Selective CB2 Agonists Research Articles

Microglial cannabinoid receptor type II stimulation improves cognitive impairment and neuroinflammation in Alzheimer's disease mice by controlling astrocyte activation.

Alzheimer's disease (AD) is the most common form of dementia and is characterized by the accumulation of amyloid β (Aβ) and phosphorylated tau. Neuroinflammation, mainly mediated by glial activation, plays an important role in AD progression. Although there is growing evidence for the anti-neuroinflammatory and neuroprotective effects of the cannabinoid system modulation, the detailed mechanism remains unclear. To address these issues, we analyzed the expression levels of cannabinoid receptor type II (Cnr2/Cb2) in AppNL-G-F/NL-G-F mice and human AD precuneus, which is vulnerable to amyloid deposition in AD, and the effects of JWH 133, a selective CB2 agonist, on neuroinflammation in primary glial cells and neuroinflammation and cognitive impairment in AppNL-G-F/NL-G-F mice. The levels of Cnr2/Cb2 were upregulated in microglia isolated from the cerebral cortex of AppNL-G-F/NL-G-F mice. CNR2 expression was also increased in RNAs derived from human precuneus with advanced AD pathology. Chronic oral administration of JWH 133 significantly ameliorated the cognitive impairment of AppNL-G-F/NL-G-F mice without neuropsychiatric side effects. Microglia and astrocyte mRNAs were directly isolated from the mouse cerebral cortex by magnetic-activated cell sorting, and the gene expression was determined by quantitative PCR. JWH 133 administration significantly decreased reactive astrocyte markers and microglial C1q, an inducer for the reactive astrocytes in AppNL-G-F/NL-G-F mice. In addition, JWH133 administration inhibited the expression of p-STAT3 (signal transducer and activator of transcription 3) in astrocytes in AppNL-G-F/NL-G-F mice. Furthermore, JWH 133 administration suppressed dystrophic presynaptic terminals surrounding amyloid plaques. In conclusion, stimulation of microglial CB2 ameliorates cognitive dysfunction in AppNL-G-F/NL-G-F mice by controlling astrocyte activation and inducing beneficial neuroinflammation, and our study has implications that CB2 may represent an attractive therapeutic target for the treatment of AD and perhaps other neurodegenerative diseases involving neuroinflammation.

Cannabinoid Receptor type 2 agonist GP1a attenuates macrophage activation induced by M. bovis-BCG by inhibiting NF-ĸB signaling.

Tuberculosis (TB) is one of the leading causes of death worldwide and a major public health problem. Immune evasion mechanisms and antibiotic resistance highlight the need to better understand this disease and explore alternative treatment approaches. Mycobacterial infection modulates the macrophage response and metabolism to persist and proliferate inside the cell. Cannabinoid receptor type 2 (CB2) is expressed mainly in leukocytes and modulates the course of inflammatory diseases. Therefore, our study aimed to evaluate the effects of the CB2-selective agonist GP1a on irradiated M. bovis-BCG (iBCG)-induced J774A.1 macrophage activation. We observed increased expression of CB2 in macrophages after iBCG stimulation. The pretreatment with CB2-agonists, GP1a, JWH-133, and GW-833972A (10 µM), reduced iBCG-induced TNF-α and IL-6 release by these cells. Moreover, the CB2-antagonist AM630 (200nM) treatment confirmed the activity of GP1a on CB2 by scale down its effect on cytokine production. GP1a pretreatment (10 µM) also inhibited the iBCG-induced production of inflammatory mediators as prostaglandin (PG)E2 and nitric oxide (NO) by macrophages. Additionally, GP1a pretreatment also reduced the transcription of proinflammatory genes (inos, il1b, cox2) and genes related to lipid metabolism (dgat1, acat1, plin2, atgl, cd36). Indeed, lipid droplet accumulation was reduced by GP1a treatment which was partially blockade by AM630 pretreatment. Finally, GP1a pretreatment reduced the activation of the NF-κB signaling pathway. In conclusion, the activation of CB2 by GP1a modulated the macrophage response to iBCG by reducing inflammatory mediator levels and metabolic reprogramming.

