Enantiomeric Agonists of the Type 2 Cannabinoid Receptor Reduce Retinal Damage during Proliferative Vitreoretinopathy and Inhibit Hyperactive Microglia In Vitro.

Microglia are resident immune cells of the central nervous system (CNS) and propagate inflammation following damage to the CNS, including the retina. Proliferative vitreoretinopathy (PVR) is a condition that can emerge following retinal detachment and is characterized by severe inflammation and microglial proliferation. The type 2 cannabinoid receptor (CB2) is an emerging pharmacological target to suppress microglial-mediated inflammation when the eyes or brain are damaged. CB2-knockout mice have exacerbated inflammation and retinal pathology during experimental PVR. We aimed to assess the anti-inflammatory effects of CB2 stimulation in the context of retinal damage and also explore the mechanistic roles of CB2 in microglia function. To target CB2, we used a highly selective agonist, HU-308, as well as its enantiomer, HU-433, which is a putative selective agonist. First, ß-arrestin2 and Gαi recruitment was measured to compare activation of human CB2 in an in vitro heterologous expression system. Both agonists were then utilized in a mouse model of PVR, and the effects on retinal damage, inflammation, and cell death were assessed. Finally, we used an in vitro model of microglia to determine the effects of HU-308 and HU-433 on phagocytosis, cytokine release, migration, and intracellular signaling. We observed that HU-308 more strongly recruited both ß-arrestin2 and Gαi compared to HU-433. Stimulation of CB2 with either drug effectively blunted LPS- and IFNγ-mediated signaling as well as NO and TNF release from microglia. Furthermore, both drugs reduced IL-6 accumulation, total caspase-3 cleavage, and retinal pathology following the induction of PVR. Ultimately, this work supports that CB2 is a valuable target for drugs to suppress inflammation and cell death associated with infection or sterile retinopathy, although the magnitude of effector recruitment may not be predictive of anti-inflammatory capacity.

Topical analgesics for acute corneal pain: current options and emerging therapeutics.

Acute corneal pain is a common complaint that causes significant distress to patients and continues to challenge therapeutic avenues for pain management. Current topical treatment options have marked limitations in terms of both efficacy and safety, thus often prompting the adjunctive use of systemic analgesics, including opioids. In general, there have not been extensive advancements in pharmacologic options for the management of corneal pain over the past several decades. Despite this, multiple promising therapeutic avenues exist which hold the potential to transform the ocular pain landscape, including druggable targets within the endocannabinoid system. This review will summarize the current evidence base for topical nonsteroidal anti-inflammatory drugs, anticholinergic agents, and anesthetics before focusing on several potential avenues in the setting of acute corneal pain management, including autologous tear serum, topical opioids and endocannabinoid system modulators.

Rodent models of ketamine-induced cystitis.

AIMS: Long-term or recreational use of ketamine affects the urinary system and can result in ketamine-induced cystitis (KIC). Rodent models of KIC are important to study KIC pathophysiology and are paramount to the future development of therapies for this painful condition. This review aims to provide a summary of rodent models of KIC, focusing on disease induction, experimental methods, and pathological features of the model. METHOD: A literature search was performed using the National Center for Biotechnology Information (NCBI) Pubmed database up to March 2021. 20 articles met the inclusion criteria and were finally selected. RESULTS: There are considerable variations in the rodent models used for studying KIC in terms of the strain of the animal being used; dose, duration, and route of ketamine administration to induce KIC, and assessment of pathological features. CONCLUSION: KIC remains difficult to fully recapitulate in humans. Improved characterization of KIC models and the experimental parameters and meticulous discussion on translational limitations are required to improve the translational value of research using rodent models of KIC.

Discovery of a Biased Allosteric Modulator for Cannabinoid 1 Receptor: Preclinical Anti-Glaucoma Efficacy.

