CB1 Agonist Research Articles

Discovery of 4-(1,2,4-Oxadiazol-5-yl)azepan-2-one Derivatives as a New Class of Cannabinoid Type 2 Receptor Agonists for the Treatment of Inflammatory Pain.

Selectively targeting the cannabinoid receptor CB2 is an attractive therapeutic strategy for the treatment of inflammatory pain without psychiatric side effects mediated by the cannabinoid receptor CB1. Herein, we report the discovery of 4-(1,2,4-oxadiazol-5-yl)azepan-2-one derivatives as a new class of CB2 agonists. Systematic structure-activity relationship investigations resulted in the identification of the most potent compound 25r. This compound displayed high selectivity for CB2 against CB1 (CB2 EC50 = 21.0 nM, Emax = 87%, CB1 EC50 > 30 μM, ratio CB1/CB2 > 1428) with favorable pharmacokinetic properties. Especially, 25r demonstrated significant efficacy in the analgesic model of rodent inflammatory pain. All the results suggest that compound 25r could serve as a lead compound for treating inflammatory pain and deserves further in-depth studies.

Reward enhances resilience to chronic social defeat stress in mice: Neural ECs and mGluR5 mechanism via neuroprotection in VTA and DRN.

Stress often leads to emotional disorders such as depression. The reward might render this effect through the enhancement of stress resilience. However, the effect of reward on stress resilience under different intensities of stress needs more evidence, and its potential neural mechanism has been poorly revealed. It has been reported that the endogenous cannabinoid system (ECs) and downstream metabolic glutamate receptor 5 (mGluR5) are closely related to stress and reward, which might be the potential cerebral mechanism between reward and stress resilience, but there is a lack of direct evidence. This study aims to observe the effect of reward on stress resilience under different intensities of stress and further explore potential cerebral mechanisms underlying this effect. Using the chronic social defeat stress model, we applied reward (accompanied by a female mouse) under different intensities of stress in mice during the modeling process. The impact of reward on stress resilience and the potential cerebral mechanism were observed after modeling through behavioral tests and biomolecules. The results showed that stronger stress led to higher degrees of depression-like behavior. Reward reduced depression-like behavior and enhanced stress resilience (all p-value <0.05) (more social interaction in the social test, less immobility time in the forced swimming test, etc.), with a stronger effect under the large stress. Furthermore, the mRNA expression levels of CB1 and mGluR5, the protein expression level of mGluR5, and the expression level of 2-AG (2-arachidonoylglycerol) in both ventral tegmental area (VTA) and dorsal raphe nucleus (DRN) were significantly upregulated by reward after modeling (all p-value <0.05). However, the protein expression of CB1 in VTA and DRN and the expression of AEA (anandamide) in VTA did not differ significantly between groups. Intraperitoneal injection of a CB1 agonist (URB-597) during social defeat stress significantly reduced depression-like behavior compared with a CB1 inhibitor (AM251) (all p-value <0.05). Interestingly, in DRN, the expression of AEA in the stress group was lower than that of the control group, with or without reward (all p-value <0.05). These findings demonstrate that combined social and sexual reward has a positive effect on stress resilience during chronic social defeat stress, potentially by influencing the ECs and mGluR5 in VTA and DRN.

Behavioral, biochemical and histopathological toxic profiles induced by sub-chronic cannabimimetic WIN55, 212–2 administration in mice

WIN55, 212–2 mesylate is a synthetic cannabinoid (SC) agonist of CB1 and CB2 receptors with much higher affinity to CB1 receptor than tetrahydrocannabinol and many potential therapeutic effects. Few studies have evaluated SCs effects on more complex animal behavior and sex differences in cannabinoids toxicology. The current study was undertaken for determination of behavioral (Open Field test), biochemical (liver and kidney function test plus GABA & Glutamate levels), histopathological and CB1 immunohistochemistry risks of sub-chronic administration of SC WIN55, 212–2 mesylate in male and female mice. A total of 40 healthy adult mice were randomly divided into four groups (5 mice each): a negative control group, a vehicle group, a low dose (0.05 mg/kg) group and a high dose group (0.1 mg/kg) for each gender.Open Field Test revealed dose and gender-dependent anxiogenic effect with reduced locomotor activity in both sexes especially the higher doses with female mice being less compromised. GABA and glutamate levels increased significantly in both dose groups compared to controls alongside female mice versus males. No significant biochemical alterations were found in all groups with minimal histopathological changes. The CB1 receptors immunohistochemistry revealed a significant increase in the number of CB1 positive neurons in both low and high dose groups against controls with higher expression in female brains.ConclusionsThere were sexual dimorphism effects induced by sub-chronic exposure to WIN55, 212–2 with lesser female mice affection and dose-dependent influences.

