification of the channel have been put forward but the issue remains unresolved.

Background and purpose: Trabecular meshwork (TM) is an ocular tissue involved in the regulation of aqueous humour outflow and intraocular pressure (IOP). CB₁receptors (CB₁) are present in TM and cannabinoid administration decreases IOP. CB₁signalling was investigated in a cell line derived from human TM (hTM).Experimental approach: CB₁signalling was investigated using ratiometric Ca²⁺imaging, western blotting and infrared In‐Cell Western analysis.Key results: WIN55212‐2, a synthetic aminoalkylindole cannabinoid receptor agonist (10–100 μM) increased intracellular Ca²⁺in hTM cells. WIN55,212‐2‐mediated Ca²⁺increases were blocked by AM251, a CB₁antagonist, but were unaffected by the CB₂antagonist, AM630. The WIN55,212‐2‐mediated increase in [Ca²⁺]_iwas pertussis toxin (PTX)‐insensitive, therefore, independent of G_i/ocoupling, but was attenuated by a dominant negative Gα_q/11subunit, implicating a G_q/11signalling pathway. The increase in [Ca²⁺]_iwas dependent upon PLC activation and mobilization of intracellular Ca²⁺stores. A PTX‐sensitive increase in extracellular signal‐regulated kinase (ERK1/2) phosphorylation was also observed in response to WIN55,212‐2, indicative of a G_i/osignalling pathway. CB₁‐G_q/11coupling to activate PLC‐dependent increases in Ca²⁺appeared to be specific to WIN55,212‐2 and were not observed with other CB₁agonists, including CP55,940 and methanandamide. CP55940 produced PTX‐sensitive increases in [Ca²⁺]_iat concentrations ≥15 μM, and PTX‐sensitive increases in ERK1/2 phosphorylation.Conclusions and implications: This study demonstrates that endogenous CB₁couples to both G_q/11and G_i/oin hTM cells in an agonist‐dependent manner. Cannabinoid activation of multiple CB₁signalling pathways in TM tissue could lead to differential changes in aqueous humour outflow and IOP.British Journal of Pharmacology(2007)152, 1111–1120; doi:10.1038/sj.bjp.0707495; published online 8 October 2007