Selective Cannabinoid CB1 Receptor Research Articles

Activation of CB2 Receptors by (-)-Cannabichromene but Not (+)-Cannabichromene.

Introduction: Cannabichromene (CBC) is a minor constituent of cannabis that is a selective cannabinoid CB2 receptor agonist and activator of TRPA1. To date, it has not been shown whether (-)-CBC, (+)-CBC, or both can mediate these effects. In this study, we investigate the activity of the CBC enantiomers at CB1, CB2, and Transient receptor potential ankyrin 1 (TRPA1) receptors in vitro. Materials and Methods: CBC enantiomers were purified from synthetic CBC by chiral chromatography, and their optical activity was confirmed by spectroscopy. Human CB1 and CB2 receptor activity was measured using a fluorescent assay of membrane potential in stably transfected AtT20 cells. TRPA1 activation was measured using a fluorescent assay of intracellular calcium in stably transfected HEK293 cells. Results: The (-)-CBC activated CB2 with an EC50 of 1.5 µM, to a maximum of 60% of (-)CP55940. (+)-CBC did not activate CB2 at concentrations up to 30 µM. Only 30 µM (-)-CBC produced detectable activation of CB1, (+)-CBC was inactive. Both (-)-CBC and (+)-CBC activated TRPA1; at 30 µM (-)-CBC produced an activation 50% of that of the reference agonist cinnamaldehyde (300 µM), 30 µM (+)-CBC activated TRPA1 to 38% of the cinnamaldehyde maximum. Discussion: It is unclear whether (-)-CBC is the sole or even the predominant enantiomer of CBC enzymatically synthesized in cannabis. This study shows that (-)-CBC is the active isomer at CB2 receptors, while both isomers activate TRPA1. The results suggest that medicinal preparations of CBC that target cannabinoid receptors would be most effective when (-)-CBC is the dominant isomer.

A Highly Potent, Orally Bioavailable Pyrazole-Derived Cannabinoid CB2 Receptor- Selective Full Agonist for In Vivo Studies.

The cannabinoid CB2 receptor (CB2R) is a potential therapeutic target for distinct forms of tissue injury and inflammatory diseases. To thoroughly investigate the role of CB2R in pathophysiological conditions and for target validation in vivo, optimal pharmacological tool compounds are essential. Despite the sizable progress in the generation of potent and selective CB2R ligands, pharmacokinetic parameters are often neglected for in vivo studies. Here, we report the generation and characterization of a tetra-substituted pyrazole CB2R full agonist named RNB-61 with high potency (K i 0.13-1.81 nM, depending on species) and a peripherally restricted action due to P-glycoprotein-mediated efflux from the brain. 3H and 14C labeled RNB-61 showed apparent K d values of <4 nM toward human CB2R in both cell and tissue experiments. The 6,800-fold selectivity over CB1 receptors and negligible off-targets in vitro, combined with high oral bioavailability and suitable systemic pharmacokinetic (PK) properties, prompted the assessment of RNB-61 in a mouse ischemia-reperfusion model of acute kidney injury (AKI) and in a rat model of chronic kidney injury/inflammation and fibrosis (CKI) induced by unilateral ureteral obstruction. RNB-61 exerted dose-dependent nephroprotective and/or antifibrotic effects in the AKI/CKI models. Thus, RNB-61 is an optimal CB2R tool compound for preclinical in vivo studies with superior biophysical and PK properties over generally used CB2R ligands.

A highly potent, orally bioavailable pyrazole-derived cannabinoid CB2 receptor-selective full agonist for in vivo studies.

The cannabinoid CB2 receptor (CB2R) is a potential therapeutic target for distinct forms of tissue injury and inflammatory diseases. To thoroughly investigate the role of CB2R in pathophysiological conditions and for target validation in vivo, optimal pharmacological tool compounds are essential. Despite the sizable progress in the generation of potent and selective CB2R ligands, pharmacokinetic parameters are often neglected for in vivo studies. Here, we report the generation and characterization of a tetra-substituted pyrazole CB2R full agonist named RNB-61 with high potency (K i 0.13-1.81 nM, depending on species) and a peripherally restricted action due to P-glycoprotein mediated efflux from the brain. 3H and 14C labelled RNB-61 showed apparent K d values < 4 nM towards human CB2R in both cell and tissue experiments. The >6000-fold selectivity over CB1 receptors and negligible off-targets in vitro, combined with high oral bioavailability and suitable systemic pharmacokinetic (PK) properties, prompted the assessment of RNB-61 in a mouse ischemia-reperfusion model of acute kidney injury (AKI) and in a rat model of chronic kidney injury/inflammation and fibrosis (CKI) induced by unilateral ureteral obstruction. RNB-61 exerted dose-dependent nephroprotective and/or antifibrotic effects in the AKI/CKI models. Thus, RNB-61 is an optimal CB2R tool compound for preclinical in vivo studies with superior biophysical and PK properties over generally used CB2R ligands.

