CB1 Activation Research Articles

The cannabinoid agonist WIN-2 affects acquisition but not consolidation of a spatial information in training and retraining processes: Relation with transcriptional regulation of the endocannabinoid system?

The endocannabinoid system is capable of modulating multiple physiological brain functions including learning and memory. Moreover, there is evidence that the processes of acquisition and consolidation have distinct biological basis. We used the cannabinoid agonist WIN 55,212-2 (WIN-2) to investigate whether chronic CB1 activation affects acquisition and consolidation differently by evaluating gene expression in the hippocampus (HIP) and prefrontal cortex (PFC). Swiss mice were treated with WIN-2 (2 mg/kg) and submitted to the Morris water maze to evaluate different aspects of memory. We observed short-term memory impairment in acquisition of the spatial task while consolidation remained unchanged. In the PFC, animals that received WIN-2 prior to the task exhibited increased expression of the 2-AG synthesis enzyme diacylglycerol lipase and decreased levels of the degradation enzyme monoacylglycerol lipase, while mice that were treated after the task for the evaluation of consolidation exhibited the opposite profile. With respect to genes related to AEA metabolism, no correlation between the molecular and behavioral data could be established. In this sense, the cognitive impairment in the acquisition promoted by WIN-2 treatment may be related to a possible increase in the concentration of 2-AG in the PFC. Overall, this study confirms the relevance of the endocannabinoid system in the modulation of cognitive processes. A better understanding of the mechanisms underlying endocannabinoids roles in cognition could provide guidance for the development of treatments to reduce the cognitive deficits caused by drug abuse.

Double blinded, vehicle controlled, crossover study on the efficacy of a topical endocannabinoid membrane transporter inhibitor in atopic Beagles.

The endocannabinoid system is important for skin homeostasis and alterations are linked to inflammatory diseases like atopic dermatitis (AD). Importantly, activation of cannabinoid receptor CB2 decreases pruritus and inflammation in mouse models. Reduction of inactivation of endogenous cannabinoids could, therefore, be a therapeutic option for AD. Dogs spontaneously develop AD, which closely mimics the human disease making them suitable to test new therapies. Our study aimed to test the effects of a topical endocannabinoid membrane transporter inhibitor (WOL067-531, 1% gel) on pruritus and dermatitis in a canine model of AD. Nineteen Beagles allergic to dust mites (DM) were randomized to receive either active ingredient or vehicle on inguinal area while challenged epicutaneously with DM twice weekly for 28days. Treatment was administered twice daily and started after three challenges (day 8). Dermatitis and pruritus were scored weekly by personnel blinded to treatment allocation. Dermatitis was scored using a validated scoring system and pruritus was scored using camera recordings. After a 4-week washout, dogs were crossed over and the study was repeated. On days 15 and 22, dermatitis scores were significantly increased after DM challenge in the vehicle group (16.34, p = 0.0089 and 7.42, p = 0.04845, respectively) but not in the active ingredient group (p = 0.3177 and p = 0.3190, respectively). Significant decrease on pruritus both on inguinal area and overall (p = 0.048 and p = 0.032, respectively) occurred in the active ingredient group. No adverse effects were noted. In conclusion, the newly developed topical endocannabinoid membrane transporter inhibitor (WOL067-531) minimized allergic flares and pruritus in a canine model of AD.

Abstract 497: Studying Cardiovascular Effects of Marijuana on Healthy Individuals Using Human Derived Induced Pluripotent Stem Cells

