CB1 Receptor Agonist Research Articles

Etomidate Modulates the Tactile Stimulation-Evoked Field Potential Responses in Cerebellar Granule Cell Layer in vivo in Mice

Etomidate (ET) produces sedation by binding on the γ-aminobutyric acid type A (GABAA) receptors. We previously found that ET inhibited cerebellar Purkinje cells activity via both GABAA and glycine receptors in vivo in mice, suggesting that ET modulated sensory information synaptic transmission in cerebellar cortex. In this study, we investigated the effect of ET on the sensory stimulation-evoked responses in the cerebellar granule layer (GL) in urethane-anesthetized mice, using electrophysiological and pharmacological methods. Our results showed that cerebellar surface perfusion of ET (100 μmol/L) significantly decreased amplitude and area under the curve (AUC) of the sensory stimulation-evoked excitatory component (N1) in the cerebellar GL. Application of GABAA receptor antagonist, SR95531 (20 μmol/L) significantly attenuated, but not abolished the ET-induced decrease in amplitude and AUC of facial stimulation-evoked responses. However, application of a mixture of SR95531 (20 μmol/L) and cannabinoid 1 receptor (CB1) antagonist, AM-251 (5 μmol/L), completely blocked the ET-induced decrease in amplitude and AUC of facial stimulation-evoked responses. Furthermore, application of the CB1 receptor agonist, WIN55212-2, induced a decrease in amplitude and AUC of N1 in the absence of GABAA receptors activity, as well occluded the ET-induced depression of N1. Moreover, the ET-induced changes in amplitude and AUC of N1 in absence of GABAA receptors activity were abolished by a specific protein kinase A (PKA) inhibitor, KT5720. These results indicate that ET facilitates CB1 receptors in the absence of GABAA receptors activity, resulting in a depression of the sensory stimulation-evoked synaptic transmission via PKA signaling pathway in mouse cerebellar GL.

Synthesis and evaluation of various heteroaromatic benzamides as analogues of –ylidene-benzamide cannabinoid type 2 receptor agonists

The CB2 receptor is an attractive target for the treatment of a wide range of diseases and pathological conditions. Compounds that selectively activate the CB2 receptor are desirable as this avoids CB1-mediated psychoactive effects. Heteroarylidene-benzamides have demonstrated efficacy as selective CB2 receptor agonists. We aimed to expand the structure-activity relationship studies of this series of compounds by investigating the heteroaromatic core via the synthesis and in vitro evaluation of a small library of various heteroaromatic benzamide analogues. As heteroaromatic amides are privileged scaffolds in drug design, methods to synthesise them are of interest. Concise and reliable synthetic strategies were developed to access these novel analogues. The –ylidene-benzamide moiety is shown to be essential for CB activity as all amide derivatives exhibit no functional activity at either CB2 or CB1 receptors.

Cannabidiol and the Remainder of the Plant Extract Modulate the Effects of Δ9-Tetrahydrocannabinol on Fear Memory Reconsolidation.