We apply the magic methyl effect to improve the potency/efficacy of GAT211, the prototypic 2-phenylindole-based cannabinoid type-1 receptor (CB1R) agonist-positive allosteric modulator (ago-PAM). Introducing a methyl group at the α-position of nitro group generated two diastereomers, the greater potency and efficacy of erythro, (±)-9 vs threo, (±)-10 constitutes the first demonstration of diastereoselective CB1R-allosteric modulator interaction. Of the (±)-9 enantiomers, (-)-(S,R)-13 evidenced improved potency over GAT211 as a CB1R ago-PAM, whereas (+)-(R,S)-14 was a CB1R allosteric agonist biased toward G protein- vs ß-arrestin1/2-dependent signaling. (-)-(S,R)-13 and (+)-(R,S)-14 were devoid of undesirable side effects (triad test), and (+)-(R,S)-14 reduced intraocular pressure with an unprecedentedly long duration of action in a murine glaucoma model. (-)-(S,R)-13 docked into both a CB1R extracellular PAM and intracellular allosteric-agonist site(s), whereas (+)-(R,S)-14 preferentially engaged only the latter. Exploiting G-protein biased CB1R-allosteric modulation can offer safer therapeutic candidates for glaucoma and, potentially, other diseases.

Endothelin B receptor dysfunction mediates elevated myogenic tone in cerebral arteries from aged male Fischer 344 rats.

The human brain requires adequate cerebral blood flow to meet the high demand for nutrients and to clear waste products. With age, there is a chronic reduction in cerebral blood flow in small resistance arteries that can eventually limit proper brain function. The endothelin system is a key mediator in the regulation of cerebral blood flow, but the contributions of its constituent receptors in the endothelial and vascular smooth muscle layers of cerebral arteries have not been well defined in the context of aging. We isolated posterior cerebral arteries from young and aged Fischer 344 rats, as well as ETB receptor knock-out rats and mounted the vessels in plexiglass pressure myograph chambers to measure myogenic tone in response to increasing pressure and targeted pharmacological treatments. We used an ETA receptor antagonist (BQ-123), an ETB receptor antagonist (BQ-788), endothelin-1, an endothelin-1 synthesis inhibitor (phosphoramidon), and vessel denudation to dissect the roles of each receptor in aging vasculature. Aged rats exhibited a higher myogenic tone than young rats, and the tone was sensitive to the ETA antagonist, BQ-123, but insensitive to the ETB antagonist, BQ-788. By contrast, the tone in the vessels from young rats was raised by BQ-788 but unaffected by BQ-123. When the endothelial layer that is normally enriched with ETB1 receptors was removed from young vessels, myogenic tone increased. However, denudation of the endothelial layer did not influence vessels from aged animals. This indicated that endothelial ETB1 receptors were not functional in the vessels from aged rats. There was also an increase in ETA receptor expression with age, whereas ETB receptor expression remained constant between young and aged animals. These results demonstrate that in young vessels, ETB1 receptors maintain a lower myogenic tone, but in aged vessels, a loss of ETB receptor activity allows ETA receptors in vascular smooth muscle cells to raise myogenic tone. Our findings have potentially important clinical implications for treatments to improve cerebral perfusion in older adults with diseases characterized by reduced cerebral blood flow.

Heteromer formation between cannabinoid type 1 and dopamine type 2 receptors is altered by combination cannabinoid and antipsychotic treatments.

The cannabinoid type 1 (CB1 ) receptor and the dopamine type 2 (D2 ) receptor are co-localized on medium spiny neuron terminals in the globus pallidus where they modulate neural circuits involved in voluntary movement. Physical interactions between the two receptors have been shown to alter receptor signaling in cell culture. The objectives of the current study were to identify the presence of CB1 /D2 heteromers in the globus pallidus of C57BL/6J male mice, define how CB1 /D2 heteromer levels are altered following treatment with cannabinoids and/or antipsychotics, and determine if fluctuating levels of CB1 /D2 heteromers have functional consequences. Using in situ proximity ligation assays, we observed CB1 /D2 heteromers in the globus pallidus of C57BL/6J mice. The abundance of the heteromers increased following treatment with the nonselective cannabinoid receptor agonist, CP55,940. In contrast, treatment with the typical antipsychotic haloperidol reduced the number of CB1 /D2 heteromers, whereas the atypical antipsychotic olanzapine treatment had no effect. Co-treatment with CP55,940 and haloperidol had similar effects to haloperidol alone, whereas co-treatment with CP55,940 and olanzapine had similar effects to CP55,940. The observed changes were found to have functional consequences as the differential effects of haloperidol and olanzapine also applied to γ-aminobutyric acid release in STHdhQ7/Q7 cells and motor function in C57BL/6J male mice. This work highlights the clinical relevance of co-exposure to cannabinoids and different antipsychotics over acute and prolonged time periods.