Marijuana is the most widely used illicit drug worldwide. Epidemiological studies indicate that marijuana increases the risk of coronary artery disease (CAD). Adverse cerebrovascular and peripheral vascular effects are also associated with marijuana use. In addition, three synthetic cannabis drugs have been approved by the FDA for treating chemotherapy-induced nausea and vomiting, which also show cardiovascular side effects. Thus, both medical and recreational marijuana have adverse cardiovascular side effects. Cannabinoid CB1 receptor signaling is involved in a variety of pathophysiological processes and selective CB1 antagonists show therapeutic potential. However, the current repertoire of CB1 antagonists has psychiatric side effects and limited application. Therefore, developing new CB1 antagonists are an unmet and growing clinical need with marijuana use on the rise. Here we found compound JW-1, an isoflavone abundantly presenting in soybeans, partially docked into the CB1 receptor and inhibited CB1 activity, suggesting that compound JW-1 was a novel CB1 antagonist. Human endothelial cells were more sensitive to Δ 9 -tetrahydrocannabinol (Δ 9 -THC) than cardiomyocytes and cardiac fibroblasts. To determine the mechanism of Δ 9 -THC pathological effects on the vasculature, we generated human induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs) from 5 healthy individuals. CB1 receptor was expressed in all hiPSC-ECs, whilst CB2 expression was low. Δ 9 -THC induced inflammation and oxidative stress via NF-κB signaling activated in hiPSC-ECs. Knockdown of CB1 receptor with siRNA, abrogation of receptor expression with CRISPRi and compound JW-1 treatment could rescue the effect of Δ 9 -THC. Furthermore, compound JW-1 blocked Δ 9 -THC-induced endothelial dysfunction in mice models. Our investigations reveal that Δ 9 -THC causes endothelial dysfunction via the CB1 receptor. Compound JW-1 is a novel CB1 antagonist that can be used for preventing Δ 9 -THC-induced side effects.

Evaluation of protective effects of non-selective cannabinoid receptor agonist WIN 55,212-2 against the nitroglycerine-induced acute and chronic animal models of migraine: A mechanistic study

AimMigraine is a neurological debilitating disorder. Previous studies have shown that cannabinoid receptor agonists have analgesic effects in various models of pain. In this study, therefore, we investigated anti-nociceptive effects of WIN 55,212-2, and the role of either CB1 or CB2 receptors in nitroglycerine (NTG)-induced animal model of migraine. MethodsThe present study was conducted on both male and female rats receiving NTG (10 mg/kg, i.p.) to induce acute (single dose of NTG) and chronic (repetitive doses of NTG) models of migraine. Additionally, three groups received WIN 55,212-2 (0.33, 1, 3 mg/kg, i.p.) 45 min before behavioral tests. Additionally, AM251 and AM630 (CB1 and CB2 receptor antagonist, respectively, 1 mg/kg, i.p.) were used to evaluate the possible involvement of CB1 and CB2 receptors during the protective effects of WIN 55,212-2. Key findingsWe found that NTG (10 mg/kg, i.p.) in both acute and chronic models increased sensitivity to pain. In acute model, we found that WIN 55,212-2 (almost high doses) decreases the level of pain mainly through CB1 receptor due to CB1 antagonist abrogates its protective effects, however, in formalin test CB2 receptors also had crucial roles in both phases at 3 mg/kg of WIN 55,212-2. In chronic model, WIN 55,212-2 (0.33, 1 and 3 mg/kg) significantly attenuated NTG-induced hyperalgesia through both CB1 and CB2 receptors. SignificanceOur data supported the argument that activation of CB1 and CB2 receptors by WIN 55,212-2 may be considered a new medication for migraine, however in lack of each receptor leads to different responses from deletion to the reduction of analgesic effects.

Cannabinoid system involves in the analgesic effect of protocatechuic acid.

Protocatechuic acid is an antioxidant which is shown to have analgesic activity in limited studies. However, the mechanisms of action remain unclear. It is aimed to investigate the possible contribution of cannabinoid system that supresses the nociceptive process by the activation of CB1 and CB2 receptors in central and peripheral levels of pain pathways, to the analgesic activity of protocatechuic acid. The analgesic activity of protocatechuic acid was determined at the doses of 75, 150 and 300mg/kg (i.p.) by acetic acid-induced writhing and tail-immersion tests in mice. The results were compared to the analgesic effect of 300mg/kg (i.p.) dipyrone and non-specific CB receptor agonist 5mg/kg (i.p.) WIN 55,212-2. For investigating the contribution of cannabinoid system to protocatechuic acid analgesia; pre-treatment with 8mg/kg (i.p.) CB1 antagonist AM251 and 8mg/kg (i.p.) CB2 antagonist AM630 were performed separately before 300mg/kg protocatechuic acid administration. It was determined that protocatechuic acid has dose-dependent analgesic effect independently from locomotor activity and is comparable with effects of dipyrone and WIN 55,212-2. Pre-treatment with CB1 receptor antagonist AM251 significantly antagonized the protocatechuic acid-induced analgesia in the tail-immersion and writhing tests, whereas pre-treatment of CB2 receptor antagonist AM630 was found to be effective only in the tail-immersion test. It is concluded that cannabinoid modulation contributes to the analgesic effect of protocatechuic acid in spinal level rather than peripheral. CB1 receptor stimulation rather than CB2 receptor stimulation mediates the analgesic effect of protocatechuic acid in both levels, especially peripheral. Graphical abstract Protocatechuic acid inhibits pain response via cannabinoidergic system.

