Evidence for a GPR18 Role in Chemotaxis, Proliferation, and the Course of Wound Closure in the Cornea.

PURPOSE: We previously showed that cannabinoid-related GPR18 receptors are present in the murine corneal epithelium, but their function remains unknown. The related CB1 receptors regulate corneal healing, possibly via chemotaxis. We therefore examined a potential role for GPR18 in corneal epithelial chemotaxis and wound healing. METHODS: We examined GPR18 messenger RNA (mRNA) and protein expression in the cornea. We additionally examined GPR18 action in cultured bovine corneal epithelial cells (bCECs) using Boyden and tracking assays, as well as proliferation and signaling. Finally, we examined wound closure in murine corneal explants. RESULTS: GPR18 mRNA was upregulated with injury in the mouse cornea. GPR18 protein was present in basal epithelial cells of the mouse and cow and redistributed to the wound site upon injury. GPR18 ligand N-arachidonoylglycine induced bCEC chemotaxis. The endocannabinoid arachidonoylethanolamine also induced chemotaxis via fatty acid amide hydrolase-mediated metabolism to N-arachidonoylglycine. GPR18 receptor activation additionally induced bCEC proliferation. In an explant model, the GPR18 antagonist O-1918 slowed corneal epithelial cell migration and the rate of corneal wound closure. CONCLUSIONS: Corneal GPR18 activation induced both chemotaxis and proliferation in corneal epithelial cells in vitro and impacted wound healing. GPR18 may contribute to the maintenance of corneal integrity.

Cannabinoid CB2R receptors are upregulated with corneal injury and regulate the course of corneal wound healing.

CB2R receptors have demonstrated beneficial effects in wound healing in several models. We therefore investigated a potential role of CB2R receptors in corneal wound healing. We examined the functional contribution of CB2R receptors to the course of wound closure in an in vivo murine model. We additionally examined corneal expression of CB2R receptors in mouse and the consequences of their activation on cellular signaling, migration and proliferation in cultured bovine corneal epithelial cells (CECs). Using a novel mouse model, we provide evidence that corneal injury increases CB2R receptor expression in cornea. The CB2R agonist JWH133 induces chemorepulsion in cultured bovine CECs but does not alter CEC proliferation. The signaling profile of CB2R activation is activating MAPK and increasing cAMP accumulation, the latter perhaps due to Gs-coupling. Lipidomic analysis in bovine cornea shows a rise in acylethanolamines including the endocannabinoid anandamide 1 h after injury. In vivo, CB2R deletion and pharmacological block result in a delayed course of wound closure. In summary, we find evidence that CB2R receptor promoter activity is increased by corneal injury and that these receptors are required for the normal course of wound closure, possibly via chemorepulsion.

Cannabinoid CB2 Agonist AM1710 Differentially Suppresses Distinct Pathological Pain States and Attenuates Morphine Tolerance and Withdrawal.

AM1710 (3-(1,1-dimethyl-heptyl)-1-hydroxy-9-methoxy-benzo(c) chromen-6-one), a cannabilactone cannabinoid receptor 2 (CB2) agonist, suppresses chemotherapy-induced neuropathic pain in rodents without producing tolerance or unwanted side effects associated with CB1 receptors; however, the signaling profile of AM1710 remains incompletely characterized. It is not known whether AM1710 behaves as a broad-spectrum analgesic and/or suppresses the development of opioid tolerance and physical dependence. In vitro, AM1710 inhibited forskolin-stimulated cAMP production and produced enduring activation of extracellular signal-regulated kinases 1/2 phosphorylation in human embryonic kidney (HEK) cells stably expressing mCB2. Only modest species differences in the signaling profile of AM1710 were observed between HEK cells stably expressing mCB2 and hCB2. In vivo, AM1710 produced a sustained inhibition of paclitaxel-induced allodynia in mice. In paclitaxel-treated mice, a history of AM1710 treatment (5 mg/kg per day × 12 day, i.p.) delayed the development of antinociceptive tolerance to morphine and attenuated morphine-induced physical dependence. AM1710 (10 mg/kg, i.p.) did not precipitate CB1 receptor-mediated withdrawal in mice rendered tolerant to Δ9-tetrahydrocannabinol, suggesting that AM1710 is not a functional CB1 antagonist in vivo. Furthermore, AM1710 (1, 3, 10 mg/kg, i.p.) did not suppress established mechanical allodynia induced by complete Freund's adjuvant (CFA) or by partial sciatic nerve ligation (PSNL). Similarly, prophylactic and chronic dosing with AM1710 (10 mg/kg, i.p.) did not produce antiallodynic efficacy in the CFA model. By contrast, gabapentin suppressed allodynia in both CFA and PSNL models. Our results indicate that AM1710 is not a broad-spectrum analgesic agent in mice and suggest the need to identify signaling pathways underlying CB2 therapeutic efficacy to identify appropriate indications for clinical translation.