Background: Δ9-Tetrahydrocannabinol (THC, a CB1 receptor agonist) and Cannabidiol (CBD, a non-competitive antagonist of endogenous CB1 and CB2 ligands) are two primary components of Cannabis species, and may modulate fear learning in mammals. The CB1 receptor is widely distributed throughout the cortex and some limbic regions typically associated with fear learning. Humans with posttraumatic disorder (PTSD) have widespread upregulation of CB1 receptor density and reduced availability of endogenous cannabinoid anandamide, suggesting a role for the endocannabinoid system in PTSD. Pharmacological blockade of memory reconsolidation following recall of a conditioned response modulates the expression of learned fear and may represent a viable target for the development of new treatments for PTSD. In this study, we focused on assessing the impact of the key compounds of the marijuana plant both singly and, more importantly, in concert on attenuation of learned fear. Specifically, we assessed the impact of THC, CBD, and/or the remaining plant materials (post-extraction; background material), on reconsolidation of learned fear.Method: Male Sprague-Dawley rats received six 1.0 mA continuous foot shocks (contextual training). Twenty-four hours later, rats were re-exposed to the context. Immediately following memory retrieval (recall) rats received oral administration of low dose THC, high dose THC, CBD, CBD + low THC, CBD + high THC [as isolated phytochemicals and, in separate experiments, in combination with plant background material (BM)]. Rodents were tested for freezing response context re-exposure at 24 h and 7 days following training.Results: CBD alone, but not THC alone, significantly attenuated fear memory reconsolidation when administered immediately after recall. The effect persisted for at least 7 days. A combination of CBD and THC also attenuated the fear response. Plant BM also significantly attenuated reconsolidation of learned fear both on its own and in combination with THC and CBD. Finally, THC attenuated reconsolidation of learned fear only when co-administered with CBD or plant BM.Conclusion: CBD may provide a novel treatment strategy for targeting fear-memories. Furthermore, plant BM also significantly attenuated the fear response. However, whereas THC alone had no significant effects, its effects were modulated by the addition of other compounds. Future research should investigate some of the other components present in the plant BM (such as terpenes) for their effects alone, or in combination with isolated pure cannabinoids, on fear learning.

CB2 Receptor Agonist JWH133 Activates AMPK to Inhibit Growth of C6 Glioma Cells.

It has been reported that endocannabinoid receptor type 2 (CB2) agonist JWH133 inhibits the growth of C6 glioma cells, but the underlying mechanism has not yet been fully elucidated. We showed that JWH133 inhibited C6 cells growth, reduced cAMP production and inhibited PKA activity through CB2 receptor. Decrease of PKA activity stimulated CaMKKβ, and subsequently elevated phosphorylation of AMPKα at threonine 172 site. The activation of AMPKα induced changes of downstream proteins, including increase of P53 phosphorylation and P21 production, as well as decrease of mTOR phosphorylation, that eventually inhibited C6 cells growth.

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.

Strategies to develop selective CB2 receptor agonists from indole carboxamide synthetic cannabinoids

Activation of the CB2 receptor is an attractive therapeutic strategy for the treatment of a wide range of inflammatory diseases. However, receptor subtype selectivity is necessary in order to circumvent the psychoactive effects associated with activation of the CB1 receptor. We aimed to use potent, non-selective synthetic cannabinoids designer drugs to develop selective CB2 receptor agonists. Simple structural modifications such as moving the amide substituent of 3-amidoalkylindole synthetic cannabinoids to the 2-position and bioisosteric replacement of the indole core to the 7-azaindole scaffold are shown to be effective and general strategies to impart receptor subtype selectivity. 2-Amidoalkylindole 16 (EC50 CB1 > 10 μM, EC50 CB2 = 189 nM) and 3-amidoalkyl-7-azaindole 21 (EC50 CB1 > 10 μM, EC50 = 49 nM) were found to be potent and selective agonists with favourable physicochemical properties. Docking studies were used to elucidate the molecular basis for the observed receptor subtype selectivity for these compounds.

Structure-activity relationships of thiazole and benzothiazole derivatives as selective cannabinoid CB2 agonists with in vivo anti-inflammatory properties

The strong therapeutic potential of CB2 receptor agonists for use as anti-inflammatory agents that lack psychiatric side effects has attracted substantial interest. We herein describe the rational design and synthesis of novel thiazole and benzothiazole derivatives and the evaluation of their binding affinity and functional activity on CB1 and CB2 receptors. The series with the general formula N-(3-pentylbenzo [d]thiazol-2(3H)-ylidene) carboxamide (compounds 6a-6d) exhibited the highest affinity and selectivity towards CB2 receptors with Kis in the picomolar or low nanomolar range, and selectivity indices (Ki hCB1/Ki hCB2) reaching up to 429 fold. Notably, these compounds also demonstrated an agonistic functional activity in cellular assays with EC50s in the low nanomolar range. More interestingly, compound 6d, the 3-(trifluoromethyl)benzamide derivative, exhibited remarkable protection against DSS-induced acute colitis in mice model.

