Cannabinoid Research Articles

Cannabinoid WIN55,212-2 reprograms monocytes and macrophages to inhibit LPS-induced inflammation.

Chronic or uncontrolled activation of myeloid cells including monocytes, macrophages and dendritic cells (DCs) is a hallmark of immune-mediated inflammatory disorders. There is an urgent need for the development of novel drugs with the capacity to impair innate immune cell overactivation under inflammatory conditions. Compelling evidence pointed out cannabinoids as potential therapeutic tools with anti-inflammatory and immunomodulatory capacity. WIN55,212-2, a non-selective synthetic cannabinoid agonist, displays protective effects in several inflammatory conditions by mechanisms partially depending on the generation of tolerogenic DCs able to induce functional regulatory T cells (Tregs). However, its immunomodulatory capacity on other myeloid cells such as monocytes and macrophages remains incompletely understood. Human monocyte-derived DCs (hmoDCs) were differentiated in the absence (conventional hmoDCs) or presence of WIN55,212-2 (WIN-hmoDCs). Cells were stimulated with LPS, cocultured with naive T lymphocytes and their cytokine production and ability to induce T cell responses were analysed by ELISA or flow cytometry. To evaluate the effect of WIN55,212-2 in macrophage polarization, human and murine macrophages were activated with LPS or LPS/IFNγ, in the presence or absence of the cannabinoid. Cytokine, costimulatory molecules and inflammasome markers were assayed. Metabolic and chromatin immunoprecipitation assays were also performed. Finally, the protective capacity of WIN55,212-2 was studied in vivo in BALB/c mice after intraperitoneal injection with LPS. We show for the first time that the differentiation of hmoDCs in the presence of WIN55,212-2 generates tolerogenic WIN-hmoDCs that are less responsive to LPS stimulation and able to prime Tregs. WIN55,212-2 also impairs the pro-inflammatory polarization of human macrophages by inhibiting cytokine production, inflammasome activation and rescuing macrophages from pyroptotic cell death. Mechanistically, WIN55,212-2 induced a metabolic and epigenetic shift in macrophages by decreasing LPS-induced mTORC1 signaling, commitment to glycolysis and active histone marks in pro-inflammatory cytokine promoters. We confirmed these data in ex vivo LPS-stimulated peritoneal macrophages (PMΦs), which were also supported by the in vivo anti-inflammatory capacity of WIN55,212-2 in a LPS-induced sepsis mouse model. Overall, we shed light into the molecular mechanisms by which cannabinoids exert anti-inflammatory properties in myeloid cells, which might well contribute to the future rational design of novel therapeutic strategies for inflammatory disorders.

Anandamide increases the physical activity of adult zebrafish (Danio rerio)

Anandamide (AEA) is a fatty acid neurotransmitter that binds to the CB1 and CB2 cannabinoid receptors in the central and peripheral nervous systems. AEA functions with the dopaminergic system and its expression has been shown to increase during activity. Cannabinoids have been used as treatments to reduce pain and discomfort, so an increase in AEA during activity may have a similar function. To determine the effects of AEA on energy expenditure, behavioral changes after exposure to AEA were observed in adult zebrafish (Danio rerio). The fish were separated into 4 groups: low dose AEA (10 µM), high dose AEA (100 µM), a purported positive control (0.5% ethanol), and a negative control (untreated). Activity was measured by counting the number of times an individual fish crossed a horizontal or vertical line drawn on the aquarium using 5-min recorded intervals (line breaks). Vertical line breaks were significantly different between the untreated group and both of the AEA-treated groups (p < 0.0001), however horizontal line breaks were not significantly different (p = 0.14). An increase in the number of vertical line crossings indicate that the fish in both AEA groups were swimming along the horizontal plane more often than the other groups, indicating an increase in physical activity. There was no difference in the activity of the ethanol-treated group compared to the untreated group. These results demonstrate that the administration of a cannabinoid agonist such as AEA may increase overall activity levels in adult zebrafish.