Endocannabinoid System in Hepatic Glucose Metabolism, Fatty Liver Disease, and Cirrhosis.

There is growing evidence that glucose metabolism in the liver is in part under the control of the endocannabinoid system (ECS) which is also supported by its presence in this organ. The ECS consists of its cannabinoid receptors (CBRs) and enzymes that are responsible for endocannabinoid production and metabolism. ECS is known to be differentially influenced by the hepatic glucose metabolism and insulin resistance, e.g., cannabinoid receptor type 1(CB1) antagonist can improve the glucose tolerance and insulin resistance. Interestingly, our own study shows that expression patterns of CBRs are influenced by the light/dark cycle, which is of significant physiological and clinical interest. The ECS system is highly upregulated during chronic liver disease and a growing number of studies suggest a mechanistic and therapeutic impact of ECS on the development of liver fibrosis, especially putting its receptors into focus. An opposing effect of the CBRs was exerted via the CB1 or CB2 receptor stimulation. An activation of CB1 promoted fibrogenesis, while CB2 activation improved antifibrogenic responses. However, underlying mechanisms are not yet clear. In the context of liver diseases, the ECS is considered as a possible mediator, which seems to be involved in the synthesis of fibrotic tissue, increase of intrahepatic vascular resistance and subsequently development of portal hypertension. Portal hypertension is the main event that leads to complications of the disease. The main complication is the development of variceal bleeding and ascites, which have prognostic relevance for the patients. The present review summarizes the current understanding and impact of the ECS on glucose metabolism in the liver, in association with the development of liver cirrhosis and hemodynamics in cirrhosis and its complication, to give perspectives for development of new therapeutic strategies.

Ultrastructural localization of cannabinoid CB1 and mGluR5 receptors in the prefrontal cortex and amygdala.

Stimulation of the postsynaptic metabotropic glutamate receptor mGluR5 triggers retrograde signaling of endocannabinoids that activate presynaptic cannabinoid CB1 receptors on juxtaposing axon terminals. To better understand the synaptic structure that supports mGluR5 mediation of CB1 activation in the prefrontal cortex (PFC) and basolateral amygdala (BLA), we examined electron microscopic dual immunolabeling of these receptors in the prelimbic PFC (prPFC) and BLA of adult male rats. CB1 immunoreactivity was detected in axon terminals that were typically large, complex, and contained dense-core and clear synaptic vesicles. Of terminals forming discernible synaptic specializations, 95% were symmetric inhibitory-type in the prPFC and 90% were inhibitory in the BLA. CB1-immunoreactive terminals frequently contacted dendrites containing mGluR5 adjacent to unlabeled terminals forming excitatory-type synapses. Because most CB1-containing terminals form inhibitory-type synapses, the unlabeled axon terminals forming asymmetric synapses are the likely source of the mGluR5 ligand glutamate. In the prPFC, serial section analysis revealed that GABAergic CB1-containing axon terminals targeted dendrites adjacent to glutamatergic axon terminals, often near dendritic bifurcations. These observations provide ultrastructural evidence that cortical CB1 receptors are strategically positioned for integration of synaptic signaling in response to stimulation of postsynaptic mGluR5 receptors and facilitation of heterosynaptic communication between multiple neurons.

Arachidonoyl‐2‐chloroethylamide alleviates inflammatory pain in a sex‐dependent manner using the formalin test