The Endogenous Cannabinoid and the Nitricoxidergic Systems in the Modulation of Stress Responses.

The effects on stress-induced analgesia (SIA) from endogenous cannabinoid system (ECS) and nitric oxide (NO) interaction after 1 h of restraint stress were evaluated in male Wistar rats. The animals were subjected to 1 h of restraint and then injected with different combinations of cannabinoid receptor type 1 agonist anandamide (AEA) or antagonist AM251 along with an NO donor, NO precursor, or inhibitor of NO synthase. Nociception was evaluated using paw pressure (PP) or hot plate (HP) tests. AEA was administered immediately after the end of restraint-SIA (r-SIA). Administration of NO precursor reversed the pronociceptive effect of the CB1 agonist on r-SIA. Both the CB1 antagonist and the NOS inhibitor neutralized the pro-analgesic effect of L-arginine (L-arg). Administration of an NO donor, instead, increased r-SIA. Our experiments confirmed that the endogenous cannabinoid and the NO-ergic systems interact in the modulation of r-SIA. This interaction probably implies NO as a second messenger of the ECS.

In Vitro and In Silico Studies of Neolignans from Magnolia grandiflora L. Seeds against Human Cannabinoids and Opioid Receptors.

Magnolia grandiflora L. (Magnoliaceae) is a plant of considerable medicinal significance; its flowers and seeds have been used in various traditional remedies. Radioligand binding assays of n-hexane seeds extract showed displacement of radioligand for cannabinoid (CB1 and CB2) and opioid δ (delta), κ (kappa), and µ (mu) receptors. Bioactivity-guided fractionation afforded 4-O-methylhonokiol (1), magnolol (2), and honokiol (3), which showed higher binding to cannabinoid rather than opioid receptors in radioligand binding assays. Compounds 1-3, together with the dihydro analog of 2 (4), displayed selective affinity towards CB2R (Ki values of 0.29, 1.4, 1.94, and 0.99 μM, respectively), compared to CB1R (Ki 3.85, 17.82, 14.55, and 19.08 μM, respectively). An equal mixture of 2 and 3 (1:1 ratio) showed additive displacement activity towards the tested receptors compared to either 2 or 3 alone, which in turn provides an explanation for the strong displacement activity of the n-hexane extract. Due to the unavailability of an NMR or X-ray crystal structure of bound neolignans with the CB1 and CB2 receptors, a docking study was performed to predict ligand-protein interactions at a molecular level and to delineate structure-activity relationships (SAR) of the neolignan analogs with the CB1 and CB2 receptors. The putative binding modes of neolignans 1-3 and previously reported related analogs (4, 4a, 5, 5a, 6, 6a, and 6b) into the active site of the CB1 and CB2 receptors were assessed for the first time via molecular docking and binding free-energy (∆G) calculations. The docking and ∆G results revealed the importance of a hydroxyl moiety in the molecules that forms strong H-bonding with Ser383 and Ser285 within CB1R and CB2R, respectively. The impact of a shift from a hydroxyl to the methoxy group on experimental binding affinity to CB1R versus CB2R was explained through ∆G data and the orientation of the alkyl chain within the CB1R. This comprehensive SAR, influenced by the computational study and the observed in vitro displacement binding affinities, has indicated the potential of magnolia neolignans for developing new CB agonists for potential use as analgesics, anti-inflammatory agents, or anxiolytics.

Hair Regrowth with Novel Hemp Extract: A Case Series.