Nabilone administration in refractory chronic diarrhea: a case series

BackgroundDaily cannabis assumption is currently associated with several physical and mental health problems, however in the past it was prescribed for a multitude of symptoms, including vomiting, abdominal pain and diarrhea. Through the years, the endocannabinoid system has been recognized in the homeostatic mechanisms of the gut, as well as in the physiological control of intestinal motility and secretion. Accordingly, cannabinoids may be a promising therapy against several gastrointestinal conditions, such as abdominal pain and motility-related disorders.Case presentationWe retrospectively analysed the efficacy and safety of a CB1-receptor agonist administered in six patients with refractory chronic diarrhea, between April 2008 and July 2016. After three months of therapy, oral nabilone improved the health of nearly all patients, with visible improvements in reducing diarrheal symptoms and weight gain. Most of the benefits persisted through the three-month follow-up. Only one patient interrupted the treatment after one month, due to severe fatigue and mental confusion; the symptoms disappeared in the follow-up period.ConclusionsThese findings encourage the study of cannabinoids acting on CB1 receptors in chronic gastrointestinal disorders, especially in refractory chronic diarrhea, offering a chance for a substantial improvement in the quality of life of selected patients, with a reasonable safety profile.

Omega-3 Endocannabinoid-Epoxides Are Novel Anti-inflammatory and Anti-Pain Lipid Metabolites (FS15-01-19)

ObjectivesOmega-3 fatty acid derived endocannabinoids are metabolized by cytochrome P450s to form bioactive endocannabinoid epoxides that are anti-inflammatory MethodsLipidomics, LC-MS/MS, microglial cells culture, lipid synthesis, extractions, enzymology ResultsCannabinoids are found in marijuana and also are produced naturally in the body from ω-3 and ω-6 fatty acids. Exocannabinoids in marijuana, are known to be responsible for some of its euphoric effects, but they also exhibit anti-inflammatory benefits. Our study revealed a cascade of enzymatic reactions that convert ω-3 fatty acids into anti-inflammatory endocannabinoid epoxides that act through the same receptors in the body as marijuana (PNAS 2017). Endocannabinoids are ligands for cannabinoid receptor 1 and 2 (CB1 and CB2). CB1 receptor agonists exhibit psychotropic properties while CB2 receptor agonists have anti-inflammatory effects. Consequently, there is a strong interest in the discovery of CB2 selective agonists to mitigate inflammatory pathologies. The work details the discovery and characterization of naturally occurring ω-3–derived endocannabinoid epoxides that are formed via enzymatic oxidation of ω-3 endocannabinoids by cytochrome P450 epoxygenases. These dual functional ω-3 endocannabinoid epoxides exhibit preference towards binding to CB2 receptor and are anti-inflammatory and vasodilatory and reciprocally modulate platelet aggregation. Some of the other regioisomers of ω-3 endocannabinoid epoxides are partial agonists of CB1 and stop tumor cell metastasis (J. Med. Chem 2018). By virtue of their physiological properties, they are expected to play important roles in neuroinflammation and pain. ConclusionsThis finding demonstrates how omega-3 fatty acids can produce some of the same medicinal qualities as marijuana, but without a psychotropic effect. In summary, the ω-3 endocannabinoid epoxides are found at concentrations comparable to those of other endocannabinoids and are expected to play critical roles during inflammation in vivo Funding SourcesAmerican Heart Association Scientist Development award, National Institute of Health (NIH) R01, NIH R03, USDA, National Multiple Sclerosis Society.