β-Caryophyllene Inhibits Endothelial Tube Formation by Modulating the Secretome of Hypoxic Lung Cancer Cells-Possible Role of VEGF Downregulation.

β-Caryophyllene (BCP), a bicyclic sesquiterpene that is a component of the essential oils of various spice and food plants, has been described as a selective CB2 cannabinoid receptor agonist. In the present study, the effect of BCP on angiogenesis was investigated. It was found that conditioned media (CM) from BCP-treated hypoxic A549 lung cancer cells exhibited a concentration-dependent inhibitory effect on human umbilical vein endothelial cell (HUVEC) tube formation induced by CM from vehicle-treated hypoxic A549 cells. There was an associated concentration-dependent decrease in the proangiogenic factor vascular endothelial growth factor (VEGF) in the CM, with both BCP inhibitory effects (tube formation, VEGF secretion) being CB2 receptor-dependent. A reduction of the transcription factor hypoxia-inducible factor 1α (HIF-1α) was furthermore detected. The antiangiogenic and VEGF-lowering properties of BCP were confirmed when CM from another lung cancer cell line, H358, were tested. When directly exposed to HUVECs, BCP showed no significant effect on tube formation, but at 10 µM, impaired VEGF receptor 2 (VEGFR2) phosphorylation triggered by recombinant VEGF in a CB2 receptor-independent manner. In summary, BCP has a dual antiangiogenic effect on HUVECs, manifested in the inhibition of tube formation through modulation of the tumor cell secretome and additionally in the inhibition of VEGF-induced VEGFR2 activation. Because the CB2 agonist has no psychoactive properties, BCP should continue to be evaluated preclinically for further antitumor effects.

Memory impairment was ameliorated by corticolimbic microinjections of arachidonylcyclopropylamide (ACPA) and miRNA-regulated lentiviral particles in a streptozotocin-induced Alzheimer's rat model

The present study aimed to investigate the effect of corticolimbic cannabinoid CB1 receptors activity on memory impairment in the intracerebroventricular (ICV)-streptozotocin (STZ) animal model of Alzheimer's like-disease. This study also assessed whether the corticolimbic overexpression of miRNA-137 or -let-7a could increase the endocannabinoids by inhibiting the monoglyceride lipase (MAGL) to ameliorate STZ response. The results showed that ICV microinjection of STZ (3 mg/kg/10 μl) impaired passive avoidance memory retrieval. The chronic microinjection of arachidonylcyclopropylamide (ACPA; 10 ng/0.5 μl), a selective cannabinoid CB1 receptor agonist, into the hippocampal CA1 region, the central amygdala (CeA) or the medial prefrontal cortex (mPFC) ameliorated the amnesic effect of ICV-STZ. Intra-CA1 or -CeA microinjection of ACPA alone did not affect memory retrieval, while its microinjection into the mPFC impaired memory formation. Based on bioinformatics analysis and verification of the MAGL gene, miRNA-137 and -let-7a were chosen to target the expression levels of MAGL in the corticolimbic regions. The chronic corticolimbic microinjection of lentiviral particles containing miRNA-137 or -let-7a ameliorated ICV-STZ-induced memory impairment. The high transfection efficiency was determined for each virus using comparing fluorescent and conventional vision. Corticolimbic overexpression of miRNA-137 or -let-7a decreased the MAGL gene expression that encodes the MAGL enzyme to increase the endocannabinoids. Thus, among the molecular mechanisms and signaling pathways involved in the pathophysiology of Alzheimer's disease (AD), it is worth mentioning the role of endocannabinoids in the corticolimbic regions. CB1 receptor agonists, miRNA-137 or -let-7a, may be potential therapeutic targets against cognitive decline in AD.