Previous studies have shown that certain cannabinoids have analgesic effects. Specifically, activation of cannabinoid receptor (CB) 1 has been demonstrated to produce analgesia in multiple animal models. Arachidonyl‐2‐chloroethylamide (ACEA) is a synthetic agonist for CB1 receptors. The activation of CB1 with ACEA could enable chronic inflammatory pain patients to manage this discomfort. ACEA has not been extensively studied in animal models; therefore, not much is known about the dose effectiveness, development of tolerance, or sex‐specific effects.To study the analgesic effects of ACEA, we treated male and female C57BL6J mice prior to the formalin pain test. Administration of formalin (2.5% intraplantar) produces a biphasic pain response, modeling both acute and inflammatory pain. We believe that administration of ACEA at a dose of 0.5 mg per kg intraperitoneal will reduce inflammatory pain in this model. Also, we suggest that daily pretreatment with ACEA prior to the formalin test will cause desensitization to this compound; therefore, the initial analgesic effect will not be seen. To examine sex‐specific effects, we also tested and compared male and female mice. Moreover, vaginal lavage was performed daily on female mice to track the estrous cycle.Preliminary data suggests that females experience higher pain sensitivity compared with males when ACEA 0.5 mg per kg was used. Furthermore, our preliminary data shows that chronic administration in females results in estrous cycle changes. We concluded that there is a sex‐dependent difference in analgesic response to ACEA, and that it may be hormonally‐mediated.Support or Funding InformationFunded by NIDA DA044999‐01A1 (JG) and Texas Tech University Health Sciences Center School of Medicine #121035 (JG).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Antidotes for Acute Cannabinoid Intoxication

The cannabis constituent Δ9‐tetrahydrocannabinol (THC), now being legalized for recreational and medical use, exerts its psychotropic effects (marijuana “high”) via CB1 receptor activation. Synthetic psychoactive cannabinoids (SPCs) such as JWH‐018, illegally sold as “designer drugs”, mimic the effects of THC, a CB1 partial agonist, with higher potency, acting via CB1 receptors. Although acute toxicity is benign for consuming high doses of THC, patients admitted to the ER may exhibit symptoms of hyperemesis, irritation of conjunctival and bronchial tissue, along with impaired cognition and motor skills. SPCs act as full CB1R agonists wherein clinical manifestations associated with their consumption include, neurotoxicity represented by several negative behavioral outcomes, and even death. While such adverse events may differ among patients, current adjunctive treatments include intubation, use of benzodiazepines, antipsychotics, antihistamines, adenosine, and hypnotics, along with i.v. saline, and supportive care. Currently, there are no effective treatments available for directly blocking and reversing the effects of cannabinoid poisoning. To address this critical public‐health issue, we have initiated a drug discovery program to develop antidotes that can counteract the intoxicating/adverse effects of CB1 activation and be therapeutically useful in an emergency setting. Although CB1 antagonists such as rimonabant appear to be promising pharmacotherapeutics, this class of drugs are known to produce nausea and unacceptable pro‐depressant effects. We have now discovered a novel class of potent, selective and brain penetrant CB1 antagonists, displaying better safety profiles as compared to rimonabant. In male CD‐1 mice, these novel CB1 antagonists represented by AM11503 (10 mg/kg, i.v), antagonized the hypothermic effect induced by THC (30 mg/kg, i.p.) and was equipotent to rimonabant (10 mg/kg, i.v.) in rapidly normalizing (≤ 60 min, post‐antagonist) the hypothermia induced by an acute dose of JWH‐018 (6 mg/kg, i.p). To mimic an ER‐like situation, antagonists were given i.v., 60 min post‐ JWH‐018, where the agonist effect is at its maximum. AM11503 (10 mg/kg, i.v or 18 mg/kg, i.p) displayed no side‐effects when compared to rimonabant and AM251, which, at similar doses, induced significant stereotypic behavior (pruritic responses). Remarkably, when tested for their ability to block the neurotoxic effects of SPCs, AM11503 (10 mg/kg, i.v.) fully blocked tremors and convulsions induced by “suprapharmacological” doses of JWH‐018 (18 mg/kg, i.p). Lead optimization efforts along with safety and efficacy studies are further underway. Here, we established proof‐of‐concept and present preliminary data towards developing the first specific antidote for cannabinoid intoxication.Support or Funding InformationSupported by NIDA/NIH grant DA045882 (to KV)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dual mechanism of TRKB activation by anandamide through CB1 and TRPV1 receptors.