Role of Central Cannabinoidergic System on Ghrelin-Induced Hypophagia in Layer-Type Neonatal Chicken

Background: Feeding behavior is regulated via a complex network which interacts through diverse signals from central and peripheral tissues. OBJECTIVES: The main purpose of the current study was to determine the role of central cannabinoidergic (CBergic) system on ghrelin-induced hypophagia in 3-h food deprived (FD3) neonatal chicken. METHODS: In experiment 1, chicks were ICV injected with control solution, ghrelin (0.6 nmol), SR141716A (selective CB1 receptors antagonist, 6.25 µg) and ghrelin + SR141716A. In experiment 2, chickens received ICV injection of (A) control solution, ghrelin (0.6 nmol), AM630 (selective CB2 receptors antagonist, 1.25 µg) and ghrelin + AM630. In experiment 3, chickens were ICV injected with control solution, 2-AG (selective CB1 receptors agonist, 2µg), GSK1614343 (selective ghrelin receptors antagonist, 6 nmol) and 2-AG + GSK1614343. In experiment 4, the birds received control solution, CB65 (selective CB2 receptors agonist, 6.25 µg), CB65 + GSK1614343. Then the cumulative food intake was measured until 120 min post injection. RESULTS: According to the results, ICV injection of the ghrelin, significantly decreased cumulative food intake (P<0.05). Co-injection of the ghrelin + SR141716A and/or ghrelin + AM630 significantly amplified ghrelin-induced hypophagia compared to control group (P<0.05). Hyperphagia observed by ICV injection of the 2-AG (2 µg) (P<0.05). Co-injection of the 2-AG + GSK1614343 increased food intake compared to control group (P<0.05). ICV injection of the CB65 (1.25 µg) significantly increased food intake (P<0.05). Also, co-injection of the CB65 + GSK1614343 significantly amplified cumulative food intake in FD3 neonatal layer-type chicken (P<0.05). CONCLUSIONS: These results suggested ghrelin-induced hypophagia mediates via CB1 and CB2 receptors in neonatal layer-type chicken

CUMYL-4CN-BINACA Is an Efficacious and Potent Pro-Convulsant Synthetic Cannabinoid Receptor Agonist.

Synthetic cannabinoid receptor agonists (SCRAs) are the largest class of new psychoactive substances (NPS). New examples are detected constantly, and some are associated with a series of adverse effects, including seizures. CUMYL-4CN-BINACA (1-(4-cyanobutyl)-N-(2-phenylpropan-2-yl)indazole-3-carboxamide) is structurally related to potent, cumylamine-derived SCRAs such as 5F-CUMYL-PINACA, but is unusual due to a terminal aliphatic nitrile group not frequently encountered in SCRAs or pharmaceuticals. We report here that CUMYL-4CN-BINACA is a potent CB1 receptor agonist (K i = 2.6 nM; EC50 = 0.58 nM) that produces pro-convulsant effects in mice at a lower dose than reported for any SCRA to date (0.3 mg/kg, i.p). Hypothermic and pro-convulsant effects in mice could be reduced or blocked, respectively, by pretreatment with CB1 receptor antagonist SR141716, pointing to at least partial involvement of CB1 receptors in vivo. Pretreatment with CB2 receptor antagonist AM-630 had no effect on pro-convulsant activity. The pro-convulsant properties and potency of CUMYL-4CN-BINACA may underpin the toxicity associated with this compound in humans.

Activation of cannabinoid type 2 receptor protects skeletal muscle from ischemia-reperfusion injury partly via Nrf2 signaling

AimsCannabinoid type 2 (CB2) receptor activation has been shown to attenuate IRI in various organs. NF-E2-related factor (Nrf2) is an anti-oxidative factor that plays multiple roles in regulating cellular redox homeostasis and modulating cell proliferation and differentiation. The protective effects of CB2 receptor activation on skeletal muscle IRI and the underlying mechanism that involves Nrf2 signaling remain unknown. Main methodsWe evaluated the in vivo effect of CB2 receptor activation by the CB2 receptor agonist AM1241 on IR-induced skeletal muscle damage and early myogenesis. We also assessed the effects of CB2 receptor activation on C2C12 myoblasts differentiation and H2O2-induced C2C12 myoblasts damage in vitro, with a focus on the mechanism of Nrf2 signaling. Key findingsOur results showed that CB2 receptor activation reduced IR-induced histopathological lesions, edema, and oxidative stress 1 day post-injury and accelerated early myogenesis 4 days post-injury in mice. Nrf2 knockout mice that were treated with AM1241 exhibited deteriorative skeletal muscle oxidative damage and myogenesis. In vitro, pretreatment with AM1241 significantly increased the expression of Nrf2 and its nuclear translocation, attenuated the decrease in H2O2-induced C2C12 cell viability, and decreased reactive oxygen species generation and apoptosis. CB2 receptor activation also significantly enhanced C2C12 myoblasts differentiation, which was impaired by silencing Nrf2. SignificanceOverall, CB2 receptor activation protected skeletal muscle against IRI by ameliorating oxidative damage and promoting early skeletal muscle myogenesis, which was partly via Nrf2 signaling.