Structural basis of selective cannabinoid CB2 receptor activation

Structural basis of selective cannabinoid CB2 receptor activation

Structural basis of selective cannabinoid CB2 receptor activation

Cannabinoid CB2 receptor (CB2R) agonists are investigated as therapeutic agents in the clinic. However, their molecular mode-of-action is not fully understood. Here, we report the discovery of LEI-102, a CB2R agonist, used in conjunction with three other CBR ligands (APD371, HU308, and CP55,940) to investigate the selective CB2R activation by binding kinetics, site-directed mutagenesis, and cryo-EM studies. We identify key residues for CB2R activation. Highly lipophilic HU308 and the endocannabinoids, but not the more polar LEI-102, APD371, and CP55,940, reach the binding pocket through a membrane channel in TM1-TM7. Favorable physico-chemical properties of LEI-102 enable oral efficacy in a chemotherapy-induced nephropathy model. This study delineates the molecular mechanism of CB2R activation by selective agonists and highlights the role of lipophilicity in CB2R engagement. This may have implications for GPCR drug design and sheds light on their activation by endogenous ligands.

The Cannabinoid Ligand Arachidonyl-2'-Chloroethylamide (ACEA) Ameliorates Depressive and Overactive Bladder Symptoms in a Corticosterone-Induced Female Wistar Rat Model.

There is growing need to increase the knowledge on the cannabinoid ligands in the treatment of overactive bladder. Among potential candidates, arachidonyl-2'-chloroethylamide (ACEA), a selective cannabinoid CB1 receptor agonist is proposed. The aim of this paper was to determine if ACEA, a selective cannabinoid CB1 receptor agonist, could reverse the effects of corticosterone (CORT), characteristic of depressive and bladder overactivity potential. The animals (48 female rats) were divided into four groups: I-control, II-received CORT, III-received ACEA, and IV-received the combination of CORT and ACEA. The conscious cystometry, forced swim test (FST), and locomotor activity measurements were performed 3 days after the last dose of ACEA, followed by ELISA measurements. In group IV, ACEA restored urodynamic parameters that were altered by CORT. CORT prolonged the immobility time in FST and the values were lowered by ACEA. ACEA normalized the expression of c-Fos in all the analyzed central micturition centers (group IV vs. group II). ACEA restored the CORT-induced changes in the biomarkers in urine (BDNF, NGF), bladder detrusor (VAChT, Rho kinase), bladder urothelium (CGRP, ATP, CRF, OCT-3, TRPV1), and hippocampus (TNF-α, IL-1β and Il-6, CRF, IL-10, BDNF, NGF). In conclusion, ACEA was proven to reverse CORT-induced changes in both cystometric and biochemical parameters that are determinants of OAB/depression, which represents an example of an existing link between OAB and depression via cannabinoid receptors.

Cross state-dependent memory retrieval between cannabinoid CB1 and serotonergic 5-HT1A receptor agonists in the mouse dorsal hippocampus

Understanding the neurobiological mechanisms of drug-related learning and memory formation may help the treatment of cognitive disorders. Dysfunction of the cannabinoid and serotonergic systems has been demonstrated in learning and memory disorders.The present paper investigates the phenomenon called state-dependent memory (SDM) induced by ACPA (a selective cannabinoid CB1 receptor agonist) and 8-OH-DPAT (a nonselective 5-HT1A receptor agonist) with special focus on the role of the 5-HT1A receptor in the effects of both ACPA and 8-OH-DPAT SDM and cross state-dependent memory retrieval between ACPA and 8-OH-DPAT in a step-down inhibitory avoidance task.The dorsal hippocampal CA1 regions of adult male NMRI mice were bilaterally cannulated, and all drugs were microinjected into the intended injection sites. A single-trial step-down inhibitory avoidance task was used to assess memory retrieval and state-dependence.Post-training and/or pre-test microinjections of ACPA (1 and 2 ng/mouse) and 8-OH-DPAT (0.5 and 1 μg/mouse) dose-dependently induced amnesia. Pre-test administration of the same doses of ACPA and 8-OH-DPAT reversed the post-training ACPA- and 8-OH-DPAT-induced amnesia, respectively. This phenomenon has been named SDM. 8-OH-DPAT (1 μg/mouse) reversed the amnesia induced by ACPA (0.5, 1, and 2 ng/mouse) and induced ACPA SDM. ACPA (2 ng/mouse) reversed the amnesia induced by 8-OH-DPAT (0.25, 0.5, and 1 μg/mouse) and induced 8-OH-DPAT SDM. Pre-test administration of a 5-HT1A receptor antagonist, (S)-WAY 100,135 (0.25 and 0.5 μg/mouse), 5 min before ACPA and 8-OH-DPAT dose-dependently inhibited ACPA- and 8-OH-DPAT-induced SDM, respectively.The present study results demonstrated ACPA- and 8-OH-DPAT- induced SDM. Overall, the data revealed that dorsal hippocampal 5-HT1A receptor mechanisms play a pivotal role in modulating cross state-dependent memory retrieval between ACPA and 8-OH-DPAT.

