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.

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.

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.

Application of Fluorine- and Nitrogen-Walk Approaches: Defining the Structural and Functional Diversity of 2-Phenylindole Class of Cannabinoid 1 Receptor Positive Allosteric Modulators.

Cannabinoid 1 receptor (CB1R) allosteric ligands hold a far-reaching therapeutic promise. We report the application of fluoro- and nitrogen-walk approaches to enhance the drug-like properties of GAT211, a prototype CB1R allosteric agonist-positive allosteric modulator (ago-PAM). Several analogs exhibited improved functional potency (cAMP, ß-arrestin 2), metabolic stability, and aqueous solubility. Two key analogs, GAT591 (6r) and GAT593 (6s), exhibited augmented allosteric-agonist and PAM activities in neuronal cultures, improved metabolic stability, and enhanced orthosteric agonist binding (CP55,940). Both analogs also exhibited good analgesic potency in the CFA inflammatory-pain model with longer duration of action over GAT211 while being devoid of adverse cannabimimetic effects. Another analog, GAT592 (9j), exhibited moderate ago-PAM potency and improved aqueous solubility with therapeutic reduction of intraocular pressure in murine glaucoma models. The SAR findings and the enhanced allosteric activity in this class of allosteric modulators were accounted for in our recently developed computational model for CB1R allosteric activation and positive allosteric modulation.

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.

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.

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.

The In Vivo Effects of the CB1-Positive Allosteric Modulator GAT229 on Intraocular Pressure in Ocular Normotensive and Hypertensive Mice.

PURPOSE: Orthosteric cannabinoid receptor 1 (CB1) activation leads to decreases in intraocular pressure (IOP). However, use of orthosteric CB1 agonists chronically has several disadvantages, limiting their usefulness as clinically relevant drugs. Allosteric modulators interact with topographically distinct sites to orthosteric ligands and may be useful to circumvent some of these disadvantages. The purpose of this study was to investigate the effects of the novel CB1-positive allosteric modulator (PAM) GAT229 on IOP. METHODS: IOP was measured using rebound tonometry in anesthetized normotensive C57Bl/6 mice and in a genetic model of ocular hypertension [nose, eyes, ears (nee) mice] before drug administration, and at 1, 6, and 12 h thereafter. RESULTS: In normotensive mice, topical administration of 5 µL GAT229 alone at either 0.2% or 2% did not reduce IOP. However, a subthreshold dose (0.25%) of the nonselective orthosteric CB1 agonist WIN 55,212-2, when combined with 0.2% GAT229, significantly reduced IOP compared with vehicle at 6 and 12 h. Similarly, combination of subthreshold Δ9-tetrahydrocannabinol (a nonselective orthosteric CB1 agonist; 1 mg/kg) with topical 0.2% GAT229 produced IOP lowering at 6 h. In nee mice, administration of topical 0.2% GAT229 or 10 mg/kg GAT229 alone was sufficient to lower IOP at 6 and 12 h, and 12 h, respectively. CONCLUSIONS: The CB1 PAM GAT229 reduces IOP in ocular hypertensive mice and enhanced CB1-mediated IOP reduction when combined with subthreshold CB1 orthosteric ligands in normotensive mice. Administration of CB1 PAMs may provide a novel approach to reduce IOP with fewer of the disadvantages associated with orthosteric CB1 activation.

Cannabinoid 2 receptor is a novel anti-inflammatory target in experimental proliferative vitreoretinopathy.

Proliferative vitreoretinopathy (PVR) can develop after ocular trauma or inflammation and is a common complication of surgery to correct retinal detachment. Currently, there are no pharmacological treatments for PVR. Cannabinoids acting at cannabinoid 2 receptor (CB2R) can decrease inflammation and fibrosis. The objective of this study was to examine the anti-inflammatory actions of CB2R as a candidate novel therapeutic target in experimental PVR. PVR was induced by intravitreal injection of dispase in wild-type (WT) and CB2R genetic knockout (CB2R-/-) mice. Ocular pathology was studied at 24 h or one week after dispase injection. CB2R modulation was examined in WT mice, using the CB2R agonist, HU308, and the CB2R antagonist, AM630. Histopathological scoring and quantification of microglia was used to evaluate tissue pathology. Quantitative PCR and multiplex assays were used to assess changes in proinflammatory cytokines. Intravital microscopy (IVM) was used to visualize and quantify leukocyte-endothelial adhesion to the iridial microcirculation. Activation of CB2R with HU308 in WT mice with PVR decreased mean histopathological scores, the number of microglia, and leukocyte adhesion compared to vehicle-treated animals. Conversely, an increase in histopathological scores and activated microglia was observed in PVR animals after treatment with AM630. CB2R-/- mice with PVR exhibited exacerbated ocular histopathology, increased microglia numbers, and elevated protein levels of cytokines as compared to WT mice. In conclusion, our results indicate that intervention at early stage PVR with CB2R agonists reduces ocular inflammation and disease severity. CB2R may represent a therapeutic target to prevent PVR progression and vision loss. This article is part of the Special Issue entitled 'Lipid Sensing G Protein-Coupled Receptors in the CNS'.

Seeing over the horizon - targeting the endocannabinoid system for the treatment of ocular disease.

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.

The fatty acid amide hydrolase inhibitor, URB597, promotes retinal ganglion cell neuroprotection in a rat model of optic nerve axotomy.