The endocannabinoid system (ECS), discovered in the 1990s, is a system involved with maintaining cellular homeostasis by down-regulating the damaging inflammatory responses and upregulating regenerative processes. Cannabidiol (CBD), tetrahydrocannabivarin (THCV), and cannabidivarin (CBDV) are all phytocannabinoids found in varying quantities in hemp extract. These three cannabinoids have novel therapeutic effects on hair regrowth through the ECS. The method of action is different from and synergistic with current hair regrowth therapies. The three cannabinoids are fat-soluble and poorly absorbed past the epidermis, but topical application easily reaches hair follicles where they act as partial or full CB1 antagonist and agonist of transient receptor potential vanilloid-1 (TRPV1) and vanilloid receptor-4 (TRPV4). All these ECS receptors relate to hair follicle function. Blocking the CB1 receptor on the hair follicle has been shown to result in hair shaft elongation; in addition, the hair follicle cycle (anagen, catagen, and telogen phases) is controlled by TRPV1. The effects of CBD on hair growth are dose dependent and higher doses may result in premature entry into the catagen phase through a different receptor known as TRPV4. CBD has also been shown to increase Wnt signaling, which causes dermal progenitor cells to differentiate into new hair follicles and maintains anagen phase of the hair cycle. This study was conducted on subjects with androgenetic alopecia (AGA), as follow-up to a prior published study using hemp extract high in CBD without CBDV or THCV. That study showed an average 93.5% increase in hair numbers after 6 months of use. This subsequent study is being done to determine if daily topical application of a hemp-oil high in CBD, THCV, and CBDV concentrations would result in improved hair regrowth in the area of the scalp most affected by AGA. A case series study was done of 31 (15 men and 16 women, 27 Caucasian, 2 Asian, and 1 mixed race) subjects with AGA. They used a once-daily topical hemp extract formulation, averaging about 33 mg/day for 6 months. A hair count of the greatest area of alopecia was carried out before treatment was started and again after 6 months of treatment. To facilitate consistent hair count analysis, a permanent tattoo was placed at the point for maximum hair loss on the scalp. The subjects were also asked to qualitatively rate their psychosocial perception of "scalp coverage" improvement after the study was completed. The qualitative scale included "very unhappy," "unhappy," "neutral," "happy," and "very happy." The subjects were photographed in a standard manner before and after the study. The photographs were compared for improvements in "scalp coverage" by an independent physician. The qualitative scale included "none," "mild," "moderate," and "extensive" improvement of scalp coverage. The results revealed that all subjects had some regrowth. This ranged from 31.25% (from 16 to 21 hairs) to 2000% (from 1 to 21 hairs). The average increase was statistically significant 246% (15.07 hairs/cm2 increase) in men and 127% (16.06 hairs/cm2) in women. There were no reported adverse effects. All subjects rated their psychosocial perception of the effects of the hair loss, as "happy" or "very happy." Independent review of the photographs revealed evidence of "mild" to "extensive" scalp coverage improvements for all of the subjects. Although the exact mechanism of therapeutic effects is not known, THCV and CBDV are most likely functioning as full CB1 receptor neutral antagonists and CBD is most likely functioning as a partial CB1 receptor antagonist and potentially through Wnt messaging. All three cannabinoids were functioning as TRPV1 agonists. The addition of menthol through the peppermint extract is probably acting through promoting a rapid onset of anagen phase. This topical hemp formulation was superior to oral finasteride, 5% minoxidil once daily foam and CBD topical extract alone. Since this hemp extract works through novel mechanisms entirely different from both finasteride and minoxidil, it can be used in conjunction with these current drugs and would be expected to have synergistic effects. However, safety and efficacy of this combination would be to be evaluated.

Selective CB2 Receptor Agonist, HU-308, Reduces Systemic Inflammation in Endotoxin Model of Pneumonia-Induced Acute Lung Injury.