Slowly Signaling G Protein-Biased CB2 Cannabinoid Receptor Agonist LY2828360 Suppresses Neuropathic Pain with Sustained Efficacy and Attenuates Morphine Tolerance and Dependence.

The CB2 cannabinoid agonist LY2828360 lacked both toxicity and efficacy in a clinical trial for osteoarthritis. Whether LY2828360 suppresses neuropathic pain has not been reported, and its signaling profile is unknown. In vitro, LY2828360 was a slowly acting but efficacious G protein-biased CB2 agonist, inhibiting cAMP accumulation and activating extracellular signal-regulated kinase 1/2 signaling while failing to recruit arrestin, activate inositol phosphate signaling, or internalize CB2 receptors. In wild-type (WT) mice, LY2828360 (3 mg/kg per day i.p. × 12 days) suppressed chemotherapy-induced neuropathic pain produced by paclitaxel without producing tolerance. Antiallodynic efficacy of LY2828360 was absent in CB2 knockout (KO) mice. Morphine (10 mg/kg per day i.p. × 12 days) tolerance developed in CB2KO mice but not in WT mice with a history of LY2828360 treatment (3 mg/kg per day i.p. × 12 days). LY2828360-induced antiallodynic efficacy was preserved in WT mice previously rendered tolerant to morphine (10 mg/kg per day i.p. × 12 days), but it was absent in morphine-tolerant CB2KO mice. Coadministration of LY2828360 (0.1 mg/kg per day i.p. × 12 days) with morphine (10 mg/kg per day × 12 days) blocked morphine tolerance in WT but not in CB2KO mice. WT mice that received LY2828360 coadministered with morphine exhibited a trend (P = 0.055) toward fewer naloxone-precipitated jumps compared with CB2KO mice. In conclusion, LY2828360 is a slowly signaling, G protein-biased CB2 agonist that attenuates chemotherapy-induced neuropathic pain without producing tolerance and may prolong effective opioid analgesia while reducing opioid dependence. LY2828360 may be useful as a first-line treatment in chemotherapy-induced neuropathic pain and may be highly efficacious in neuropathic pain states that are refractive to opioid analgesics.

Synthesis of Photoswitchable Δ9-Tetrahydrocannabinol Derivatives Enables Optical Control of Cannabinoid Receptor 1 Signaling.

The cannabinoid receptor 1 (CB1) is an inhibitory G protein-coupled receptor abundantly expressed in the central nervous system. It has rich pharmacology and largely accounts for the recreational use of cannabis. We describe efficient asymmetric syntheses of four photoswitchable Δ9-tetrahydrocannabinol derivatives (azo-THCs) from a central building block 3-Br-THC. Using electrophysiology and a FRET-based cAMP assay, two compounds are identified as potent CB1 agonists that change their effect upon illumination. As such, azo-THCs enable CB1-mediated optical control of inwardly rectifying potassium channels, as well as adenylyl cyclase.

Two Janus Cannabinoids That Are Both CB2 Agonists and CB1 Antagonists.