Cannabinoid-1 receptor activation modulates lipid mobilization and adipogenesis in the adipose tissue of dairy cows

Amplified adipose tissue (AT) lipolysis and suppressed lipogenesis characterize the periparturient period of dairy cows. The intensity of lipolysis recedes with the progression of lactation; however, when lipolysis is excessive and prolonged, disease risk is exacerbated and productivity compromised. Interventions that minimize lipolysis while maintaining adequate supply of energy and enhancing lipogenesis may improve periparturient cows' health and lactation performance. Cannabinoid-1 receptor (CB1R) activation in rodent AT enhances the lipogenic and adipogenic capacity of adipocytes, yet the effects in dairy cow AT remain unknown. Using a synthetic CB1R agonist and an antagonist, we determined the effects of CB1R stimulation on lipolysis, lipogenesis, and adipogenesis in the AT of dairy cows. Adipose tissue explants were collected from healthy, nonlactating and nongestating (NLNG; n = 6) or periparturient (n = 12) cows at 1 wk before parturition and at 2 and 3 wk postpartum (PP1 and PP2, respectively). Explants were treated with the β-adrenergic agonist isoproterenol (1 μM) in the presence of the CB1R agonist arachidonyl-2'-chloroethylamide (ACEA) ± the CB1R antagonist rimonabant (RIM). Lipolysis was quantified based on glycerol release. We found that ACEA reduced lipolysis in NLNG cows; however, it did not exhibit a direct effect on AT lipolysis in periparturient cows. Inhibition of CB1R with RIM in postpartum cow AT did not alter lipolysis. To evaluate adipogenesis and lipogenesis, preadipocytes isolated from NLNG cows' AT were induced to differentiate in the presence or absence of ACEA ± RIM for 4 and 12 d. Live cell imaging, lipid accumulation, and expressions of key adipogenic and lipogenic markers were assessed. Preadipocytes treated with ACEA had higher adipogenesis, whereas ACEA+RIM reduced it. Adipocytes treated with ACEA and RIM for 12 d exhibited enhanced lipogenesis compared with untreated cells (control). Lipid content was reduced in ACEA+RIM but not with RIM alone. Collectively, our results support that lipolysis may be reduced by CB1R stimulation in NLNG cows but not in periparturient cows. In addition, our findings demonstrate that adipogenesis and lipogenesis are enhanced by activation of CB1R in the AT of NLNG dairy cows. In summary, we provide initial evidence which supports that the sensitivity of the AT endocannabinoid system to endocannabinoids, and its ability to modulate AT lipolysis, adipogenesis, and lipogenesis, vary based on dairy cows' lactation stage.

Cannabinoid Receptor 1 Agonist ACEA and Cannabinoid Receptor 2 Agonist GW833972A Attenuates Cell-Mediated Immunity by Different Biological Mechanisms

Cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2) are components in the endocannabinoid system that play significant roles in regulating immune responses. There are many agonists for the cannabinoid receptors; however, their effects on T cell regulation have not been elucidated. In the present study, we determined the effects of the CB1 selective agonist ACEA and the CB2 selective agonist GW833972A on T cell responses. It was found that both agonists impaired anti-CD3 monoclonal antibody induced T cell proliferation. However, ACEA and GW833972A agonists down-regulated the expression of activation markers on CD4+ and CD8+ T cells and co-stimulatory molecules on B cells and monocytes in different manners. Moreover, only GW833972A suppressed the cytotoxic activities of CD8+ T cells without interfering in the cytotoxic activities of CD4+ T cells and NK cells. In addition, the CB2 agonist, but not CB1 agonist, caused the reduction of Th1 cytokine production. Our results demonstrated that the CB1 agonist ACEA and CB2 agonist GW833972A attenuated cell-mediated immunity in different mechanisms. These agonists may be able to be used as therapeutic agents for inducing T cell hypofunction in inflammatory and autoimmune diseases.