Acute respiratory distress syndrome (ARDS) and sepsis are risk factors contributing to mortality in patients with pneumonia. In ARDS, also termed acute lung injury (ALI), pulmonary immune responses lead to excessive pro-inflammatory cytokine release and aberrant alveolar neutrophil infiltration. Systemic spread of cytokines is associated with systemic complications including sepsis, multi-organ failure, and death. Thus, dampening pro-inflammatory cytokine release is a viable strategy to improve outcome. Activation of cannabinoid type II receptor (CB2) has been shown to reduce cytokine release in various in vivo and in vitro studies. Herein, we investigated the effect of HU-308, a specific CB2 agonist, on systemic and pulmonary inflammation in a model of pneumonia-induced ALI. C57Bl/6 mice received intranasal endotoxin or saline, followed by intravenous HU-308, dexamethasone, or vehicle. ALI was scored by histology and plasma levels of select inflammatory mediators were assessed by Luminex assay. Intravital microscopy (IVM) was performed to assess leukocyte adhesion and capillary perfusion in intestinal and pulmonary microcirculation. HU-308 and dexamethasone attenuated LPS-induced cytokine release and intestinal microcirculatory impairment. HU-308 modestly reduced ALI score, while dexamethasone abolished it. These results suggest administration of HU-308 can reduce systemic inflammation without suppressing pulmonary immune response in pneumonia-induced ALI and systemic inflammation.

In Vitro Characterization of 6-Methyl-3-(2-nitro-1-(thiophen-2-yl)ethyl)-2-phenyl-1H-indole (ZCZ011) at the Type 1 Cannabinoid Receptor: Allosteric Agonist or Allosteric Modulator?

Orthosteric activation of CB1 is known to cause a plethora of adverse side effects in vivo. Allosteric modulation is an exciting therapeutic approach and is hoped to offer improved therapeutic potential and a reduced on-target side effect profile compared to orthosteric agonists. This study aimed to systematically characterize the in vitro activity of the positive allosteric modulator ZCZ011, explicitly considering its effects on receptor regulation. HEK293 cells expressing hCB1 receptors were used to characterize ZCZ011 alone and in combination with orthosteric agonists. Real-time BRET approaches were employed for G protein dissociation, cAMP signaling, and β-arrestin translocation. Characterization also included ERK1/2 phosphorylation (PerkinElmer AlphaLISA) and receptor internalization. ZCZ011 is an allosteric agonist of CB1 in all pathways tested, with a similar signaling profile to that of the partial orthosteric agonist Δ9-tetrahydrocannabinol. ZCZ011 also showed limited positive allosteric modulation in increasing the potency and efficacy of THC-induced ERK1/2 phosphorylation, β-arrestin translocation, and receptor internalization. However, no positive allosteric modulation was observed for ZCZ011 in combination with either CP55940 or AMB-FUBINACA, in G protein dissociation, nor cAMP inhibition. Our study suggests that ZCZ011 is an allosteric agonist, with effects that are often difficult to differentiate from those of orthosteric agonists. Together with its pronounced agonist activity, the limited extent of ZCZ011 positive allosteric modulation suggests that further investigation into the differences between allosteric and orthosteric agonism is required, especially in receptor regulation end points.

Peripheral sensory neuron CB2 cannabinoid receptors are necessary for both CB2-mediated antinociceptive efficacy and sparing of morphine tolerance in a mouse model of anti-retroviral toxic neuropathy

Painful peripheral neuropathy is a common neurological complication associated with human immunodeficiency virus (HIV) infection and anti-retroviral therapy. We characterized the impact of two CB2 cannabinoid agonists (AM1710 and LY2828360 – ligands differing in signaling bias and CNS penetration) on neuropathic nociception induced by the antiretroviral agent Zalcitabine (2',3'-dideoxycytidine; ddC). We also used a conditional knockout approach to identify cell types mediating CB2 agonist-induced antinociceptive efficacy and sparing of morphine tolerance. AM1710 and LY2828360 alleviated ddC-induced neuropathic nociception in mice of both sexes. These benefits were absent in global CB2 knockout mice, which exhibited robust morphine antinociception. Like morphine, AM1710 blunted ddC-induced increases in proinflammatory cytokine (IL-1β, TNF-α) and chemokine (CCL2) mRNA expression levels. We generated advillinCre/+;CB2f/f conditional knockout mice to ascertain the role of CB2 localized to primary sensory neurons in CB2-mediated therapeutic effects. Antinociceptive efficacy of both AM1710 and LY2828360, but not reference analgesics, were absent in advillinCre/+;CB2f/f mice, which exhibited robust ddC-induced neuropathy. In ddC-treated CB2f/f mice, LY2828360 suppressed development of morphine tolerance and reversed established morphine tolerance, albeit with greater efficacy in male compared to female mice. LY2828360 failed to block or reverse morphine tolerance in advillinCre/+;CB2f/f mice. The present studies indicate that CB2 activation may alleviate HIV-associated antiretroviral neuropathy and identify a previously unreported mechanism through which CB2 activation produces antinociceptive efficacy. Our results also provide the first evidence that a CB2 agonist can reverse established morphine tolerance and demonstrate that CB2 localized to peripheral sensory neurons mediates the opioid tolerance sparing efficacy of CB2 agonists.