BackgroundAdministration of anandamide (AEA) or 2-arachidonoylglycerol (2AG) induces CB1 coupling and activation of TRKB receptors, regulating the neuronal migration and maturation in the developing cortex. However, at higher concentrations AEA also engages vanilloid receptor TRPV1, usually with opposed consequences on behavior.Methods and ResultsUsing primary cell cultures from the cortex of rat embryos (E18) we determined the effects of AEA on phosphorylated TRKB (pTRK). We observed that AEA (at 100 and 200 nM) induced a significant increase in pTRK levels. Such effect of AEA at 100 nM was blocked by pretreatment with the CB1 antagonist AM251 (200 nM) and, at the higher concentration of 200 nM by the TRPV1 antagonist capsazepine (200 nM), but mildly attenuated by AM251. Interestingly, the effect of AEA or capsaicin (a TRPV1 agonist, also at 200 nM) on pTRK was blocked by TRKB.Fc (a soluble form of TRKB able to bind BDNF) or capsazepine, suggesting a mechanism dependent on BDNF release. Using the marble-burying test (MBT) in mice, we observed that the local administration of ACEA (a CB1 agonist) into the prelimbic region of prefrontal cortex (PL-PFC) was sufficient to reduce the burying behavior, while capsaicin or BDNF exerted the opposite effect, increasing the number of buried marbles. In addition, both ACEA and capsaicin effects were blocked by previous administration of k252a (an antagonist of TRK receptors) into PL-PFC. The effect of systemically injected CB1 agonist WIN55,212-2 was blocked by previous administration of k252a. We also observed a partial colocalization of CB1/TRPV1/TRKB in the PL-PFC, and the localization of TRPV1 in CaMK2+ cells.ConclusionTaken together, our data indicate that anandamide engages a coordinated activation of TRKB, via CB1 and TRPV1. Thus, acting upon CB1 and TRPV1, AEA could regulate the TRKB-dependent plasticity in both pre- and postsynaptic compartments.

Maternal high-fat diet impairs leptin signaling and up-regulates type-1 cannabinoid receptor with sex-specific epigenetic changes in the hypothalamus of newborn rats

Maternal nutritional imbalances trigger developmental adaptations involving early epigenetic mechanisms associated with adult chronic disease. Maternal high-fat (HF) diet promotes obesity and hypothalamic leptin resistance in male rat offspring at weaning and adulthood. Leptin resistance is associated with over activation of the endocannabinoid system (ECS). The ECS mainly consists of endocannabinoids derived from n-6 fatty acids and cannabinoid receptors (CB1 coded by Cnr1 and CB2 coded by Cnr2). The CB1 activation in hypothalamus stimulates feeding and appetite for fat while CB2 activation seems to play an immunomodulatory role. We demonstrated that maternal HF diet increases hypothalamic CB1 in male offspring while increases CB2 in female offspring at birth, prior to obesity development. However, the molecular mechanisms behind these changes remain unexplored. We hypothesized that maternal HF diet would down-regulate leptin signaling and up-regulate Cnr1 mRNA levels in the hypothalamus of the offspring at birth, associated with sex-specific changes in epigenetic markers and sex steroid signaling. To test our hypothesis, we used progenitor female rats that received control diet (C, 9% fat) or isocaloric high-fat diet (HF, 28% fat) from 8 weeks before mating until delivery. Blood, hypothalamus and carcass from C and HF male and female offspring were collected for biochemical and molecular analyses at birth. Maternal HF diet down-regulated the transcriptional factor STAT3 in the hypothalamus of male and female offspring, but induced hypoleptinemia only in males and decreased phosphorylated STAT3 only in female offspring. Because leptin acts through STAT3 pathway to inhibit central ECS, our results suggest that leptin pathway impairment might contribute to increased levels of Crn1 mRNA in hypothalamus of both sex offspring. Besides, maternal HF diet increased the histone acetylation percentage of Cnr1 promoter in male offspring and increased the androgen receptor binding to the Cnr1 promoter, which can contribute to higher expression of Cnr1 in newborn HF offspring. Maternal HF diet increased plasma n6 to n3 fatty acid ratio in male offspring, which is an important risk factor to metabolic diseases and might indicate an over activation of endocannabinoid signaling. Thus, although maternal HF diet programs a similar phenotype in adult offspring of both sexes (obesity, hyperphagia and higher preference for fat), here we showed that molecular mechanisms involving leptin signaling, ECS, epigenetic markers and sex hormone signaling were modified prior to obesity development and can differ between newborn male and female offspring. These observations may provide molecular insights into sex-specific targets for anti-obesity therapies.