Synthesis and In Vitro Characterization of Selective Cannabinoid CB2 Receptor Agonists: Biological Evaluation against Neuroblastoma Cancer Cells.

1,8-naphthyridine-3-carboxamide structures were previously identified as a promising scaffold from which to obtain CB2R agonists with anticancer and anti-inflammatory activity. This work describes the synthesis and functional characterization of new 1,8-naphthyridin-2(1H)-one-3-carboxamides with high affinity and selectivity for CB2R. The new compounds were able to pharmacologically modulate the cAMP response without modulating CB2R-dependent β-arrestin2 recruitment. These structures were also evaluated for their anti-cancer activity against SH-SY5Y and SK-N-BE cells. They were able to reduce the cell viability of both neuroblastoma cancer cell lines with micromolar potency (IC50 of FG158a = 11.8 μM and FG160a = 13.2 μM in SH-SY5Y cells) by a CB2R-mediated mechanism. Finally, in SH-SY5Y cells one of the newly synthesized compounds, FG158a, was able to modulate ERK1/2 expression by a CB2R-mediated effect, thus suggesting that this signaling pathway might be involved in its potential anti-cancer effect.

Behavioral and binding studies on the quinolinyl ester indoles 5F-PB22 (5F-QUPIC) and BB-22 (QUCHIC) in the mouse model

Novel Psychoactive Substances newly introduced on the drug market are constantly changing patterns of drug consumption. Among these, Synthetic Cannabinoids (SCs) mainly dominated European seizures in the last years. Quinolin-8-yl 1-(5-fluoropentyl)-1H-indole-3-carboxylate (5F-PB22) and quinolin-8-yl 1-(cyclohexylmethyl)-1H-indole-3-carboxylate (BB-22) are SCs whose abuse has been linked to a range of fatal intoxications and hospitalizations. Therefore, this study aims to investigate and compare in vitro and in vivo pharmacodynamics activity of these quinolinyl ester indoles. In vitro competition binding experiments performed on CD-1 murine and human cannabinoid CB1 and CB2 receptors revealed a sub-nanomolar affinity and potency of 5F-PB22 and BB-22. In vivo studies demonstrated that the acute systemic administration of 5F-PB22 and BB-22 (0.001-6 mg/kg) deeply impaired sensorimotor and motor responses, core temperature, breath rate and nociceptive threshold of CD-1 male mice. Pre-treatment with the selective cannabinoid CB1 receptor antagonist/inverse agonist AM-251 (6 mg/kg) fully prevented the effects of both cannabinoids (at 1 mg/kg) suggesting a CB1 receptor mediated action. Relying on these findings, this study showed for the first time the pharmaco-toxicological effects of these substances confirming their potential burden on human health. Furthermore, it revealed that the difference of the chemical structures of the two SCs results in their potency related disparities.

Development of fluorinated and methoxylated benzothiazole derivatives as highly potent and selective cannabinoid CB2 receptor ligands