Endothelin receptor heteromerization inhibits ß-arrestin function in HEK293 cells.

The endothelin receptor A (ETA) and endothelin receptor B (ETB) are G protein-coupled receptors that are co-expressed in vascular smooth muscle cells. Endothelin-1 (ET-1) activates endothelin receptors to cause microvascular vasoconstriction. Previous studies have shown that heteromerization between ETA and ETB prolongs Ca2+ transients, leading to prolongation of Gαq-dependent signaling and sustained vasoconstriction. We hypothesized that these effects are in part mediated by the resistance of ETA/ETB heteromers to ß-arrestin recruitment and subsequent desensitization. Using bioluminescence resonance energy transfer 2 (BRET2), we found that ETB has a relatively equal affinity to form either homomers or heteromers with ETA when co-expressed in the human embryonic kidney 293 (HEK293) cells. When co-expressed, activation of ETA and ETB by ET-1 caused a heteromer-specific reduction and delay in ß-arrestin-2 recruitment with a corresponding reduction and delay in ET-1-induced ETA/ETB co-internalization. Furthermore, the co-expression of ETA and ETB inhibited ET-1-induced ß-arrestin-1-dependent extracellular signal-regulated kinase (ERK) phosphorylation while prolonging ET-1-induced Gαq-dependent ERK phosphorylation. ETA/ETB heteromerization mediates the long-lasting vasoconstrictor response to ET-1 by the prolongation of Gαq-dependent signaling and inhibition of ß-arrestin function.

Allosteric Cannabinoid Receptor 1 (CB1) Ligands Reduce Ocular Pain and Inflammation.

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.

Indomethacin Enhances Type 1 Cannabinoid Receptor Signaling.

In addition to its known actions as a non-selective cyclooxygenase (COX) 1 and 2 inhibitor, we hypothesized that indomethacin can act as an allosteric modulator of the type 1 cannabinoid receptor (CB1R) because of its shared structural features with the known allosteric modulators of CB1R. Indomethacin enhanced the binding of [3H]CP55940 to hCB1R and enhanced AEA-dependent [35S]GTPγS binding to hCB1R in Chinese hamster ovary (CHO) cell membranes. Indomethacin (1 µM) also enhanced CP55940-dependent ßarrestin1 recruitment, cAMP inhibition, ERK1/2 and PLCß3 phosphorylation in HEK293A cells expressing hCB1R, but not in cells expressing hCB2R. Finally, indomethacin enhanced the magnitude and duration of CP55940-induced hypolocomotion, immobility, hypothermia, and anti-nociception in C57BL/6J mice. Together, these data support the hypothesis that indomethacin acted as a positive allosteric modulator of hCB1R. The identification of structural and functional features shared amongst allosteric modulators of CB1R may lead to the development of novel compounds designed for greater CB1R or COX selectivity and compounds designed to modulate both the prostaglandin and endocannabinoid systems.

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.

Combining SRET2 and BiFC to Study GPCR Heteromerization and Protein-Protein Interactions.

G protein-coupled receptors (GPCRs) are the target for many drugs. Evidence continues to accumulate demonstrating that multiple receptors form homo- and heteromeric complexes, which in turn dynamically couple with G proteins, and other interacting proteins. Here, we describe a method to simultaneously determine the identity of up to four distinct constituents of GPCR complexes using a combination of sequential bioluminescence resonance energy transfer 2-fluorescence resonance energy transfer (SRET2) with bimolecular fluorescence complementation (BiFC). The method is amenable to moderate throughput screening of changes in response to ligands and time-course analysis of protein-protein oligomerization.