A2 ACUTE COLITIS CAUSES CHANGES TO THE EFFICACY OF CANNABINOID-1 AND MU-OPIOID RECEPTOR AGONISTS ON INHIBITING ABDOMINAL PAIN

Abstract Background Abdominal pain is a debilitating symptom in patients with inflammatory bowel disease. Previously we have shown that both cannabinoid-1 receptor (CB1R) and mu opioid receptor (MOR) agonists inhibit mechanosensitivity of colonic nociceptive nerves in healthy mice. However, it is unknown whether CBR and MOR agonists continue to have effects during colitis. Purpose To determine the effects of CBR and MOR agonists on colonic nociceptive nerves during acute colitis. Method Colitis was induced in male and female C57BL/6 mice using 2.5% dextran sodium sulfate in drinking water. Visceromotor response (VMR) to colorectal distention (CRD) (volume range 20-80 µL) was measured using telemetric transmitters. Mice were injected intraperitoneally with vehicle, ACEA, a selective CB1R agonist and/or morphine, a MOR agonist, 30-minutes prior to distention. Extracellular afferent nerve recordings were obtained from ex vivo flat sheet preparations of mouse distal colon. Mechanosensitivity of single afferent axons was assessed via mechanical probing of the colon with a 1g von Frey hair before and after superfusion of ACEA and/or DAMGO (MOR agonist). Data were analyzed using a one- or two-way ANOVA with Bonferroni test. N denotes number of mice; n denotes number of single afferent axons. Result(s) ACEA (3 mg/kg), a dose that significantly inhibited VMR in healthy mice, did not inhibit VMR in mice with colitis (p=0.55, N=6). At a dose that previously had no effect in healthy controls, morphine (0.3 mg/kg) significantly inhibited VMR to CRD, when compared to vehicle (34% reduction, p&amp;lt;0.01, N = 8). A combination of a sub-analgesic dose of ACEA (0.3 mg/kg) with morphine (0.3 mg/kg) significantly reduced VMR (p&amp;lt;0.01, N=5); at 60 µL and 80 µL distention there was a 44% and 49% reduction, respectively (p&amp;lt;0.05 for both). Interestingly, the effect of the combination of ACEA and morphine was larger than that of morphine alone (0.3 mg/kg), but this did not reach statistical significance (-62% vs. -34%; p=0.06, N=5-8). In extracellular afferent nerve recordings, compared to the previous findings in healthy mice, a higher concentration of ACEA (10 µM) was required to inhibit mechanosensitivity (15.2 vs. 11.6 Hz; p&amp;lt;0.05, n=11, N=6) whereas 100nM (p&amp;gt;0.99, n=7, N=5) and 1µM (p=0.25, n=10, N=6) had no effect. DAMGO (1 nM), which previously had no effect in healthy controls, had a tendency to reduce colonic mechanosensitivity, but this did not reach statistical significance (p=0.12, n=7 units, N=5). Interestingly, a combination of sub-analgesic concentrations of ACEA (100 nM) and DAMGO (1 nM) significantly reduced colonic mechanosensitivity (18.4 vs. 12.0 Hz; p&amp;lt;0.05, n=7, N=5). Conclusion(s) At doses that previously inhibited nociception in healthy mice, CB1R agonists may have lost their analgesic effect during acute colitis. Conversely, less of MOR agonists may be needed to achieve analgesia. Interestingly, sub-analgesic doses of CB1R agonists potentiate the analgesic effect of the MOR agonist during colitis. Please acknowledge all funding agencies by checking the applicable boxes below NRC Disclosure of Interest None Declared

The protective effect of cannabinoids against colorectal cancer cachexia through modulation of inflammation and immune responses

Cancer cachexia is a multifactorial disorder characterized by weight loss and muscle wasting, and there are currently no FDA-approved medications. In the present study, upregulation of six cytokines was observed in serum samples from patients with colorectal cancer (CRC) and in mouse models. A negative correlation between the levels of the six cytokines and body mass index in CRC patients was seen. Gene Ontology analysis revealed that these cytokines were involved in regulating T cell proliferation. The infiltration of CD8+ T cells was found to be associated with muscle atrophy in mice with CRC. Adoptive transfer of CD8+ T cells isolated from CRC mice resulted in muscle wasting in recipients. The Genotype-Tissue Expression database showed that negative correlations between the expression of cachexia markers and cannabinoid receptor 2 (CB2) in human skeletal muscle tissues. Pharmacological treatment with Δ9-tetrahydrocannabinol (Δ9-THC), a selective CB2 agonist or overexpression of CB2 attenuated CRC-associated muscle atrophy. In contrast, knockout of CB2 with a CRISPR/Cas9-based strategy or depletion of CD8+ T cells in CRC mice abolished the Δ9-THC-mediated effects. This study demonstrates that cannabinoids ameliorate CD8+ T cell infiltration in CRC-associated skeletal muscle atrophy via a CB2-mediated pathway. Serum levels of the six-cytokine signature might serve as a potential biomarker to detect the therapeutic effects of cannabinoids in CRC-associated cachexia.