The upregulation of the CB2 receptors in neuroinflammation and cancer and their potential visualization with PET (positron emission tomography) could provide a valuable diagnostic and therapy-monitoring tool in such disorders. However, the availability of reliable CB2-selective imaging probes is still lacking in clinical practice. We have recently identified a benzothiazole-2-ylidine amide hit (6a) as a highly potent CB2 ligand. With the aim of enhancing its CB2 over CB1 selectivity and introducing structural sites suitable for radiolabeling, we herein describe the development of fluorinated and methoxylated benzothiazole derivatives endowed with extremely high CB2 binding affinity and an exclusive selectivity to the CB2 receptor. Compounds 14, 15, 18, 19, 21, 24 and 25 displayed subnanomolar CB2Ki values (ranging from 0.16 nM to 0.68 nM) and interestingly, all of the synthesized compounds completely lacked affinity at the CB1 receptor (Ki > 10,000 nM for all compounds), indicating their remarkably high CB2 over CB1 selectivity factors. The fluorinated analogs, 15 and 21, were evaluated for their in vitro metabolic stability in mouse and human liver microsomes (MLM and HLM). Both 15 and 21 displayed an exceptionally high stability (98% and 91% intact compounds, respectively) after 60 min incubation with MLM. Contrastingly, a 5- and 2.8-fold lower stability was demonstrated for compounds 15 and 21, respectively, upon incubation with HLM for 60 min. Taken together, our data present extremely potent and selective CB2 ligands as credible leads that can be further exploited for 18F- or 11C-radiolabeling and utilization as PET tracers.

The bidirectional effect of prelimbic 5-hydroxytryptamine type-4 (5-HT4) receptors on ACPA-mediated aversive memory impairment in adult male Sprague-Dawley rats.

Objective(s):This study aimed at investigating the effect of serotonergic 5-HT4 receptor agonist/antagonist on memory consolidation deficit induced by ACPA (a potent, selective CB1 cannabinoid receptor agonist) in the pre-limbic (PL) cortex. Materials and Methods: We used the step-through passive avoidance test to evaluate memory consolidation of male Sprague-Dawley (SD) rats. Bilateral post-training microinjections of the drugs were done in a volume of 0.6 μl/rat into the PL area (0.3 μl per side).Results:The results showed a significant interaction between RS67333 hydrochloride (5-HT4 receptor agonist) or RS23597-190 hydrochloride (5-HT4 receptor antagonist) and ACPA on consolidation of aversive memory. RS67333 hydrochloride (0.5 μg/rat) enhanced consolidation of memory and its co-administration at the ineffective dose of 0.005 μg/rat with ineffective (0.001 μg/rat) or effective (0.1 μg/rat) doses of ACPA improved and prevented impairment of memory caused by ACPA, respectively. In other words, RS67333 had a bidirectional effect on ACPA-caused amnesia. While RS23597-190 hydrochloride had no effect on memory at the doses used (0.005, 0.01, 0.1, or 0.5 μg/rat); but its concomitant use with an effective dose of ACPA (0.1 μg/rat) potentiated amnesia. None of the drugs had an effect on locomotor activity.Conclusion:This study revealed that activation or deactivation of the 5-HT4 receptors in the PL may mediate the IA memory impairment induced by ACPA indicating a modulatory role for the 5-HT4 serotonergic receptors.

Characterization of Subtype Selective Cannabinoid CB2 Receptor Agonists as Potential Anti-Inflammatory Agents.

Activation of the CB2 receptor has been shown to have anti-inflammatory and antinociceptive effects without causing psychoactive effects. Previously, we reported that the compound ethyl 2(2-(N-(2,3-dimethylphenyl) phenylsulfonamido)acetamido)benzoate (ABK5) is a CB2 subtype selective agonist with anti-inflammatory and antinociceptive effects. In the present study, we tested four ABK5 derivatives, ABK5-1, ABK5-2, ABK5-5, and ABK5-6, to analyze the structure of ABK5 to obtain CB2-selective agonists with higher affinity and efficacy. Affinity, subtype selectivity, and G-protein coupling were determined by radioligand binding assays. Selected compounds were then subjected to evaluation of anti-inflammatory effects using two different cell lines, Jurkat (ABK5-1 and 5-2) and BV-2 cells (ABK5-1), which are models of T cells and microglia, respectively. ABK5-1, ABK5-2, and ABK5-6 had comparable CB2 binding affinity with ABK5 (and stimulated G-protein coupling), while only ABK5-1 and ABK5-2 maintained CB2-subtype selectivity. ABK5-5 did not bind CB2 in the detectable range. RT-PCR and ELISA analysis showed that the two compounds also inhibit IL-2 and TNF-α production, and they were more efficacious than ABK5 in inhibiting TNF-α production. CXCL-12 mediated chemotaxis was also evaluated by the transwell migration assay, and both ABK5-1 and ABK5-2 inhibited chemotaxis with a stronger effect observed in ABK5-1. In the microglia cell line BV-2, ABK5-1 inhibited IL-1β and IL-6 production, which suggests this compound has anti-inflammatory effects through targeting multiple immune cells, and may be a candidate for treatment of inflammation.