Positive allosteric modulation of the type 1 cannabinoid receptor reduces the signs and symptoms of Huntington's disease in the R6/2 mouse model.

Huntington's disease (HD) is an inherited progressive neurodegenerative disease characterized by motor, cognitive, and behavioural changes. One of the earliest changes to occur in HD is a reduction in cannabinoid 1 receptor (CB1) levels in the striatum, which is strongly correlated with HD pathogenesis. CB1 positive allosteric modulators (PAM) enhance receptor affinity for, and efficacy of activation by, orthosteric ligands, including the endocannabinoids anandamide and 2-arachidonoylglycerol. The goal of this study was to determine whether the recently characterized CB1 allosteric modulators GAT211 (racemic), GAT228 (R-enantiomer), and GAT229 (S-enantiomer), affected the signs and symptoms of HD. GAT211, GAT228, and GAT229 were evaluated in normal and HD cell models, and in a transgenic mouse model of HD (7-week-old male R6/2 mice, 10 mg/kg/d, 21 d, i.p.). GAT229 was a CB1 PAM that improved cell viability in HD cells and improved motor coordination, delayed symptom onset, and normalized gene expression in R6/2 HD mice. GAT228 was an allosteric agonist that did not enhance endocannabinoid signaling or change symptom progression in R6/2 mice. GAT211 displayed intermediate effects between its enantiomers. The compounds used here are not drugs, but probe compounds used to determine the potential utility of CB1 PAMs in HD. Changes in gene expression, and not protein, were quantified in R6/2 HD mice because HD pathogenesis is associated with dysregulation of mRNA levels. The data presented here provide the first proof of principle for the use of CB1 PAMs to treat the signs and symptoms of HD.

Modulation of the Endocannabinoid System Following Central Nervous System Injury.

Central nervous system (CNS) injury, such as stroke or trauma, is known to increase susceptibility to various infections that adversely affect patient outcomes (CNS injury-induced immunodepression-CIDS). The endocannabinoid system (ECS) has been shown to have immunoregulatory properties. Therefore, the ECS might represent a druggable target to overcome CIDS. Evidence suggests that cannabinoid type 2 receptor (CB2R) activation can be protective during the early pro-inflammatory phase after CNS injury, as it limits neuro-inflammation and, therefore, attenuates CIDS severity. In the later phase post CNS injury, CB2R inhibition is suggested as a promising pharmacologic strategy to restore immune function in order to prevent infection.

Allosteric and orthosteric pharmacology of cannabidiol and cannabidiol-dimethylheptyl at the type 1 and type 2 cannabinoid receptors.

BACKGROUND AND PURPOSE: We sought to understand why (-)-cannabidiol (CBD) and (-)-cannabidiol-dimethylheptyl (CBD-DMH) exhibit distinct pharmacology, despite near identical structures. EXPERIMENTAL APPROACH: HEK293A cells expressing either human type 1 cannabinoid (CB1 ) receptors or CB2 receptors were treated with CBD or CBD-DMH with or without the CB1 and CB2 receptor agonist CP55,940, CB1 receptor allosteric modulator Org27569 or CB2 receptor inverse agonist SR144528. Ligand binding, cAMP levels and ßarrestin1 recruitment were measured. CBD and CBD-DMH binding was simulated with models of human CB1 or CB2 receptors, based on the recently published crystal structures of agonist-bound (5XRA) or antagonist-bound (5TGZ) human CB1 receptors. KEY RESULTS: At CB1 receptors, CBD was a negative allosteric modulator (NAM), and CBD-DMH was a mixed agonist/positive allosteric modulator. CBD and Org27569 shared multiple interacting residues in the antagonist-bound model of CB1 receptors (5TGZ) but shared a binding site with CP55,940 in the agonist-bound model of CB1 receptors (5XRA). The binding site for CBD-DMH in the CB1 receptor models overlapped with CP55,940 and Org27569. At CB2 receptors, CBD was a partial agonist, and CBD-DMH was a positive allosteric modulator of cAMP modulation but a NAM of ßarrestin1 recruitment. CBD, CP55,940 and SR144528 shared a binding site in the CB2 receptor models that was separate from CBD-DMH. CONCLUSION AND IMPLICATIONS: The pharmacological activity of CBD and CBD-DMH in HEK293A cells and their modelled binding sites at CB1 and CB2 receptors may explain their in vivo effects and illuminates the difficulties associated with the development of allosteric modulators for CB1 and CB2 receptors. LINKED ARTICLES: This article is part of a themed section on 8th European Workshop on Cannabinoid Research. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.10/issuetoc.