Model-free and kinetic modelling approaches for characterising non-equilibrium pharmacological pathway activity: Internalisation of cannabinoid CB1 receptors.

Receptor internalisation is by nature kinetic. Application of a standard equilibrium dose response model to describe the properties of a ligand inducing internalisation, while commonly used, are therefore problematic. Here, we propose two quantitative approaches to address this issue-(a) a model-free method and (b) a kinetic modelling approach-and systematically evaluate the performance of these methods against traditional equilibrium methods to characterise the internalisation profiles of cannabinoid CB1 receptor agonists. Kinetic internalisation assays were conducted using a concentration series of six CB1 receptor ligands. Internalisation rate analysis and snapshot equilibrium analysis were performed. A model-free method was developed based on the mean residence time of internalisation. A kinetic internalisation model was developed under the quasi-steady state assumption. Rates of receptor internalisation depended on both agonist and concentration. Agonist potencies from snapshot equilibrium analysis increased with stimulation time, and there was no single time point at which internalisation profiles could infer agonist properties in a comparative manner. The model-free method yielded a time-invariant measure of potency/efficacy for internalisation. The kinetic model adequately described the internalisation of CB1 receptors over time and provided robust estimates of both potency and efficacy. Applying equilibrium analysis to a non-equilibrium pathway cannot provide a reliable estimate of agonist potency. Both the model-free and kinetic modelling approaches characterised the internalisation profiles of CB1 receptor agonists. The kinetic model provides additional advantages as a method to capture changes in receptor number during other functional assays.

Preparation of bivalent agonists for targeting the mu opioid and cannabinoid receptors

In order to obtain novel pharmacological tools and to investigate a multitargeting analgesic strategy, the CB1 and CB2 cannabinoid receptor agonist JWH-018 was conjugated with the opiate analgesic oxycodone or with an enkephalin related tetrapeptide. The opioid and cannabinoid pharmacophores were coupled via spacers of different length and chemical structure. In vitro radioligand binding experiments confirmed that the resulting bivalent compounds bound both to the opioid and to the cannabinoid receptors with moderate to high affinity. The highest affinity bivalent derivatives 11 and 19 exhibited agonist properties in [35S]GTPγS binding assays. These compounds activated MOR and CB (11 mainly CB2, whereas 19 mainly CB1) receptor-mediated signaling, as it was revealed by experiments using receptor specific antagonists. In rats both 11 and 19 exhibited antiallodynic effect similar to the parent drugs in 20 μg dose at spinal level. These results support the strategy of multitargeting G-protein coupled receptors to develop lead compounds with antinociceptive properties.

Major Contribution of Somatostatin-Expressing Interneurons and Cannabinoid Receptors to Increased GABA Synaptic Activity in the Striatum of Huntington's Disease Mice.