Δ9-Tetrahydrocannabinol elicited 22-kHz ultrasonic vocalization changes after air puff stimulus through CB1 receptor in adult rats

Δ9-Tetrahydrocannabinol (THC) is known to have various pharmacological effects mediated through activation of cannabinoid CB1 and CB2 receptors in rodents. In adult rats, 22- and 50-kHz ultrasonic vocalizations (USVs) serve as an effective communication system and as indicators of negative and positive states, respectively. The present study was performed to determine whether THC affects USVs in adult rats, and to determine the roles of cannabinoid receptors in these effects. THC (1, 3 mg/kg) was administered intraperitoneally to adult male Wistar rats 60 min before measurement of USVs. The CB1 antagonist, SR141716 (3, 6 mg/kg), or CB2 antagonist, AM630 (1, 10 mg/kg), was administered intraperitoneally 10 min before THC. USVs were measured during a 5-minute period without air puff stimulus or with air puff stimulus. THC did not affect 22- or 50-kHz USVs without air puff stimulus. On the other hand, THC significantly increased the number of 22-kHz USVs, but not 50-kHz USVs, after air puff stimulus. Moreover, SR141716 at 6 mg/kg, but not AM630 at either dose, inhibited the increase in number of 22-kHz USVs induced by THC after air puff stimulus. These results suggest that THC induced changes in sensitivity to aversive air puff stimuli through CB1 receptors, and as a result increased emission of 22-kHz USVs in rats.

Hypothalamic endocannabinoid signalling modulates aversive responses related to panic attacks

Recurrent panic attacks, comprising emotional and cardiovascular aversive responses, are common features in panic disorder, a subtype of anxiety disorder. The underlying brain circuitry includes nuclei of the hypothalamus, such as the dorsomedial hypothalamus (DMH). The endocannabinoid system has been proposed to modulate several biological processes in the hypothalamus. Thus, we tested the hypothesis that hypothalamic endocannabinoid signalling controls aversive responses in an animal model of panic attacks. Local infusion of NMDA into the DMH of rats induced panic-like behaviour. This effect was prevented by local, but not intraperitoneal, injection of a 2-arachidonoylglycerol (2-AG) hydrolysis inhibitor (MAGL inhibitor, URB602). The anandamide hydrolysis inhibitor (FAAH inhibitor), URB597, was ineffective. The anti-aversive action of URB602 was reversed by CB1 and CB2 antagonists (AM251 and AM630, respectively), and mimicked by CB1 and CB2 agonists (ACEA and JWH133, respectively). URB602 also prevented the cardiovascular effects of DMH-stimulation in anaesthetised animals. None of the treatments modified blood corticosterone levels. In conclusion, facilitation of 2-AG-signalling in the DMH modulates panic-like responses. The possible mechanisms comprise activation of both CB1 and CB2 receptors in this brain region.

Δ9-Tetrahydrocannabinol and Cannabidiol Differentially Regulate Intraocular Pressure.

PurposeIt has been known for nearly 50 years that cannabis and the psychoactive constituent Δ9-tetrahydrocannabinol (THC) reduce intraocular pressure (IOP). Elevated IOP remains the chief hallmark and therapeutic target for glaucoma, a major cause of blindness. THC likely acts via one of the known cannabinoid-related receptors (CB1, CB2, GPR18, GPR119, GPR55) but this has never been determined explicitly. Cannabidiol (CBD) is a second major constituent of cannabis that has been found to be without effect on IOP in most studies.MethodsEffects of topically applied THC and CBD were tested in living mice by using tonometry and measurements of mRNA levels. In addition the lipidomic consequences of CBD treatment were tested by using lipid analysis.ResultsWe now report that a single topical application of THC lowered IOP substantially (∼28%) for 8 hours in male mice. This effect is due to combined activation of CB1 and GPR18 receptors each of which has been shown to lower ocular pressure when activated. We also found that the effect was sex-dependent, being stronger in male mice, and that mRNA levels of CB1 and GPR18 were higher in males. Far from inactive, CBD was found to have two opposing effects on ocular pressure, one of which involved antagonism of tonic signaling. CBD prevents THC from lowering ocular pressure.ConclusionsWe conclude that THC lowers IOP by activating two receptors—CB1 and GPR18—but in a sex-dependent manner. CBD, contrary to expectation, has two opposing effects on IOP and can interfere with the effects of THC.