Pharmacologic antagonism of CB1 receptors improves electrophysiological alterations in Purkinje cells exposed to 3-AP

IntroductionAlthough ataxia is associated with cerebellar dysfunction, little is known about the effects of 3-AP exposure on Purkinje cell electrophysiological properties. Here, we evaluated these parameters in cerebellar vermis brain slices.MethodsPurkinje cells were exposed to artificial cerebrospinal fluid (aCSF) (control) or to 1 mM 3-acetylpyridine (3-AP) in the recording chamber. The effects of a cannabinoid agonist (WIN; 7.5 nmol) and a cannabinoid antagonist (AM; 20 nmol) were evaluated under both conditions.ResultsExposure to 3-AP induced dramatic changes in cellular excitability that likely would affect Purkinje cell output. In whole-cell current clamp recordings, 3-AP-exposed Purkinje cells demonstrated a significantly higher frequency of action potentials, a larger afterhyperpolarization (AHP), and a larger rebound of action potentials. In addition, 3-AP caused a significant decrease in the interspike interval (ISI), half-width, and first spike latency. Remarkably, the action potential frequency, AHP amplitude, rebound, ISI, action potential halfwidth, and first spike latency were no longer different from controls in 3-AP cells treated with AM. Sag percentage, on the other hand, showed no significant difference under any treatment condition, indicating that cannabinoids' actions on 3-AP-mediated Purkinje cell changes may not include effects on neuronal excitability through changes of Ih.ConclusionsThese data show that cannabinoid antagonists reduce the excitability of Purkinje cells following exposure to 3-AP and suggest their potential as therapeutics in cerebellar dysfunctions.

Abstract P4-04-02: Evaluation of Dronabinol to Decrease Opioid Use for Cancer- Induced Bone Pain

Abstract Purpose Bone metastases (BM) from breast cancer cause significant complications including pain, hypercalcemia, spinal cord compression, and pathologic fractures, collectively referred to as skeletal related events (SREs). Of all SREs, bone pain impacts the quality of life (QOL) most significantly. Cancer- induced bone pain (CIBP) is difficult to treat with limited treatment options and requires a multimodal approach. Management of CIBP is primarily by opioids which have notable side effects like sedation, constipation, and concern for addiction. In addition, pre-clinical evidence suggests that opioids accelerate bone loss and increase risk of fractures. There is an unmet need for novel analgesic therapy interventions to optimize QOL for patients with BM from metastatic breast cancer. Preclinical studies have shown that the endogenous cannabinoid (CB) system is involved in pain modulation, bone regulation, immunity, and restraint of cancer pathogenesis. CB2 receptor activation has been shown to inhibit proinflammatory cytokines/chemokines in pre-clinical models. Treatment with selective CB2 agonist in mice with BM led to significant antinociception, decreased cancer-induced bone degradation, and reduced side effects of morphine. In addition to alleviating pain, CB2 agonists were shown to enhance bone growth/strength in these mice. Based on this pre-clinical data, we hypothesized that the addition of a CB2 agonist will improve pain symptoms and decrease opioid requirement in patients with bone metastases from breast cancer. We proceeded to conduct a pilot study by repurposing a clinically approved CB2/CB1 agonist, dronabinol. Methods We conducted a prospective, single site study among patients with BM from breast cancer at our center (NCT03661892). Patients had to have been on opioids for CIBP for at least 4 weeks and not using marijuana or CBD products. Patients were treated with 10mg dronabinol BID for 8 weeks. Our primary objective was to determine the proportion who decrease their opioid use by ≥ 20%. The null hypothesis value was 5% of women would have a 20% decrease. With 14 participants, we could detect an increase from 5% to 29% (n=4) with 80% statistical power using a one-sided alpha level of 0.05. Participants completed Brief Pain Inventory and the European Organization for Research and Treatment of Cancer quality of life questionnaires pre and post treatment. Results Twenty participants consented with 14 patients completing the study and evaluable for primary analysis. No patients received any palliative radiation therapy or other therapies for bone pain within 3 months prior to enrollment to this study. Four patients decreased their opioid use by ≥ 20% meeting the primary objective. Patients reported significant improvement in pain severity, interference scores, quality of life and insomnia based on the questionnaires. There were no grade 4 side effects and only 1 patient had grade 3 adverse event (dizziness) related to study drug. Of the 14 patients who completed the study, 9 desired to continue dronabinol therapy after completion. Conclusion Our pilot study shows that the addition of dronabinol resulted in decreased opioid requirements for CIBP in patients with metastatic breast cancer. Patient-reported outcomes also demonstrated improved pain and QOL with the addition of dronabinol. While we did not see any significant AEs tolerability may be of concern due to CB1 psychoactive effects. Our results are promising and warrant further investigation into evaluating CB2 agonists for improved pain control from CIBP and to decrease opioid use. Citation Format: Jennifer Segar, Kiah Farr, Mary Junak, Denise Roe, Sima Ehsani, Sao Jiralerspong, Ibrahim Mohab, Todd Vanderah, Pavani Chalasani. Evaluation of Dronabinol to Decrease Opioid Use for Cancer- Induced Bone Pain [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-04-02.