The cannabinoid signaling system includes two G protein-coupled receptors, CB1 and CB2 These receptors are widely distributed throughout the body and have each been implicated in many physiologically important processes. Although the cannabinoid signaling system has therapeutic potential, the development of receptor-selective ligands remains a persistent hurdle. Because CB1 and CB2 are involved in diverse processes, it would be advantageous to develop ligands that differentially engage CB1 and CB2 We now report that GW405833 [1-(2,3-dichlorobenzoyl)-5-methoxy-2-methyl-3-[2-(4-morpholinyl)ethyl]-1H-indole] and AM1710 [1-hydroxy-9-methoxy-3-(2-methyloctan-2-yl)benzo[c]chromen-6-one], described as selective CB2 agonists, can antagonize CB1 receptor signaling. In autaptic hippocampal neurons, GW405833 and AM1710 both interfered with CB1-mediated depolarization-induced suppression of excitation, with GW405833 being more potent. In addition, in CB1-expressing human embryonic kidney 293 cells, GW405833 noncompetitively antagonized adenylyl cyclase activity, extracellular signal-regulated kinase 1/2 phosphorylation, phosphatidylinositol 4,5-bisphosphate signaling, and CB1 internalization by CP55940 (2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]-5-(2-methyloctan-2-yl)phenol). In contrast, AM1710 behaved as a low-potency competitive antagonist/inverse agonist in these signaling pathways. GW405833 interactions with CB1/arrestin signaling were complex: GW405833 differentially modulated arrestin recruitment in a time-dependent fashion, with an initial modest potentiation at 20 minutes followed by antagonism starting at 1 hour. AM1710 acted as a low-efficacy agonist in arrestin signaling at the CB1 receptor, with no evident time dependence. In summary, we determined that GW405833 and AM1710 are not only CB2 agonists but also CB1 antagonists, with distinctive and complex signaling properties. Thus, experiments using these compounds must take into account their potential activity at CB1 receptors.

Functional Selectivity of CB2 Cannabinoid Receptor Ligands at a Canonical and Noncanonical Pathway.

The CB2 cannabinoid receptor (CB2) remains a tantalizing, but unrealized therapeutic target. CB2 receptor ligands belong to varied structural classes and display extreme functional selectivity. Here, we have screened diverse CB2 receptor ligands at canonical (inhibition of adenylyl cyclase) and noncanonical (arrestin recruitment) pathways. The nonclassic cannabinoid (-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol (CP55940) was the most potent agonist for both pathways, while the classic cannabinoid ligand (6aR,10aR)-3-(1,1-Dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran JWH133) was the most efficacious agonist among all the ligands profiled in cyclase assays. In the cyclase assay, other classic cannabinoids showed little [(-)-trans-Δ(9)-tetrahydrocannabinol and (-)-(6aR,7,10,10aR)-tetrahydro-6,6,9-trimethyl-3-(1-methyl-1-phenylethyl)-6H-dibenzo[b,d]pyran-1-ol] (KM233) to no efficacy [(6aR,10aR)-1-methoxy-6,6,9-trimethyl-3-(2-methyloctan-2-yl)-6a,7,10,10a-tetrahydrobenzo[c]chromene(L759633) and (6aR,10aR)-3-(1,1-dimethylheptyl)-6a,7,8,9,10,10a-hexahydro-1-methoxy-6,6-dimethyl-9-methylene-6H-dibenzo[b,d]pyran]L759656. Most aminoalkylindoles, including [(3R)-​2,​3-​dihydro-​5-​methyl-​3-​(4-​morpholinylmethyl)pyrrolo[1,​2,​3-​de]-​1,​4-​benzoxazin-​6-​yl]-​1-​naphthalenyl-​methanone,​ monomethanesulfonate (WIN55212-2), were moderate efficacy agonists. The cannabilactone 3-(1,1-dimethyl-heptyl)-1-hydroxy-9-methoxy-benzo(c)chromen-6-one (AM1710) was equiefficacious to CP55940 to inhibit adenylyl cyclase, albeit with lower potency. In the arrestin recruitment assays, all classic cannabinoid ligands failed to recruit arrestins, indicating a bias toward G-protein coupling for this class of compound. All aminoalkylindoles tested, except for WIN55212-2 and (1-​pentyl-​1H-​indol-​3-​yl)(2,​2,​3,​3-​tetramethylcyclopropyl)-​methanone (UR144), failed to recruit arrestin. WIN55212-2 was a low efficacy agonist for arrestin recruitment, while UR144 was arrestin biased with no significant inhibition of cyclase. Endocannabinoids were G-protein biased with no arrestin recruitment. The diarylpyrazole antagonist 5-​(4-​chloro-​3-​methylphenyl)-​1-​[(4-​methylphenyl)methyl]-​N-​[(1S,​2S,​4R)-​1,​3,​3-​trimethylbicyclo[2.2.1]hept-​2-​yl]-​1H-​pyrazole-​3-​carboxamide (SR144258) was an inverse agonist in cyclase and arrestin recruitment assays while the aminoalkylindole 6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)methanone (AM630) and carboxamide N-(1,3-benzodioxol-5-ylmethyl)-1,2-dihydro-7-methoxy-2-oxo-8-(pentyloxy)-3-quinolinecarboxamide (JTE907) were inverse agonists in cyclase but low efficacy agonists in arrestin recruitment assays. Thus, CB2 receptor ligands display strong and varied functional selectivity at both pathways. Therefore, extreme care must be exercised when using these compounds to infer the role of CB2 receptors in vivo.