β-caryophyllene, a cannabinoid receptor 2 agonist, decreases the motivational salience and conditioning place preference for palatable food in female mice.

β-caryophyllene (BCP) is a cannabinoid receptor CB2 agonist plant-derived terpenoid found in different essential oil plants, including rosemary, black pepper, copaiba and cannabis. It has GRAS (generally recognized as safe) status and is approved by the FDA (Food and Drug Administration) for food use. BCP displays agonist activity on the CB2 receptor and is a potential therapeutic target in several neuropsychiatric disorders, including anxiety and drug addiction. Unlike CB1 receptors, activation of the CB2 receptors is devoid of psychotomimetic and addictive properties. In this regard, this study aimed to evaluate the effects of BCP on incentive salience ("wanting") performance and motivational properties elicited by sweetened palatable foods in female Swiss mice. After 9days of training for incentive salience performance for a sweet reward (hazelnut cream with chocolate), food-restricted mice received a systemic injection of BCP (50 and 100 mg/kg) before testing over 3days. Moreover, independent groups of female mice were tested on sweet reward-induced conditioned place preference (CPP) for 22 consecutive days. To evaluate BCP effects on the expression of seeking behaviour for sweetened food, mice received a single intraperitoneal injection of BCP (50 mg/kg) 30min before testing on the CPP task. BCP significantly decreased the incentive performance for a sweet reward compared with the control group in a CB2 receptor-dependent manner. Also, BCP suppressed the expression of sweet reward-CPP. Altogether, these preclinical data demonstrate the potential role of BCP in treating disorders associated with food addiction-like behaviour.

The mystery behind the apprehensions of the selective cannabinoid receptor type-2 agonist BZO-HEXOXIZID (MDA-19) as a drug of abuse.

MDA-19 or BZO-HEXOXIZID (N'-[(3Z)-1-(1-hexyl)-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-benzohydrazide), in a more recent nomenclature, was first synthesized in 2008 as a selective type-2 cannabinoid receptor (CB2) agonist due to its potential to treat neuropathic pain. In Brazil, this substance was identified in a series of 53 apprehensions between September 2021 and February 2022. Nevertheless, what intrigues toxicologists is that BZO-HEXOXIZID does not exert significant type-1 cannabinoid receptor (CB1) agonism-which is responsible for the well-known psychoactivity of Δ-9-tetrahydrocannabinol. Thus, the objective of this work is to report the first apprehension and identification of BZO-HEXOXIZID in Brazil and to discuss pharmacologically the possible reasons why a CB2 agonist has been incorporated to the illicit market. Suspected seized samples were sent to the Laboratory of the Scientific Police of the State of Sao Paulo. After the screening, samples were confirmed for the presence of BZO-HEXOXIZID using chromatography gas-mass spectrometry, Fourier-transform infrared spectroscopy and nuclear magnetic resonance techniques. Of the 53 samples analyzed, 25 contained only BZO-HEXOXIZID and 28 with mixtures, of which 11 with the CB1 agonist ADB-BUTINACA. Other substances were found in association such as cocaine and caffeine. BZO-HEXOXIZID was detected in a series of seized materials for the first time in Brazil. Nevertheless, there are still unanswered questions regarding the use of this selective CB2 agonist as a drug of abuse.

The Neuroprotective Effect of NEUROMIDE, a Compound Bioidentical to Commensal Bacteria Metabolites.