Phytocannabinoids in Triple Negative Breast Cancer Treatment: Current Knowledge and Future Insights.

Triple negative breast cancer (TNBC) represents an aggressive subtype of breast cancer, which is deficient in estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression. Thus, TNBC cells are unable to respond to the conventional hormonal therapies, making chemotherapy the only therapeutic choice. Patients with TNBC develop metastasis and recurrence over time and have reduced survival compared to patients with other subtypes of breast cancer. Therefore, there is a need for innovative therapies. Data emerged from pre-clinical studies, highlighted various antitumor activities of plant-derived Cannabis sativa and synthetic cannabinoids (CBs), including delta-9-tetrahydrocannabinol (THC) and non-psychoactive cannabidiol (CBD). On the contrary, some studies indicated that CBs might also promote tumor progression. At present, clinical studies on the effects of CBs from Cannabis sativa in cancer patients are few. In the present study, we reviewed known and possible interactions between cannabinoids and TNBC therapies.

Exogenous Cannabinoids Impair Effort-Related Decision-Making via Affecting Neural Synchronization between the Anterior Cingulate Cortex and Nucleus Accumbens.

Humans and animals frequently make an endeavor-based choice based on assessing reinforcement value and response costs. The cortical-limbic-striatal pathway mediates endeavor-based choice behavior, including the nucleus accumbens (NAc) and the anterior cingulate cortex (ACC). Furthermore, cannabinoid agonists demonstratively impairs decision-making processes. In this study, neural synchronization and functional connectivity between the NAc and ACC while endeavor-related decision-making and reaching reward were evaluated. The effect of exogenous cannabinoids on this synchronization was then assessed. A T-maze decision-making task with a differential expense (low vs. high endeavor) and remuneration (low vs. high remuneration) was performed and local field potentials (LFP) from the ACC and NAc were registered simultaneously. Results showed functional connectivity during endeavor-related decision-making while the animals chose the high endeavor/high remuneration in both regions' delta/beta (1-4 and 13-30 Hertz) frequency bands. Furthermore, functional connectivity existed between both areas in delta/theta (1-4 and 4-12) frequencies while reaching a remuneration. However, neural simultaneity was not observed while the animals received cannabinoid agonists, making a decision and reaching remuneration. The obtained results demonstrated that functional connectivity and neural simultaneity between the NAc and ACC in delta/beta and delta/theta frequencies have a role in endeavor-related decision-making and reaching remuneration, respectively. The effect of exogenous cannabinoids on decision-making impairment is relevant to changes in the ACC and NAC brain wave frequencies.

Synthetic cannabinoid receptor agonists are monoamine oxidase-A selective inhibitors.