Mice Lacking Both Cannabinoid-1 and Cannabinoid-2 Receptors Are Resistant to Diet-Induced Obesity

Exogenous cannabinoids are a group of related compounds derived from the Cannabis plant, commonly known as marijuana. The discovery of cannabinoid receptors (CB1R and CB2R) has provided a platform for investigating the health effects of cannabinoids. The CB1R is predominantly expressed in the central nervous system, whereas CB2R is primarily expressed in the immune system. Mounting evidence has shown that activation of CB1 is associated with increased food intake and obesity, whereas activation of CB2 is associated with decreased levels of cytokines associated with insulin resistance and type 2 diabetes (T2D). Epidemiological studies, however, have shown a decreased prevalence of obesity and T2D among cannabis users. Here we use high-fat diet (HFD, 45% of calories from fat vs. control LFD with 10% of calories from fat) to study the metabolic changes in male mice lacking receptors of the cannabinoid system (CB-DKO) when compared to wild type (WT) C57BL/6 mice. Eight-weeks old CB-DKO mice displayed lower body fat percent and higher percent of lean mass tissue when compared to WT mice. After 12 weeks, WT mice gained an average of 22 grams on HFD compared to 9 grams on LFD. CB-DKO mice did not respond to HFD, gaining an average of 5 grams on both HFD and LFD. This corresponded to a 5% increase in body fat in CB-DKO mice compared to 18% increase in WT mice. Glucose tolerance test indicated increased insulin sensitivity in CB-DKO mice. Automated measurements revealed no significant differences in food intake or locomotion between CB-DKO and WT mice, whereas CB-DKO mice displayed higher indirect calorimetry parameters including VO2, respiratory exchange ratio, and energy expenditure. Gene expression analysis confirmed an upregulation of thermogenic genes (UCP1, CIDEA, and COX8B) suggesting browning of white adipose tissue in CB-DKO mice. These findings highlight the role of the cannabinoid system in metabolic control of adipose tissues and their potential role for insulin resistance and T2D risk. Disclosure O. Alshaarawy: None. E. Kurjan: None. N. Truong: None. L.K. Olson: None.

Assessment of Cannabinoids Agonist and Antagonist in Invasion Potential of K562 Cancer Cells

Background:The prominent hallmark of malignancies is the metastatic spread of cancer cells. Recent studies have reported that the nature of invasive cells could be changed after this phenomenon, causing chemotherapy resistance. It has been demonstrated that the up-regulated expression of matrix metalloproteinase (MMP) 2/MMP-9, as a metastasis biomarker, can fortify the metastatic potential of leukemia. Furthermore, investigations have confirmed the inhibitory effect of cannabinoid and endocannabinoid on the proliferation of cancer cells in vitro and in vivo. Methods:In the present study, the inhibitory effect of WIN 55212-2 (a CB1/CB2 receptor agonist) and AM251 (a selective CB1 receptor antagonist) on K562 cells, as a chronic myelogenous leukemia (CML) model, was evaluated using MTT and invasion assay. Expressions of MMP-2 and MMP-9 were then assessed by Western blot analysis.Results:The data obtained from MTT assay showed that WIN 55212-2 could attenuate cell proliferation; however, AM251 was less effective in this regard. Our results showed that WIN 55212-2 considerably reduced cancer cell invasiveness, while AM251 exhibited a converse effect. Moreover, CB1 activation resulted in decreased expression of MMP-2 and MMP-9. Conclusion:Our findings clarifies that CB1 receptors are responsible for anti-invasive effects in the K562 cell line.

Discovery of Selective Cannabinoid CB2 Receptor Agonists by High-Throughput Screening

The endocannabinoid system (ECS) plays a diverse role in human physiology ranging from the regulation of mood and appetite to immune modulation and the response to pain. Drug development that targets the cannabinoid receptors (CB1 and CB2) has been explored; however, success in the clinic has been limited by the psychoactive side effects associated with modulation of the neuronally expressed CB1 that are enriched in the CNS. CB2, however, are expressed in peripheral tissues, primarily in immune cells, and thus development of CB2-selective drugs holds the potential to modulate pain among other indications without eliciting anxiety and other undesirable side effects associated with CB1 activation. As part of a collaborative effort among industry and academic laboratories, we performed a high-throughput screen designed to discover selective agonists or positive allosteric modulators (PAMs) of CB2. Although no CB2 PAMs were identified, 167 CB2 agonists were discovered here, and further characterization of four select compounds revealed two with high selectivity for CB2 versus CB1. These results broaden drug discovery efforts aimed at the ECS and may lead to the development of novel therapies for immune modulation and pain management with improved side effect profiles.

Cannabinoid Receptor Type 1 Expression in the Developing Avian Retina: Morphological and Functional Correlation With the Dopaminergic System.