Simple SummaryN-Palmitoyl serinol is a commensal bacterial metabolite that acts as an endocannabinoid CB1 and GPR119 agonist. We investigated the potential of N-Palmitoyl serinol (NEUROIMIDE) to exert its neuroprotective effects via the gut–brain axis. NEUROMIDE improves cell survival and can reduce and even prevent scopolamine-induced memory impairments and passive avoidance disorder while increasing acetylcholine levels. These results suggest that the endocannabinoid system can act via the gut–brain axis.GPR119 is a novel cannabinoid receptor that is primarily expressed in the pancreas and gastrointestinal tract and has beneficial effects on glucose homeostasis exerted through the stimulation of GLP-1 secretion, as demonstrated in the rodent brain. GLP-1 also has important anti-inflammatory effects in chronic inflammatory diseases, including type 1 and 2 diabetes, asthma, psoriasis, and neurodegenerative disorders. Recently, there has been increasing interest in the effect of the gut microbiota on both the gut and the brain. However, few studies have examined how gut microbes affect brain health through the endocannabinoid system. NEUROMIDE is a compound that shares a bioidentical structure with certain commensal bacterial metabolites, acting as a CB1 and GPR119 agonist. In an in vitro system exposed to reactive oxygen species (ROS), pretreatment with NEUROMIDE resulted in a significant increase in cell viability. The ROS-exposed system also showed decreased acetylcholine and an increase in inflammatory cytokines such as IL-1β, changes that were counteracted in a dose-dependent manner in the NEUROMIDE treatment groups. To measure the effectiveness of NEUROMIDE in an in vivo system, we used scopolamine-treated mice as a neurodegenerative disease model and performed a series of passive avoidance tests to observe and quantify the cognitive impairment of the mice. Mice in the NEUROMIDE treatment group had increased latency time, thus indicating an improvement in their cognitive function. Furthermore, the NEUROMIDE treatment groups showed dose-dependent increases in acetylcholine along with decreases in TNF-α and IL-1β. These experiments demonstrate that NEUROMIDE can potentially be used for neuroprotection and the improvement of cognitive ability.

The Synthetic Cannabinoid URB447 Exerts Antitumor and Antimetastatic Effect in Melanoma and Colon Cancer.

The endocannabinoid system is widespread through the body and carries out a wide variety of functions. However, its involvement in other pathologies, such as cancer, still needs further attention. We aim to investigate the role of CB2 receptor during melanoma and colorectal cancer (CRC) aggressiveness and metastatic growth in the liver. We used the synthetic cannabinoid URB447, a known CB2 agonist and CB1 antagonist drug, and studied prometastatic ability of mouse B16 melanoma and MCA38 CRC cells, by means of proliferation, apoptosis, cell cycle, migration and matrix degradation in vitro upon URB447 treatment. We reported a dose-dependent viability decrease in both tumor types. This result is partly mediated by apoptotic cell death and cell cycle arrest in G1/G0 phase, as observed through flow cytometry. Melanoma and CRC cell migration was affected in a dose-dependent fashion as observed through scratch assay, whereas the secretion of matrix degrading proteins metalloprotease 2 (MMP2) and 9 (MMP9) in tumor cells did not significantly change. Moreover, daily treatment of tumor bearing mice with URB447 decreased the development of liver metastasis in a melanoma model in vivo. This proof of concept study points out to the synthetic cannabinoid URB447 as a potential candidate for deeper studies to confirm its potential as antitumor therapy and liver metastasis treatment for CRC and melanoma.

Structure-Activity Relationship Development Efforts towards Peripherally Selective Analogs of the Cannabinoid Receptor Partial Agonist BAY 59-3074.

Selective modulation of peripheral cannabinoid receptors (CBRs) has potential therapeutic applications in medical conditions, including obesity, diabetes, liver diseases, GI disorders and pain. While there have been considerable efforts to produce selective antagonists or full agonists of CBRs, there has been limited reports on the development of partial agonists. Partial agonists targeting peripheral CBRs may have desirable pharmacological profiles while not producing centrally mediated dissociative effects. Bayer reported that BAY 59-3074 is a CNS penetrant partial agonist of both CB1 and CB2 receptors with efficacy in rat models of neuropathic and inflammatory pain. In this report, we demonstrate our efforts to synthesize analogs that would favor peripheral selectivity, while maintaining partial agonism of CB1. Our efforts led to the identification of a novel compound, which is a partial agonist of the human CB1 (hCB1) receptor with vastly diminished brain exposure compared to BAY 59-3074.