Synthetic cannabinoid receptor agonists (SCRAs) are one of the fastest growing classes of recreational drugs. Despite their growth in use, their vast chemical diversity and rapidly changing landscape of structures make understanding their effects challenging. In particular, the side effects for SCRA use are extremely diverse, but notably include severe outcomes such as cardiac arrest. These side effects appear at odds with the main putative mode of action, as full agonists of cannabinoid receptors. We have hypothesized that SCRAs may act as MAO inhibitors, owing to their structural similarity to known monoamine oxidase inhibitors (MAOI's) as well as matching clinical outcomes (hypertensive crisis) of 'monoaminergic toxicity' for users of MAOIs and some SCRA use. We have studied the potential for SCRA-mediated inhibition of MAO-A and MAO-B via a range of SCRAs used commonly in the UK, as well as structural analogues to prove the atomistic determinants of inhibition. By combining in silico and experimental kinetic studies we demonstrate that SCRAs are MAO-A-specific inhibitors and their affinity can vary significantly between SCRAs, most notably affected by the nature of the SCRA 'head' group. Our data allow us to posit a putative mechanism of inhibition. Crucially our data demonstrate that SCRA activity is not limited to just cannabinoid receptor agonism and that alternative interactions might account for some of the diversity of the observed side effects and that these effects can be SCRA-specific.

Sex- and age-specific respiratory alterations induced by prenatal exposure to the cannabinoid receptor agonist WIN 55,212-2 in rats.

Cannabis legalization has risen in many countries, and its use during pregnancy has increased. The endocannabinoid system is present in the CNS at early stages of embryonic development, and regulates functional brain maturation including areas responsible for respiratory control, data on the influence of external cannabinoids on the development of the respiratory system and possible consequences during postnatal life are limited. We evaluated the effects of prenatal exposure to synthetic cannabinoid (WIN 55,212-2 [WIN], 0.5 mg·kg-1 ·day-1 ) on the respiratory control system in neonatal (P0, P6-7 and P12-13) and juvenile (P27-28) male and female rats. WIN administration to pregnant rats interfered sex-specifically with breathing regulation of offspring, promoting a greater sensitivity to CO2 at all ages in males (except P6-7) and in juvenile females. An altered hypoxic chemoreflex was observed in P0 (hyperventilation) and P6-7 (hypoventilation) males, which was absent in females. Along with breathing alterations, brainstem analysis showed an increase in the number of catecholaminergic neurons and cannabinoid receptor type 1 (CB1 ) and changes in tissue respiration in the early males. A reduction in pulmonary compliance was observed in juvenile male rats. Preexposure to WIN enhanced spontaneous apnoea and reduced the number of serotoninergic (5-HT) neurons in the raphe magnus nucleus of P0 females. These data demonstrate that excess stimulation of the endocannabinoid system during gestation has prolonged and sex-specific consequences for the respiratory control system.

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.

Characterization of the Antitumor Potential of Extracts of Cannabis sativa Strains with High CBD Content in Human Neuroblastoma.

Cannabis has been used for decades as a palliative therapy in the treatment of cancer. This is because of its beneficial effects on the pain and nausea that patients can experience as a result of chemo/radiotherapy. Tetrahydrocannabinol and cannabidiol are the main compounds present in Cannabis sativa, and both exert their actions through a receptor-mediated mechanism and through a non-receptor-mediated mechanism, which modulates the formation of reactive oxygen species. These oxidative stress conditions might trigger lipidic changes, which would compromise cell membrane stability and viability. In this sense, numerous pieces of evidence describe a potential antitumor effect of cannabinoid compounds in different types of cancer, although controversial results limit their implementation. In order to further investigate the possible mechanism involved in the antitumoral effects of cannabinoids, three extracts isolated from Cannabis sativa strains with high cannabidiol content were analyzed. Cell mortality, cytochrome c oxidase activity and the lipid composition of SH-SY5Y cells were determined in the absence and presence of specific cannabinoid ligands, with and without antioxidant pre-treatment. The cell mortality induced by the extracts in this study appeared to be related to the inhibition of the cytochrome c oxidase activity and to the THC concentration. This effect on cell viability was similar to that observed with the cannabinoid agonist WIN55,212-2. The effect was partially blocked by the selective CB1 antagonist AM281, and the antioxidant α-tocopherol. Moreover, certain membrane lipids were affected by the extracts, which demonstrated the importance of oxidative stress in the potential antitumoral effects of cannabinoids.