Receptor mechanism of infarct-limiting effect of adaptation to normobaric hypoxia

The aim of the study was to investigate the involvement of bradykinin, cannabinoid and vanilloid (TRPV1 channel) receptors in the implementation of the infarct-limiting effect of chronic normobaric hypoxia (CNH). Materials and methods . The study was performed on male Wistar rats ( n = 117) weighing 250–300 g. Adaptation to CNH was modeled for 21 days at 12% pO 2 , 0.3% pCO 2 and normal atmospheric pressure. A day after adaptation of rats to CNH coronary artery occlusion (45 min) and reperfusion (2 h) was performed. In the study the following compounds were used: selective cannabinoid CB1 receptor antagonist rimonabant (1 mg/kg), selective cannabinoid CB2 receptor antagonist AM630 (2.5 mg/kg), selective bradykinin B2 receptor antagonist HOE140 (50 μg/kg), and vanilloid receptor (TRPV1 channel) antagonist capsazepine (3 mg/kg). All antagonists were administered 15 min before coronary artery occlusion. Results. Adaptation to normobaric hypoxia promoted the formation of the pronounced infarct-limiting effect. The blockade of B2 receptor eliminated the infarct-limiting effect of CNH. Blockade of cannabinoid or vanilloid receptors did not affect the infarct-limiting effect of CNH. Conclusion . The infarct-limiting effect of CNH depends on the activation of B2 receptor, and the adaptive increase in cardiac tolerance to ischemia/reperfusion does not depend on cannabinoid or vanilloid receptors.

Anti-inflammatory and antinociceptive effects of the selective cannabinoid CB2 receptor agonist ABK5

Cannabinoid receptors are a potential target for anti-inflammatory and pain therapeutics. There are two subtypes, CB1 and CB2, and Δ9-tetrahydrocannabinol activates both of them, providing an analgesic effect but also psychoactive side effects. The psychoactive side effects are considered to be caused by activation of CB1, but not CB2. ABK5 is a CB2 subtype selective agonist that has a very different structure from known cannabinoid receptor agonists. Here, we report anti-inflammatory effects of ABK5 using the T-cell line Jurkat cells, and antinociceptive effect in an inflammatory pain model in rats. Production of the cytokines IL-2 and TNF-α was measured in stimulated Jurkat cells and MOLT-4 cells, and CXCL12-mediated chemotaxis of Jurkat cells was evaluated by a transwell migration assay. Anti-inflammatory and antinociceptive effects of ABK5 were also evaluated in a hindpaw CFA model in rats. ABK5 significantly decreased production of IL-2 and TNF-α measured as both mRNA and protein levels, and reduced chemotaxis towards CXCL12. It also attenuated edema and increased mechanical threshold in the hindpaw of CFA-treated rats. These results suggest that ABK5 is a good lead compound for the development of potential anti-inflammatory and analgesic agents.

Antidepressant-Like Effect of Terpineol in an Inflammatory Model of Depression: Involvement of the Cannabinoid System and D2 Dopamine Receptor.

Depression has a multifactorial etiology that arises from environmental, psychological, genetic, and biological factors. Environmental stress and genetic factors acting through immunological and endocrine responses generate structural and functional changes in the brain, inducing neurogenesis and neurotransmission dysfunction. Terpineol, monoterpenoid alcohol, has shown immunomodulatory and neuroprotective effects, but there is no report about its antidepressant potential. Herein, we used a single lipopolysaccharide (LPS) injection to induce a depressive-like effect in the tail suspension test (TST) and the splash test (ST) for a preventive and therapeutic experimental schedule. Furthermore, we investigated the antidepressant-like mechanism of action of terpineol while using molecular and pharmacological approaches. Terpineol showed a coherent predicted binding mode mainly against CB1 and CB2 receptors and also against the D2 receptor during docking modeling analyses. The acute administration of terpineol produced the antidepressant-like effect, since it significantly reduced the immobility time in TST (100–200 mg/kg, p.o.) as compared to the control group. Moreover, terpineol showed an antidepressant-like effect in the preventive treatment that was blocked by a nonselective dopaminergic receptor antagonist (haloperidol), a selective dopamine D2 receptor antagonist (sulpiride), a selective CB1 cannabinoid receptor antagonist/inverse agonist (AM281), and a potent and selective CB2 cannabinoid receptor inverse agonist (AM630), but it was not blocked by a nonselective adenosine receptor antagonist (caffeine) or a β-adrenoceptor antagonist (propranolol). In summary, molecular docking suggests that CB1 and CB2 receptors are the most promising targets of terpineol action. Our data showed terpineol antidepressant-like modulation by CB1 and CB2 cannabinoid receptors and D2-dopaminergic receptors to further corroborate our molecular evidence.