Where's my entourage? The curious case of 2-oleoylglycerol, 2-linolenoylglycerol, and 2-palmitoylglycerol.

2-Arachidonoylglycerol (2-AG) is the most abundant endogenous cannabinoid in the brain and an agonist at two cannabinoid receptors (CB1 and CB2). The synthesis, degradation and signaling of 2-AG have been investigated in detail but its relationship to other endogenous monoacylglycerols has not been fully explored. Three congeners that have been isolated from the CNS are 2-linoleoylglycerol (2-LG), 2-oleoylglycerol (2-OG), and 2-palmitoylglycerol (2-PG). These lipids do not orthosterically bind to cannabinoid receptors but are reported to potentiate the activity of 2-AG, possibly through inhibition of 2-AG degradation. This phenomenon has been dubbed the 'entourage effect' and has been proposed to regulate synaptic activity of 2-AG. To clarify the activity of these congeners of 2-AG we tested them in neuronal and cell-based signaling assays. The signaling profile for these compounds is inconsistent with an entourage effect. None of the compounds inhibited neurotransmission via CB1 in autaptic neurons. Interestingly, each failed to potentiate 2-AG-mediated depolarization-induced suppression of excitation (DSE), behaving instead as antagonists. Examining other signaling pathways we found that 2-OG interferes with agonist-induced CB1 internalization while 2-PG modestly internalizes CB1 receptors. However in tests of pERK, cAMP and arrestin recruitment, none of the acylglycerols altered CB1 signaling. Our results suggest 1) that these compounds do not serve as entourage compounds under the conditions examined, and 2) that they may instead serve as functional antagonists. Our results suggest that the relationship between 2-AG and its congeners is more nuanced than previously appreciated.

Cannabinoid-induced chemotaxis in bovine corneal epithelial cells.

PURPOSE: Cannabinoid CB1 receptors are found in abundance in the vertebrate eye, with most tissue types expressing this receptor. However, the function of CB1 receptors in corneal epithelial cells (CECs) is poorly understood. Interestingly, the corneas of CB1 knockout mice heal more slowly after injury via a mechanism proposed to involve protein kinase B (Akt) activation, chemokinesis, and cell proliferation. The current study examined the role of cannabinoids in CEC migration in greater detail. METHODS: We determined the role of CB1 receptors in corneal healing. We examined the consequences of their activation on migration and proliferation in bovine CECs (bCECs). We additionally examined the mRNA profile of cannabinoid-related genes and CB1 protein expression as well as CB1 signaling in bovine CECs. RESULTS: We now report that activation of CB1 with physiologically relevant concentrations of the synthetic agonist WIN55212-2 (WIN) induces bCEC migration via chemotaxis, an effect fully blocked by the CB1 receptor antagonist SR141716. The endogenous agonist 2-arachidonoylglycerol (2-AG) also enhances migration. Separately, mRNA for most cannabinoid-related proteins are present in bovine corneal epithelium and cultured bCECs. Notably absent are CB2 receptors and the 2-AG synthesizing enzyme diglycerol lipase-α (DAGLα). The signaling profile of CB1 activation is complex, with inactivation of mitogen-activated protein kinase (MAPK). Lastly, CB1 activation does not induce bCEC proliferation, but may instead antagonize EGF-induced proliferation. CONCLUSIONS: In summary, we find that CB1-based signaling machinery is present in bovine cornea and that activation of this system induces chemotaxis.