Dual Cannabinoid and Orexin Regulation of Anhedonic Behaviour Caused by Prolonged Restraint Stress.

The endocannabinoid and orexin systems share many biological functions, including wakefulness, stress response, reward processing, and mood. While these systems work against one another with respect to arousal, chronic stress-induced downregulation of both systems often leads to anhedonia or the inability to experience pleasure from natural rewards. In the current study, a 24 h restraint stress test (24 h RST) reduced sucrose preference in adult male and female C57BL/6 mice. Prior to the stressor, subsets of mice were intraperitoneally administered cannabinoid and orexin receptor agonists, antagonists, and combinations of these drugs. Restraint mice that received the cannabinoid receptor type 1 (CB1R) antagonist SR141716A, orexin receptor type 2 (OX2R) agonist YNT-185, and the combination of SR141716A and YNT-185, exhibited less anhedonia compared to vehicle/control mice. Thus, the 24 h RST likely decreased orexin signaling, which was then restored by YNT-185. Receptor colocalization analysis throughout mesocorticolimbic brain regions revealed increased CB1R-OX1R colocalization from SR141716A and YNT-185 treatments. Although a previous study from our group showed additive cataleptic effects between CP55,940 and the dual orexin receptor antagonist (TCS-1102), the opposite combination of pharmacological agents proved additive for sucrose preference. Taken together, these results reveal more of the complex interactions between the endocannabinoid and orexin systems.

Neuroimaging revealed long-lasting glucose metabolism changes to morphine withdrawal in rats pretreated with the cannabinoid agonist CP-55,940 during periadolescence

This study evaluates the long-term effects of a six and 14-week morphine withdrawal in rats pretreated with a cannabinoid agonist (CP-55,940, CP) during periadolescence. Wistar rats (33 males; 32 females) were treated with CP or its vehicle (VH) from postnatal day (PND) 28-38. At PND100, rats performed morphine self-administration (MSA, 15d/12h/session). Eight groups were defined according to pretreatment (CP), treatment (morphine), and sex. Three [18F]FDG-PET brain images were acquired: after MSA, and after six and 14 weeks of withdrawal. PET data were analyzed with SPM12. Endocannabinoid (EC) markers were evaluated in frozen brain tissue at endpoint. Females showed a higher mean number of self-injections than males. A main Sex effect on global brain metabolism was found. FDG uptake in males was discrete, whereas females showed greater brain metabolism changes mainly in areas of the limbic system after morphine treatment. Moreover, the morphine-induced metabolic pattern in females was exacerbated when CP was previously present. In addition, the CP-Saline male group showed reduced CB1R, MAGL expression, and NAPE/FAAH ratio compared to the control group, and morphine was able to reverse CB1R and MAGL expression almost to control levels. In conclusion, females showed greater and longer-lasting metabolic changes after morphine withdrawal than males, indicating a higher vulnerability and a different sensitivity to morphine in subjects pre-exposed to CP. In contrast, males primarily showed changes in EC markers. Together, our results suggest that CP pre-exposure contributes to the modulation of brain metabolism and EC systems in a sex-dependent manner.

Understanding synthetic drug analogues among the homeless population from the perspectives of the public: thematic analysis of Twitter data

ABSTRACT Objectives The last few years have seen the rapid emergence of synthetic drug derivatives known as new psychoactive substances (NPS) among the homeless population. Previous research has focused on understanding the issues from homeless or stakeholder perspectives but not the public’s. The purpose of this research is to understand the perspectives of the public and service providers regarding NPS use among the homeless population using thematic analysis of Twitter data. Method Tweets from Twitter were extracted and imported into NVivo 12 for thematic analysis. Tweets were included if they were written and related to NPS among the homeless. Excluded tweets were those related to interventional studies or with personalized information. Results The findings showed that two NPS were discussed on Twitter being novel synthetic opioids (NSOs) and synthetic cannabinoid receptor agonists (SCRAs). Thematic analysis of Twitter discussions revealed that individuals held a negative attitude toward the government and the services provided to the homeless NPS users, for both NSOs and SCRAs. Conclusion In summary, NSOs were more frequently discussed on the Twitter platform than SCRAs. NSOs were consumed by homeless population in the United States and Canada, and SCRA by the homeless in the United Kingdom.