Novel halogenated synthetic cannabinoids impair sensorimotor functions in mice

JWH-018-Cl, JWH-018-Br and AM-2201 (JWH-018 halogenated-derivatives; JWH-018-R compounds) are synthetic cannabinoid agonists illegally marketed as “Spice”, “K2”, “herbal blend” and research chemicals for their cannabis-like psychoactive effects. In rodents, JWH-018 and its halogenated derivatives reproduce the typical effects of Δ9-tetrahydrocannabinol (Δ9-THC), i.e. hypothermia, analgesia, hypolocomotion and akinesia. Yet, the effects of JWH-018-R compounds on sensorimotor functions are still unknown. This study was designed to investigate the effect of an acute intraperitoneal (i.p.) administration of JWH-018-R compounds (0.01–6 mg/kg) on sensorimotor functions in mice and to compare them to those caused by the reference compound JWH-018 and Δ9-THC. A well validated battery of behavioral tests was used to investigate the effects of these synthetic cannabinoids on the visual, auditory and tactile responses in mice, while the pre-pulse inhibition (PPI) test was used to investigate their effect on sensorimotor gating. The effect of the synthetic cannabinoids on spontaneous locomotion was also measured by a video tracking analysis to assess potential cannabinoid-induced motor impairment. Results showed that, similarly to JWH-018, systemic administration of JWH-018-R compounds inhibits sensorimotor and PPI responses at lower doses (0.01-0.1 mg/kg) and reduced spontaneous locomotion at intermediate/high doses (1–6 mg/kg). All effects were prevented by the administration of the selective cannabinoid CB1 receptor antagonist/inverse agonist AM-251 thus confirming a CB1 receptor-mediated action. Finding that lower doses of JWH-018-R compounds selectively impair sensorimotor and PPI responses without affecting locomotion should be carefully considered to better understand the potential danger that halogenated-derivatives of JWH-018 may pose to public health, with particular reference to decreased performance in driving and hazardous works.

Levetiracetam combined with ACEA, highly selective cannabinoid CB1 receptor agonist changes neurogenesis in mouse brain

The aim of the study was to evaluate the impact of second generation antiepileptic drug levetiracetam (LEV) with arachidonyl-2′-chloroethylamide (ACEA) on proliferating neural precursor cells in mouse brain. Additionally, we established the relationship between treatment with ACEA in combination with LEV and hippocampal neurogenesis in mouse brain. All experiments were performed on male CB57/BL mice injected i.p. with LEV (10 mg/kg), ACEA (10 mg/kg) and PMSF (30 mg/kg) for 10 days. Experiments were provided in two stages: stage 1- an acute response of proliferating neural precursor cells to ACEA and LEV administration (Ki-67 staining), stage 2 – a long term response to ACEA and LEV administration (BrDU, NeuN, GFAP staining). Results indicate that ACEA + PMSF and ACEA + PMSF + LEV significantly increased the total number of Ki-67 positive cells comparing to the control group. PMSF and LEV administered alone and in combination had no significant impact on cell proliferation compared to the control group. Results from neurogenesis study indicated that ACEA + PMSF administered alone and in combination with LEV increased the total number of BrDU cells compared to the control group, although LEV on its own decreased the number of BrDU cells. Moreover, the combination of ACEA + PMSF + LEV significantly increased the total number of newborn neurons compared to the control group. In turn, LEV significantly decreased the process of neurogenesis. Astrocytes were considerably reduced in all treated groups as compare to the control mice. These data provide substantial evidence that LEV administered chronically decreases the proliferation and differentiation of newly born cells while combination of LEV + ACEA significantly increases the level of newborn neurons in the dentate subgranular zone.

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.