Regulation of Expression of Cannabinoid CB2 and Serotonin 5HT1A Receptor Complexes by Cannabinoids in Animal Models of Hypoxia and in Oxygen/Glucose-Deprived Neurons.

Background: Cannabidiol (CBD) is a phytocannabinoid with potential in one of the most prevalent syndromes occurring at birth, the hypoxia of the neonate. CBD targets a variety of proteins, cannabinoid CB2 and serotonin 5HT1A receptors included. These two receptors may interact to form heteromers (CB2–5HT1A-Hets) that are also a target of CBD. Aims: We aimed to assess whether the expression and function of CB2–5HT1A-Hets is affected by CBD in animal models of hypoxia of the neonate and in glucose- and oxygen-deprived neurons. Methods: We developed a quantitation of signal transduction events in a heterologous system and in glucose/oxygen-deprived neurons. The expression of receptors was assessed by immuno-cyto and -histochemistry and, also, by using the only existing technique to visualize CB2–5HT1A-Hets fixed cultured cells and tissue sections (in situ proximity ligation PLA assay). Results: CBD and cannabigerol, which were used for comparative purposes, affected the structure of the heteromer, but in a qualitatively different way; CBD but not CBG increased the affinity of the CB2 and 5HT1A receptor–receptor interaction. Both cannabinoids regulated the effects of CB2 and 5HT1A receptor agonists. CBD was able to revert the upregulation of heteromers occurring when neurons were deprived of oxygen and glucose. CBD significantly reduced the increased expression of the CB2–5HT1A-Het in glucose/oxygen-deprived neurons. Importantly, in brain sections of a hypoxia/ischemia animal model, administration of CBD led to a significant reduction in the expression of CB2–5HT1A-Hets. Conclusions: Benefits of CBD in the hypoxia of the neonate are mediated by acting on CB2–5HT1A-Hets and by reducing the aberrant expression of the receptor–receptor complex in hypoxic-ischemic conditions. These results reinforce the potential of CBD for the therapy of the hypoxia of the neonate.

Effect of Delta-9-tetrahydrocannabinol and cannabidiol on milk proteins and lipid levels in HC11 cells.

Pregnant and lactating women have been discouraged from using cannabis by Health Canada. However, the increasing rate of cannabis use among pregnant women has presented an urgent need to investigate its physiological effects during the perinatal period. During pregnancy, the mammary gland (MG) undergoes remodeling, which involves alveolar differentiation of mammary epithelial cells (MECs), which is essential for breast milk production and secretion. Limited evidence has been reported on the impact of cannabis or its components, delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD), on MG development or MEC differentiation. In this study, we investigated the effects of THC and CBD on the differentiation of MECs by assessing changes in cellular viability, lipid accumulation, and gene and protein expression of major milk protein and lipid synthesizing markers. using the HC11 cells as a model. We hypothesized that THC and CBD will negatively impact the synthesis of milk proteins and lipids, as well as lipid markers in HC11 cells. Our results demonstrated that THC and CBD reduced cellular viability at concentrations above 30μM and 20μM, respectively. Relative to control, 10μM THC and 10μM CBD reduced mRNA levels of milk proteins (CSN2 and WAP), lipid synthesizing and glucose transport markers (GLUT 1, HK2, FASN, FABP4, PLIN2 and LPL), as well as whey acidic protein and lipid levels. In addition, co-treatment of a CB2 antagonist with THC, and a CB2 agonist with CBD, reversed the impact of THC and CBD on the mRNA levels of key markers, respectively. In conclusion, 10μM THC and CBD altered the differentiation of HC11 cells, in part via the CB2 receptor.

CB2 Agonist GW842166x Protected against 6-OHDA-Induced Anxiogenic- and Depressive-Related Behaviors in Mice.