The endocannabinoid, N-arachidonoylethanolamine (AEA), is degraded by the enzyme fatty acid amide hydrolase (FAAH). This study examined whether the FAAH inhibitor, URB597, increases retinal ganglion cell (RGC) survival following optic nerve axotomy in young and aged animals. URB597 alone, or together with either a CB1 or CB2 receptor antagonist, was administered daily for 1 or 2 weeks post-axotomy. Histological assessment of retinas indicated that URB597 increased RGC survival in young retina at 1 and 2 weeks post-axotomy. The increase in RGC survival at 2 weeks was accompanied by a reduction in phagocytic microglia. The CB1 antagonist, AM281, but not the CB2 antagonist, AM630, ablated URB597-mediated RGC neuroprotection. CB1 or CB2 antagonism increased phagocytic microglia in URB597 and vehicle-treated animals. In aged animals, URB597 increased RGC survival at 1 week, but not at 2 weeks post-axotomy and had no effect on microglia. Retinal Iba-1 positive microglia were also decreased in URB597-treated axotomized young animals and this decrease was mitigated by CB1 but not CB2 antagonism. As seen with phagocytotic microglia, the CB2 antagonist, AM630, increased Iba-1 positive microglia in the absence of URB597 treatment. Measurement of retinal endocannabinoid levels in URB597-treated animals at 2 weeks post-axotomy revealed a significant increase in AEA levels, accompanied by a decrease in the AEA metabolite, N-arachidonoyl glycine, in young animals but not aged animals. 2-arachidonoylglycerol levels were similar across all experimental groups. These data demonstrate that URB597-mediated retinal neuroprotective effects are mediated primarily through CB1 receptors and that URB597 neuroprotective efficacy declines with age.

Indirect sympatholytic actions at ß-adrenoceptors account for the ocular hypotensive actions of cannabinoid receptor agonists.

Intraocular pressure (IOP) is the primary risk factor for glaucoma, a blinding eye disease. Cannabinoid agonists have long been known to decrease IOP, suggesting they may be useful in glaucoma treatment. However, the specific mechanism by which cannabinoids generate this ocular hypotensive effect remains unknown. The current evidence suggests the cannabinoids reduce IOP through actions at cannabinoid 1 (CB(1)) receptors within the eye, and adrenergic receptors (ARs) may also contribute to this action of cannabinoids. Considering this, the present study aimed to elucidate the mechanism behind the ocular hypotensive properties of cannabinoids through the use of mice genetically lacking either cannabinoid receptors or ßARs. Cannabinoid agonists, ßAR antagonists, and ßAR agonists decreased IOP in wild-type mice and CB(2)(-/-) mice. In contrast, none of these compounds were found to reduce IOP in ßAR(-/-) or CB(1)(-/-) mice. Desensitization of the ßARs and depletion of catecholamines in wild-type mice also eliminated the ability of the cannabinoid agonist (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate (WIN 55,212-2) to reduce IOP, strongly implicating a role for both ßARs and catecholamines in the ocular hypotensive properties of cannabinoids. Finally, CB(1) receptors were shown to colocalize with tyrosine hydroxylase, a marker for adrenergic neurons. Taken together, these findings suggest that ßARs are required for the ocular hypotensive properties of cannabinoids, and cannabinoids reduce IOP by acting as indirect sympatholytics and inhibiting norepinephrine release within the eye.

Nonpsychotropic cannabinoids, abnormal cannabidiol and canabigerol-dimethyl heptyl, act at novel cannabinoid receptors to reduce intraocular pressure.

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 (CB(1)R) and CB(2)R 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 CB(1)R antagonist, AM251 (2.5 mg/kg), or the CB(2)R antagonist, AM630 (2.5 mg/kg). In contrast, the IOP-lowering action of WIN55,212-2 was completely blocked by the CB(1)R-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 CB(2)R antagonist, AM630. Consistent with this, the selective CB(2)R 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 CB(1)R or CB(2)R activation, via activation of CBx receptors. The enhanced decrease in IOP seen after coapplication of the CB(1)R 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 CB(1)R. These results indicate that both CBG-DMH and abn-CBD have the potential for further investigation as novel ocular hypotensive cannabinoids devoid of CB(1)R-mediated side-effects.

Ocular hypotensive effects of an intratracheally delivered liposomal delta9-tetrahydrocannabinol preparation in rats.

This study investigated the effect of an intratracheal (i.t.) administration of a liposome-entrapped Delta9-tetrahydrocannabinol (LTHC) preparation on intraocular pressure (IOP) in nonanaesthetized Brown Norway rats. The ocular hypotensive effects of i.t. LTHC were compared to that of intraperitoneal (i.p.) LTHC administration. All i.t. LTHC doses >0.05 mg/kg significantly decreased IOP (P < 0.05) within 30 min of administration, and doses of i.t. LTHC >0.1 mg/kg decreased IOP within 15 min of administration. A maximal reduction in IOP of 2.32 +/- 0.27 mmHg (n = 4) was seen with 1.0 mg/kg of i.t. LTHC. In comparison, no significant IOP drop was apparent prior to 30 min with all doses (0.01-1.0 mg/kg) of i.p. LTHC tested, although a similar maximum drop in IOP (2.15 +/- 0.12 mmHg; n = 8) was obtained with 1.0 mg/kg of LTHC. The ED(50) for i.t. and i.p. LTHC was 0.08 mg/kg and 0.12 mg/kg, respectively. The IOP-lowering effects of i.p. LTHC (0.2 mg/kg) were reduced by 14% and 80% by 0.25 mg/kg (n = 6) and 2.5 mg/kg (n = 6), respectively, of the CB1R antagonist, SR141716A. In conclusion, i.t. LTHC was superior to i.p. LTHC in producing a more rapid and potent decrease in IOP. The IOP-lowering effect of LTHC was blocked by the CB1R-selective antagonist, SR141716A, suggesting that CB1Rs contribute to the ocular hypotensive effect of Delta9-tetrahydrocannabinol.