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.

Differential regulation of Cav 3.2 and Cav 2.2 calcium channels by CB1 receptors and cannabidiol.

Cannabinoids are a promising therapeutic avenue for chronic pain. However, clinical trials often fail to report analgesic efficacy of cannabinoids. Inhibition of voltage gate calcium (Cav ) channels is one mechanism through which cannabinoids may produce analgesia. We hypothesized that cannabinoids and cannabinoid receptor agonists target different types of Cav channels through distinct mechanisms. Electrophysiological recordings from tsA-201 cells expressing either Cav 3.2 or Cav 2.2 were used to assess inhibition by HU-210 or cannabidiol (CBD) in the absence and presence of the CB1 receptor. Homology modelling assessed potential interaction sites for CBD in both Cav 2.2 and Cav 3.2. Analgesic effects of CBD were assessed in mouse models of inflammatory and neuropathic pain. HU-210 (1μM) inhibited Cav 2.2 function in the presence of CB1 receptor but had no effect on Cav 3.2 regardless of co-expression of CB1 receptor. By contrast, CBD (3μM) produced no inhibition of Cav 2.2 and instead inhibited Cav 3.2 independently of CB1 receptors. Homology modelling supported these findings, indicating that CBD binds to and occludes the pore of Cav 3.2, but not Cav 2.2. Intrathecal CBD alleviated thermal and mechanical hypersensitivity in both male and female mice, and this effect was absent in Cav 3.2 null mice. Our findings reveal differential modulation of Cav 2.2 and Cav 3.2 channels by CB1 receptors and CBD. This advances our understanding of how different cannabinoids produce analgesia through action at different voltage-gated calcium channels and could influence the development of novel cannabinoid-based therapeutics for treatment of chronic pain.

The piperazine analogue para-fluorophenylpiperazine alters timing of the physiological effects of the synthetic cannabinoid receptor agonist AMB-FUBINACA, without changing its discriminative stimulus, signalling effects, or metabolism

AMB-FUBINACA is a synthetic cannabinoid receptor agonist (SCRA), which has been associated with substantial abuse and health harm since 2016 in many countries including New Zealand. A characteristic of AMB-FUBINACA use in New Zealand has included the observation that forensic samples (from autopsies) and drugs seized by police have often been found to contain para-fluorophenylpiperazine (pFPP), a relatively little-characterised piperazine analogue that has been suggested to act through 5HT1a serotonin receptors. In the current study, we aimed to characterise the interactions of these two agents in rat physiological endpoints using plethysmography and telemetry, and to examine whether pFPP altered the subjective effects of AMB-FUBINACA in mice trained to differentiate a cannabinoid (THC) from vehicle. Though pFPP did not alter the ability of AMB-FUBINACA to substitute for THC, it did appear to abate some of the physiological effects of AMB-FUBINACA in rats by delaying the onset of AMB-FUBINACA-mediated hypothermia and shortening duration of bradycardia. In HEK cells stably expressing the CB1 cannabinoid receptor, 5HT1a, or both CB1 and 5HT1a, cAMP signalling was recorded using a BRET biosensor (CAMYEL) to assess possible direct receptor interactions. Although low potency pFPP agonism at 5HT1a was confirmed, little evidence for signalling interactions was detected in these assays: additive or synergistic effects on potency or efficacy were not detected between pFPP and AMB-FUBINACA-mediated cAMP inhibition. Experiments utilising higher potency, classical 5HT1a ligands (agonist 8OH-DPAT and antagonist WAY100635) also failed to reveal evidence for mutual CB1/5HT1a interactions or cross-antagonism. Finally, the ability of pFPP to alter the metabolism of AMB-FUBINACA in rat and human liver microsomes into its primary carboxylic acid metabolite via carboxylesterase-1 was assessed by HPLC; no inhibition was detected. Overall, the effects we have observed do not suggest that increased harm/toxicity would result from the combination of pFPP and AMB-FUBINACA.