Huntington’s disease (HD) is a heritable neurological disorder that affects cognitive and motor performance in patients carrying the mutated huntingtin (HTT) gene. In mouse models of HD, previous reports showed a significant increase in spontaneous GABAA receptor-mediated synaptic activity in striatal spiny projection neurons (SPNs). In this study, using optogenetics and slice electrophysiology, we examined the contribution of γ-aminobutyric acid (GABA)-ergic parvalbumin (PV)- and somatostatin (SOM)-expressing interneurons to the increase in GABA neurotransmission using the Q175 (heterozygote) mouse model of HD. Patch clamp recordings in voltage-clamp mode were performed on SPNs from brain slices of presymptomatic (2 months) and symptomatic (8 and 12 months) Q175 mice and wildtype (WT) littermates. While inhibitory postsynaptic currents (IPSCs) evoked in SPNs following optical activation of PV- and SOM-expressing interneurons differed in amplitude, no genotype-dependent differences were observed at all ages from both interneuron types; however, responses evoked by either type were found to have faster kinetics in symptomatic mice. Since SOM-expressing interneurons are constitutively active in striatal brain slices, we then examined the effects of acutely silencing these neurons in symptomatic mice with enhanced Natronomonas pharaonis halorhodopsin (eNpHR). Optically silencing SOM-expressing interneurons resulted in a greater decrease in the frequency of spontaneous IPSCs (sIPSCs) in a subset of SPNs from Q175 mice compared to WTs, suggesting that SOM-expressing interneurons are the main contributors to the overall increased GABA synaptic activity in HD SPNs. Additionally, the effects of activating GABAB and cannabinoid (CB1) receptors were investigated to determine whether these receptors were involved in modulating interneuron-specific GABA synaptic transmission and if this modulation differed in HD mice. When selectively activating PV- and SOM-expressing interneurons in the presence of the CB1 receptor agonist WIN-55,212, the magnitudes of the evoked IPSCs in SPNs decreased for both interneuron types although this change was less prominent in symptomatic Q175 SPNs during SOM-expressing interneuron activation. Overall, these findings show that dysfunction of SOM-expressing interneurons contributes to the increased GABA synaptic activity found in HD mouse models and that dysregulation of the endocannabinoid system may contribute to this effect.

The involvement of ventral hippocampal microglial cells, but not cannabinoid CB1 receptors, in morphine-induced analgesia in rats.

It is well known that glial cells are involved in pain processing. The purpose of the present study was to investigate the possible involvement of the ventral hippocampal (VH) glial cells in morphine-induced analgesia. A tail-flick apparatus was used to measure pain sensitivity in male Wistar rats that were bilaterally cannulated in the VH by stereotaxic surgery. The results showed that intraperitoneal (i.p.) administration of morphine (2.5-7.5mg/kg) induced analgesia in a time-dependent manner. The blockade of the VH glial cell activation by bilateral microinjection of a glial inhibitor, minocycline (5-15µg/rat) into the VH with an ineffective dose of morphine (2.5mg/kg, i.p) significantly increased morphine analgesia. Considering that the endocannabinoid system via CB1 receptors play a crucial role in pain modulation, we also assessed the possible role of the VH cannabinoid CB1 receptors in the functional interaction between minocycline and morphine in acute pain. Our results indicated that intra-VH injection of the cannabinoid CB1 receptor agonist, arachidonylcyclopropylamide (ACPA; 4-12ng/rat) had no effect on minocycline-induced potentiation of morphine analgesia. It should be considered that intra-VH microinjection of minocycline or ACPA by itself had no effect on tail-flick latency. Our findings suggest that the activation of the VH microglial cells may be involved in mediating pain sensation, because the inhibition of these cells by intra-VH injection of minocycline could potentiate morphine-induced analgesia. Although endocannabinoids have a regulatory role in glia function, the activation of CB1 receptors could not affect the potentiative effect of minocycline on morphine analgesia.

Drug-drug interactions between antiepileptics and cannabinoids

ABSTRACTIntroduction: About 40% of patients with epilepsy are associated with drug-resistant seizures, therefore there has been a continuous search for novel treatment approaches. In experimental studies, natural and synthetic cannabimimetic compounds alone or combined with antiepileptic drugs (AEDs) have been extensively studied and cannabidiol, a naturally occurring compound, has been involved in a number of clinical trials.Areas covered: The authors have performed a literature search (PubMed database up to December 2018) for studies evaluating interactions between AEDs and cannabinoid receptor ligands in experimental models of seizures. Clinical data relate to the add-on treatment with cannabidiol.Expert opinion: WIN55,212–2 mesylate (WIN, a non-selective agonist of CB1 and CB2 receptors) significantly potentiated the anticonvulsant activity of various AEDs in mice. Profound neurotoxic effects accompanied combinations of WIN with conventional AEDs. Among conventional and newer AEDs, ACEA (a selective CB1 receptor agonist) enhanced the protective action of phenobarbital and levetiracetam without accompanying adverse effects or pharmacokinetic interactions. Cannabidiol proved effective in clinical trials, reducing seizure frequency and the most frequently observed adverse effects were diarrhea, somnolence, and poor appetite. The retention rate was within 14–24% (12–14 weeks – 1 year) but it reached the level of 35% after 2 years.