CB2 Cannabinoid receptors as a therapeutic target-what does the future hold?

The past decades have seen an exponential rise in our understanding of the endocannabinoid system, comprising CB1 and CB2 cannabinoid receptors, endogenous cannabinoids (endocannabinoids), and the enzymes that synthesize and degrade endocannabinoids. The primary focus of this review is the CB2 receptor. CB2 receptors have been the subject of considerable attention, primarily due to their promising therapeutic potential for treating various pathologies while avoiding the adverse psychotropic effects that can accompany CB1 receptor-based therapies. With the appreciation that CB2-selective ligands show marked functional selectivity, there is a renewed opportunity to explore this promising area of research from both a mechanistic as well as a therapeutic perspective. In this review, we summarize our present knowledge of CB2 receptor signaling, localization, and regulation. We discuss the availability of genetic tools (and their limitations) to study CB2 receptors and also provide an update on preclinical data on CB2 agonists in pain models. Finally, we suggest possible reasons for the failure of CB2 ligands in clinical pain trials and offer possible ways to move the field forward in a way that can help reconcile the inconsistencies between preclinical and clinical data.

Benzophenanthridine alkaloid, piperonyl butoxide and (S)-methoprene action at the cannabinoid-1 receptor (CB1-receptor) pathway of mouse brain: Interference with [(3)H]CP55940 and [(3)H]SR141716A binding and modification of WIN55212-2-dependent inhibition of synaptosomal l-glutamate release.

Benzophenanthridine alkaloids (chelerythrine and sanguinarine) inhibited binding of [(3)H]SR141716A to mouse brain membranes (IC50s: <1µM). Piperonyl butoxide and (S)-methoprene were less potent (IC50s: 21 and 63µM respectively). Benzophenanthridines and piperonyl butoxide were more selective towards brain CB1 receptors versus spleen CB2 receptors. All compounds reduced Bmax of [(3)H]SR141716A binding to CB1 receptors, but only methoprene and piperonyl butoxide increased Kd (3-5-fold). Benzophenanthridines increased the Kd of [(3)H]CP55940 binding (6-fold), but did not alter Bmax. (S)-methoprene increased the Kd of [(3)H]CP55940 binding (by almost 4-fold) and reduced Bmax by 60%. Piperonyl butoxide lowered the Bmax of [(3)H]CP55940 binding by 50%, but did not influence Kd. All compounds reduced [(3)H]SR141716A and [(3)H]CP55940 association with CB1 receptors. Combined with a saturating concentration of SR141716A, only piperonyl butoxide and (S)-methoprene increased dissociation of [(3)H]SR141716A above that of SR141716A alone. Only piperonyl butoxide increased dissociation of [(3)H]CP55940 to a level greater than CP55940 alone. Binding results indicate predominantly allosteric components to the study compounds action. 4-Aminopyridine-(4-AP-) evoked release of l-glutamate from synaptosomes was partially inhibited by WIN55212-2, an effect completely neutralized by AM251, (S)-methoprene and piperonyl butoxide. With WIN55212-2 present, benzophenanthridines enhanced 4-AP-evoked l-glutamate release above 4-AP alone. Modulatory patterns of l-glutamate release (with WIN-55212-2 present) align with previous antagonist/inverse agonist profiling based on [(35)S]GTPγS binding. Although these compounds exhibit lower potencies compared to many classical CB1 receptor inhibitors, they may have potential to modify CB1-receptor-dependent behavioral/physiological outcomes in the whole animal.

The G protein-coupled cannabinoid-1 (CB1) receptor of mammalian brain: inhibition by phthalate esters in vitro.