Effects of intravitreal bevacizumab in Gram-positive and Gram-negative models of ocular inflammation.

BACKGROUND: Exogenous endophthalmitis is a potential complication of intraocular surgery and frequently results in visual impairment. Current treatment involves administration of intravitreal (IVT) antibiotics with or without vitrectomy surgery. Evidence for the use of adjunctive anti-inflammatory agents is conflicting. We set out to determine if bevacizumab, a humanized monoclonal IgG1 antibody targeted against vascular endothelial growth factor (VEGF), has anti-inflammatory properties in experimental models of Gram-positive and Gram-negative inflammation. METHODS: BALB/c mice were subjected to lipopolysaccharide- (LPS) or peptidoglycan- (PGN) induced ocular inflammation and treated with IVT bevacizumab. Iris microvasculature was imaged 6 hours following irritant/treatment using intravital microscopy (IVM) before the mice were euthanized and the eyes were enucleated immediately post-mortem. Following enucleation, levels of VEGF and 23 cytokines and chemokines (IL-1α, IL-1ß, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-17, TNF, KC, G-CSF, GM-CSF, Eotaxin, INF-γ, MCP-1, MIP-1α, MIP-1ß, RANTES) were quantified using a multiplex assay. RESULTS: Levels of VEGF were significantly increased during the inflammatory response, triggered by either PGN or LPS. Both the adherence of leukocytes to the iris vascular endothelium and the levels of pro-inflammatory cytokines and chemokines were significantly increased following administration of either irritant. Treatment with bevacizumab decreased levels of leukocyte adherence in LPS-treated eyes, however, not in PGN-treated eyes. Conversely, bevacizumab treatment decreased levels of cytokines and chemokines (TNF, IL-6, MCP-1, MIP-1α, MIP-1ß, RANTES, KC) in PGN-treated eyes, however, not in LPS-treated eyes. CONCLUSIONS: Within a 6-hour window bevacizumab had anti-inflammatory actions that were distinct in both Gram-positive (PIU) and Gram-negative (EIU) models, respectively. Given our findings, this would suggest that bevacizumab may have utility as an adjunctive therapy to IVT antibiotics and vitrectomy in the management of exogenous endophthalmitis.

Revisiting cannabinoid receptor 2 expression and function in murine retina.

The cannabinoid receptor CB2 plays a significant role in the regulation of immune function whereas neuronal expression remains a subject of contention. Multiple studies have described CB2 in retina and a recent study showed that CB2 deletion altered retinal visual processing. We revisited CB2 expression using immunohistochemistry and a recently developed CB2-eGFP reporter mouse. We examined the consequence of acute vs. prolonged CB2 deactivation on the electroretinogram (ERG) responses. We also examined lipidomics in CB2 knockout mice and potential changes in microglia using Scholl analysis. Consistent with a published report, in CB2 receptor knockout mice see an increased ERG scotopic a-wave, as well as stronger responses in dark adapted cone-driven ON bipolar cells and, to a lesser extent cone-driven ON bipolar cells early in light adaptation. Significantly, however, acute block with CB2 antagonist, AM630, did not mimic the results observed in the CB2 knockout mice whereas chronic (7 days) block did. Immunohistochemical studies show no CB2 in retina under non-pathological conditions, even with published antibodies. Retinal CB2-eGFP reporter signal is minimal under baseline conditions but upregulated by intraocular injection of either LPS or carrageenan. CB2 knockout mice see modest declines in a broad spectrum of cannabinoid-related lipids. The numbers and morphology of microglia were unaltered. In summary minimal CB2 expression is seen in healthy retina. CB2 appears to be upregulated under pathological conditions. Previously reported functional consequences of CB2 deletion are an adaptive response to prolonged blockade of these receptors. CB2 therefore impacts retinal signaling but perhaps in an indirect, potentially extra-ocular fashion.