Cannabinoid CB1 and CB2 receptor mechanisms underlie cannabis reward and aversion in rats.

Endocannabinoids are critically involved in brain reward functions, mediated by activation of CB1 receptors, reflecting their high density in the brain. However, the recent discovery of CB2 receptors in the brain, particularly in the midbrain dopamine neurons, has challenged this view and inspired us to re-examine the roles of both CB1 and CB2 receptors in the effects of cannabis. In the present study, we used the electrical intracranial self-stimulation paradigm to evaluate the effects of various cannabinoid drugs on brain reward in laboratory rats and the roles of CB1 and CB2 receptors activation in brain reward function(s). Two mixed CB1 / CB2 receptor agonists, Δ9 -tetrahydrocannabinol (Δ9 -THC) and WIN55,212-2, produced biphasic effects-mild enhancement of brain-stimulation reward (BSR) at low doses but inhibition at higher doses. Pretreatment with a CB1 receptor antagonist (AM251) attenuated the low dose-enhanced BSR, while a CB2 receptor antagonist (AM630) attenuated high dose-inhibited BSR. To confirm these opposing effects, rats were treated with selective CB1 and CB2 receptor agonists. These compounds produced significant BSR enhancement and inhibition, respectively. CB1 receptor activation produced reinforcing effects, whereas CB2 receptor activation was aversive. The subjective effects of cannabis depend on the balance of these opposing effects. These findings not only explain previous conflicting results in animal models of addiction but also explain why cannabis can be either rewarding or aversive in humans, as expression of CB1 and CB2 receptors may differ in the brains of different subjects.

Identification and biochemical analyses of selective CB2 agonists

Cannabinoid CB1 and CB2 receptors are activated by Δ9-tetrahydrocannabinol, a psychoactive component of marijuana. The cannabinoid CB1 receptor is primarily located in the brain and is responsible for the psychoactive side effects, whereas the cannabinoid CB2 receptor is located in immune cells and is an attractive target for immune-related maladies. We identify small molecules that selectively bind to the cannabinoid CB2 receptor and can be further developed into therapeutics. The affinity of three molecules, ABK5, ABK6, and ABK7, to the cannabinoid CB2 receptor was determined with radioligand competition binding. The potency of G-protein coupling was determined with GTPγS binding. The three compounds bound selectively to the cannabinoid CB2 receptor, and no binding to the cannabinoid CB1 receptor was detected up to 10 μM. Immunoblotting studies show that the amount of ERK1/2 and MEK phosphorylation increased in a Gi/o-dependent manner. Furthermore, an immune cell line (Jurkat cells) was treated with ABK5, and as a result, inhibited cell proliferation. These three compounds are novel cannabinoid CB2 receptor agonists and hold promise to be further developed to treat inflammation and the often-associated pain.

The effect of the cannabinoid receptor agonist and antagonist on the light-induced changes in the suprachiasmatic nucleus of rats

The CB1 cannabinoid receptors have been found in the rodent suprachiasmatic nucleus, and their activation suppresses the light-induced phase shift in locomotor rhythmicity of mice and hamsters. Here, we show that the CB1 receptor agonist CP55940 significantly attenuates the light-induced phase delay in rats as well. Furthermore, it blocks the light induction of c-Fos and light-induced downregulation of pERK1/2 in the SCN, and the CB1 antagonist AM251 prevents the photic induction of pERK1/2 and reduces pGSK3β after photic stimulation. Our data suggest that the modulation of the cannabinoid receptor activity may affect the photic entrainment via the setting of the SCN sensitivity to light.