The avian retina has been used as a model to study signaling by different neuro- and gliotransmitters. It is unclear how dopaminergic and cannabinoid systems are related in the retina. Here we studied the expression of type 1 and 2 cannabinoid receptors (CB1 and CB2), as well as monoacylglycerol lipase (MAGL), the enzyme that degrades 2-arachidonoylglycerol (2-AG), during retina development. Our data show that CB1 receptor is highly expressed from embryonic day 5 (E5) until post hatched day 7 (PE7), decreasing its levels throughout development. CB1 is densely found in the ganglion cell layer (GCL) and inner plexiform layer (IPL). CB2 receptor was also found from E5 until PE7 with a decrease in its contents from E9 afterwards. CB2 was mainly present in the lamination of the IPL at PE7. MAGL is expressed in all retinal layers, mainly in the IPL and OPL from E9 to PE7 retina. CB1 and CB2 were found both in neurons and glia cells, but MAGL was only expressed in Müller glia. Older retinas (PE7) show CB1 positive cells mainly in the INL and co-expression of CB1 and tyrosine hydroxylase (TH) are shown in a few cells when both systems are mature. CB1 co-localized with TH and was heavily associated to D1 receptor labeling in primary cell cultures. Finally, cyclic AMP (cAMP) was activated by the selective D1 agonist SKF38393, and inhibited when cultures were treated with WIN55, 212–2 (WIN) in a CB1 dependent manner. The results suggest a correlation between the endocannabinoid and dopaminergic systems (DSs) during the avian retina development. Activation of CB1 limits cAMP accumulation via D1 receptor activation and may influence embryological parameters during avian retina differentiation.

Optimization of the metabolic stability of a fluorinated cannabinoid receptor subtype 2 (CB2) ligand designed for PET studies

The central CB2 receptor represents a promising target for the treatment of neuroinflammatory diseases as CB2 activation mediates anti-inflammatory effects. Recently, the F-18 labeled PET radiotracer [18F]7a was reported, which shows high CB2 affinity and high selectivity over the CB1 subtype but low metabolic stability due to hydrolysis of the amide group. Based on these findings twelve bioisosteres of 7a were synthesized containing a non-hydrolysable functional group instead of the amide group. The secondary amine 23a (Ki = 7.9 nM) and the ketone 26a (Ki = 8.6 nM) displayed high CB2 affinity and CB2:CB1 selectivity in in vitro radioligand binding studies. Incubation of 7a, 23a and 26a with mouse liver microsomes and LC-quadrupole-MS analysis revealed a slightly higher metabolic stability of secondary amine 23a, but a remarkably higher stability of ketone 26a in comparison to amide 7a. Furthermore, a logD7.4 value of 5.56 ± 0.08 was determined for ketone 26a by micro shake-flask method and LC-MS quantification.

Molecular Understanding of the Activation of CB1 and Blockade of TRPV1 Receptors: Implications for Novel Treatment Strategies in Osteoarthritis.

Osteoarthritis (OA) is a joint disease in which cartilage degenerates as a result of mechanical and biochemical changes. The main OA symptom is chronic pain involving both peripheral and central mechanisms of nociceptive processing. Our previous studies have implicated the benefits of dual- over single-acting compounds interacting with the endocannabinoid system (ECS) in OA treatment. In the present study, we focused on the specific molecular alterations associated with pharmacological treatment. OA was induced in Wistar rats by intra-articular injection of 3 mg of monoiodoacetate (MIA). Single target compounds (URB597, an FAAH inhibitor, and SB366791, a TRPV1 antagonist) and a dual-acting compound OMDM198 (FAAH inhibitor/TRPV1 antagonist) were used in the present study. At day 21 post-MIA injection, rats were sacrificed 1 h after i.p. treatment, and changes in mRNA expression were evaluated in the lumbar spinal cord by RT-qPCR. Following MIA administration, we observed 2-4-fold increase in mRNA expression of targeted receptors (Cnr1, Cnr2, and Trpv1), endocannabinoid degradation enzymes (Faah, Ptgs2, and Alox12), and TRPV1 sensitizing kinases (Mapk3, Mapk14, Prkcg, and Prkaca). OMDM198 treatment reversed some of the MIA effects on the spinal cord towards intact levels (Alox12, Mapk14, and Prkcg). Apparent regulation of ECS and TRPV1 in response to pharmacological intervention is a strong justification for novel ECS-based multi-target drug treatment in OA.