Methods to Quantify Cell Signaling and GPCR Receptor Ligand Bias: Characterization of Drugs that Target the Endocannabinoid Receptors in Huntington's Disease.

G protein-coupled receptors (GPCRs) interact with multiple intracellular effector proteins such that different ligands may preferentially activate one signal pathway over others, a phenomenon known as signaling bias. Signaling bias can be quantified to optimize drug selection for preclinical research. Here, we describe moderate-throughput methods to quantify signaling bias of known and novel compounds. In the example provided, we describe a method to define cannabinoid-signaling bias in a cell culture model of Huntington's disease (HD). Decreasing type 1 cannabinoid receptor (CB1) levels is correlated with chorea and cognitive deficits in HD. There is evidence that elevating CB1 levels and/or signaling may be beneficial for HD patients while decreasing CB1 levels and/or signaling may be detrimental. Recent studies have found that Gαi/o-biased CB1 agonists activate extracellular signal-regulated kinase (ERK), increase CB1 protein levels, and improve viability of cells expressing mutant huntingtin. In contrast, CB1 agonists that are ß-arrestin1-biased were found to reduce CB1 protein levels and cell viability. Measuring agonist bias of known and novel CB1 agonists will provide important data that predict CB1-specific agonists that might be beneficial in animal models of HD and, following animal testing, in HD patients. This method can also be applied to study signaling bias for other GPCRs.

The Cannabinoids Δ8THC, CBD, and HU-308 Act via Distinct Receptors to Reduce 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-HT1A 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-HT1A 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-HT1A receptors and CB2Rs, respectively. Cannabinoids could be a novel clinical therapy for corneal pain and inflammation resulting from ocular surface injury.

Potential for endocannabinoid system modulation in ocular pain and inflammation: filling the gaps in current pharmacological options.

Challenges in the management of ocular pain are an underappreciated topic. Currently available therapeutics lack both efficacy and clear guidelines for their use, with many also possessing unacceptable side effects. Promising novel agents would offer analgesic, anti-inflammatory, and possibly neuroprotective actions; have favorable ocular safety profiles; and show potential in managing neuropathic pain. Growing evidence supports a link between the endocannabinoid system (ECS) and a range of physiological and disease processes, notably those involving inflammation and pain. Both preclinical and clinical data suggest analgesic and anti-inflammatory actions of cannabinoids and ECS-modifying drugs in chronic pain conditions, including those of neuropathic origin. This review will examine existing evidence for the anatomical and physiological basis of ocular pain, specifically, ocular surface disease and the development of chronic ocular pain. The mechanism of action, efficacy, and limitations of currently available treatments will be discussed, and current knowledge related to ECS-modulation of ocular pain and inflammatory disease will be summarized. A perspective will be provided on the future directions of ECS research in terms of developing cannabinoid therapeutics for ocular pain.

Cannabinoids in the Cardiovascular System.

Cannabinoids are known to modulate cardiovascular functions including heart rate, vascular tone, and blood pressure in humans and animal models. Essential components of the endocannabinoid system, namely, the production, degradation, and signaling pathways of endocannabinoids have been described not only in the central and peripheral nervous system but also in myocardium, vasculature, platelets, and immune cells. The mechanisms of cardiovascular responses to endocannabinoids are often complex and may involve cannabinoid CB1 and CB2 receptors or non-CB1/2 receptor targets. Preclinical and some clinical studies have suggested that targeting the endocannabinoid system can improve cardiovascular functions in a number of pathophysiological conditions, including hypertension, metabolic syndrome, sepsis, and atherosclerosis. In this chapter, we summarize the local and systemic cardiovascular effects of cannabinoids and highlight our current knowledge regarding the therapeutic potential of endocannabinoid signaling and modulation.