In addition to motor dysfunction, patients with Parkinson’s disease (PD) are often affected by neuropsychiatric disorders, such as anxiety and depression. In animal models, activation of the endocannabinoid (eCB) system produces anxiolytic and antidepressant-like behavioral effects. CB2 agonists have demonstrated neuroprotective effects against neurotoxin-induced dopamine neuron loss and deficits in motor function. However, it remains unknown whether CB2 agonism ameliorates anxiogenic- and depressive-like behaviors in PD models. Here, we report that the selective CB2 agonist GW842166x exerted neuroprotective effects against 6-hydroxydopamine (6-OHDA)-induced loss of dopaminergic terminals and dopamine release in the striatum, which were blocked by the CB2 antagonist AM630. We found that 6-OHDA-treated mice exhibited anxiogenic- and depressive-like behaviors in the open-field, sucrose preference, novelty-suppressed feeding, marble burying, and forced swim tests but did not show significant changes in the elevated plus-maze and light–dark box test. GW842166x treatments ameliorated 6-OHDA-induced anxiogenic- and depressive-like behaviors, but the effects were blocked by CB2 antagonism, suggesting a CB2-dependent mechanism. These results suggest that the CB2 agonist GW842166x not only reduces 6-OHDA-induced motor function deficits but also anxiogenic- and depressive-like behaviors in 6-OHDA mouse models of PD.

Cannabinoid Receptor-1 suppresses M2 macrophage polarization in colorectal cancer by downregulating EGFR

Cannabinoid receptors, CB1 and CB2, have been implicated as emerging targets for cancer therapy. Herein, we investigated the potential regulation mechanism of CB1 and its implications in colorectal cancer. CB1 and EGFR expression were examined in colorectal cancer cell lines. The effects of CB1 agonist ACEA and its antagonist AM251 on the proliferation, migration and invasion of colorectal cancer cells and the expression of M1 and M2 macrophage markers were examined. EGFR overexpression was performed with plasmids containing EGFR gene. Tumor xenografts were constructed to explore the effects of CB1 activation on tumorigenesis. We showed that CB1 was downregulated while EGFR was upregulated in colorectal cancer cells. The activation of CB1 suppressed the proliferation, migration and invasion of colorectal cancer cells and the differentiation of M2 macrophages, while CB1 inhibition had opposite effects. Moreover, the alterations in tumorigenesis and M2 macrophage activation induced by CB1 activation were counteracted by EGFR overexpression. Besides, CB1 silencing promoted tumor cell proliferation and M2 polarization which was counteracted by EGFR knockdown. In vivo, CB1 activation also repressed tumorigenesis and M2 macrophage activation. The present study demonstrated that CB1 activation suppressed M2 macrophage through EGFR downregulation in colorectal cancers. These findings first unveiled the potential avenue of CB1 as a targeted therapy for colorectal cancer.

Cannabinoids modulate proliferation, differentiation, and migration signaling pathways in oligodendrocytes.

Cannabinoid signaling, mainly via CB1 and CB2 receptors, plays an essential role in oligodendrocyte health and functions. However, the specific molecular signals associated with the activation or blockade of CB1 and CB2 receptors in this glial cell have yet to be elucidated. Mass spectrometry-based shotgunproteomics and in silico biology tools were used to determine which signaling pathways and molecular mechanisms are triggered in a human oligodendrocytic cell line (MO3.13) by several pharmacological stimuli: the phytocannabinoid cannabidiol (CBD); CB1 and CB2 agonists ACEA, HU308, and WIN55, 212-2; CB1 and CB2 antagonists AM251 and AM630; and endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG). The modulation of cannabinoid signaling in MO3.13 was found to affect pathways linked to cell proliferation, migration, and differentiation of oligodendrocyte progenitor cells. Additionally, we found that carbohydrate and lipid metabolism, as well as mitochondrial function, were modulated by these compounds. Comparing the proteome changes and upstream regulators among treatments, the highest overlap was between the CB1 and CB2 antagonists, followed by overlaps between AEA and 2-AG. Our study opens new windows of opportunities, suggesting that cannabinoid signaling in oligodendrocytes might be relevant in the context of demyelinating and neurodegenerative diseases.Proteomics data are available at ProteomeXchange (PXD031923).

OP0162 EFFICACY AND SAFETY OF LENABASUM IN THE PHASE 3 DETERMINE TRIAL IN DERMATOMYOSITIS

OP0162 EFFICACY AND SAFETY OF LENABASUM IN THE PHASE 3 DETERMINE TRIAL IN DERMATOMYOSITIS