This research examines the in vitro interaction of phthalate diesters and monoesters with the G protein-coupled cannabinoid 1 (CB(1)) receptor, a presynaptic complex involved in the regulation of synaptic activity in mammalian brain. The diesters, n-butylbenzylphthalate (nBBP), di-n-hexylphthalate (DnHP), di-n-butylphthalate (DnBP), di-2-ethylhexylphthalate (DEHP), di-isooctylphthalate (DiOP) and di-n-octylphthalate (DnOP) inhibited the specific binding of the CB(1) receptor agonist [(3)H]CP-55940 to mouse whole brain membranes at micromolar concentrations (IC(50)s: nBBP 27.4 µM; DnHP 33.9 µM; DnBP 45.9 µM; DEHP 47.4 µM; DiOP 55.4 µM; DnOP 75.2 µM). DnHP, DnBP and nBBP achieved full (or close to full) blockade of [(3)H]CP-55940 binding, whereas DEHP, DiOP and DnOP produced partial (55-70%) inhibition. Binding experiments with phenylmethane-sulfonylfluoride (PMSF) indicated that the ester linkages of nBBP and DnBP remain intact during assay. The monoesters mono-2-ethylhexylphthalate (M2EHP) and mono-isohexylphthalate (MiHP) failed to reach IC(50) at 150 µM and mono-n-butylphthalate (MnBP) was inactive. Inhibitory potencies in the [(3)H]CP-55940 binding assay were positively correlated with inhibition of CB(1) receptor agonist-stimulated binding of [(35)S]GTPγS to the G protein, demonstrating that phthalates cause functional impairment of this complex. DnBP, nBBP and DEHP also inhibited binding of [(3)H]SR141716A, whereas inhibition with MiHP was comparatively weak and MnBP had no effect. Equilibrium binding experiments with [(3)H]SR141716A showed that phthalates reduce the B(max) of radioligand without changing its K(d). DnBP and nBBP also rapidly enhanced the dissociation of [(3)H]SR141716A. Our data are consistent with an allosteric mechanism for inhibition, with phthalates acting as relatively low affinity antagonists of CB(1) receptors and cannabinoid agonist-dependent activation of the G-protein. Further studies are warranted, since some phthalate esters may have potential to modify CB(1) receptor-dependent behavioral and physiological outcomes in the whole animal.

The actions of benzophenanthridine alkaloids, piperonyl butoxide and (S)-methoprene at the G-protein coupled cannabinoid CB1 receptor in vitro.

This investigation focused primarily on the interaction of two benzophenanthridine alkaloids (chelerythrine and sanguinarine), piperonyl butoxide and (S)-methoprene with G-protein-coupled cannabinoid CB(1) receptors of mouse brain in vitro. Chelerythrine and sanguinarine inhibited the binding of the CB(1) receptor agonist [(3)H]CP-55940 to mouse whole brain membranes at low micromolar concentrations (IC(50)s: chelerythrine 2.20 µM; sanguinarine 1.10 µM). The structurally related isoquinoline alkaloids (berberine and papaverine) and the phthalide isoquinoline ((-)-ß-hydrastine) were either inactive or considerably below IC(50) at 30 µM. Chelerythrine and sanguinarine antagonized CP-55940-stimulated binding of [(35)S] GTPγS to the G-protein (IC(50)s: chelerythrine 2.09 µM; sanguinarine 1.22 µM). In contrast to AM251, both compounds strongly inhibited basal binding of [(35)S]GTPγS (IC(50)s: chelerythrine 10.06 µM; sanguinarine 5.19µM). Piperonyl butoxide and S-methoprene inhibited the binding of [(3)H]CP-55940 (IC(50)s: piperonyl butoxide 8.2 µM; methoprene 16.4 µM), and also inhibited agonist-stimulated (but not basal) binding of [(35)S]GTPγS to brain membranes (IC(50)s: piperonyl butoxide 22.5 µM; (S)-methoprene 19.31 µM). PMSF did not modify the inhibitory effect of (S)-methoprene on [(3)H]CP-55940 binding. Our data suggest that chelerythrine and sanguinarine are efficacious antagonists of G-protein-coupled CB(1) receptors. They exhibit lower potencies compared to many conventional CB(1) receptor blockers but act differently to AM251. Reverse modulation of CB(1) receptor agonist binding resulting from benzophenanthridines engaging with the G-protein component may explain this difference. Piperonyl butoxide and (S)-methoprene are efficacious, low potency, neutral antagonists of CB(1) receptors. Certain of the study compounds may represent useful starting structures for development of novel/more potent G-protein-coupled CB(1) receptor blocking drugs.