Long-Term Safety and Efficacy of Lenabasum, a Cannabinoid Receptor Type 2 Agonist, in Patients with Dermatomyositis with Refractory Skin Disease: Follow-Up Data from a 3-Year Open-Label Extension Study

BackgroundDermatomyositis (DM) is a rare autoimmune condition involving skin manifestations often resistant to standard treatments like immunosuppressants and antimalarials. Biopsies show elevated inflammatory cells such as CD4+ T cells, dendritic cells, and cytokines. Lenabasum, a selective CB2 agonist, has demonstrated significant benefits in treating autoimmune skin diseases. ObjectivesThis study utilizes data from the open-label extension (OLE) phase of the lenabasum phase 2 trial and additional post-OLE follow-up data. Key aims include evaluating the drug's long-term effectiveness and assessing disease manifestation recurrence. MethodsThe phase 2 lenabasum trial enrolled patients with treatment-resistant, skin-predominant DM. The OLE consisted of a 3-year period during which 20 patients were on the drug for the entire duration, with assessments every 8 weeks to evaluate drug safety and efficacy. Subsequently, a follow-up retrospective chart review was performed on patients who completed the OLE, as well as on control subjects with DM who did not participate in the lenabasum trial. ResultsBy week 68, patients exhibited reductions in CDASI activity score (-21.8), Patient Skin Activity VAS (-3.0), and Skindex-29 (-28.0) from OLE baseline. Post-OLE, 58.3% maintained stable disease, significantly higher than controls (p=0.035), with 41.7% not experiencing flares compared to 91.6% of controls. Additionally, 50% of patients reported sustained pruritus improvement. ConclusionsData from OLE and subsequent follow-up periods demonstrate lenabasum's efficacy in maintaining disease stability, reducing flares, and improving DM symptoms, suggesting it is a promising option for treatment-resistant skin-predominant DM patients.

Involvement of CB1 and CB2 receptors in neuroprotective effects of cannabinoids in experimental TDP-43 related frontotemporal dementia using male mice

BackgroundThe elevation of endocannabinoid levels through inhibiting their degradation afforded neuroprotection in CaMKIIα-TDP-43 mice, a conditional transgenic model of frontotemporal dementia. However, which cannabinoid receptors are mediating these benefits is still pending to be elucidated. MethodsWe have investigated the involvement of the CB1 and the CB2 receptor using chronic treatments with selective ligands in CaMKIIα-TDP-43 mice, analysis of their cognitive deterioration with the Novel Object Recognition test, and immunostaining for neuronal and glial markers in two areas of interest in frontotemporal dementia. ResultsOur results confirmed the therapeutic value of activating either the CB1 or the CB2 receptor, with improvements in the animal performance in the Novel Object Recognition test, preservation of pyramidal neurons, in particular in the medial prefrontal cortex, and attenuation of glial reactivity, in particular in the hippocampus. In addition, the activation of both CB1 and CB2 receptors reduced the elevated levels of TDP-43 in the medial prefrontal cortex of CaMKIIα-TDP-43 mice, an effect exerted by mechanisms that are currently under investigation. ConclusionsThese data reinforce the notion that the activation of CB1 and CB2 receptors may represent a promising therapy against TDP-43-induced neuropathology in frontotemporal dementia. Future studies will have to confirm these benefits, in particular with one of the selective CB2 agonists used here, which has been thoroughly characterized for clinical development.

In Rats, the Chemotherapeutic Drug Vincristine-Induced Neuropathic Nociception is Suppressed When Cannabinoid CB1 and CB2 Receptors are Activated

Rats were used to test the potential of cannabis to reduce mechanical hypersensitivity, or mechanical allodynia, which was brought on by vincristine chemotherapy. After that, action sites were located. Method of experimentation: After ten daily injections of vincristine, mechanical hypersensitivity developed in comparison to those that received saline at the same periods. The effects on chemotherapy-induced neuropathy were assessed for the CB1/CB2 receptor agonist WIN55, 212-2, the receptor-inactive enantiomer WIN55, 212-3, the CB2-selective agonist (R,S)-AM1241, the opiate agonist morphine, and vehicle. To determine the locations of action, WIN55, 212-2 was injected either locally in the hind paw or intrathecally (i.t.). By employing competitive antagonists for either CB1 (SR141716) or CB2 receptors (SR144528), pharmacological selectivity was demonstrated. Vincristine-evoked mechanical allodynia was decreased when WIN55, 212-2, but not WIN55, 212-3, were administered systemically. A change in the dose-response curve to the left was noticed after WIN55, 212-2 in comparison to morphine therapy. Antibodies of CB1 (SR141716) and CB2 (SR144528) inhibited WIN55, 212-2’s anti-allodynic actions. Via a CB2 mechanism, (R,S)-AM1241 reduced c-induced mechanical hypersensitivity. Without causing catalepsy, both cannabinoid agonists reduced the mechanical hypersensitivity brought on by vincristine. Cannabis-induced neuropathy may be modulated by cannabinoids at spinal sites of action. When delivered intraperitoneally, WIN55, 212-2 but not WIN55,212-3 inhibited vincristine-evoked mechanical hypersensitivity at dosages that were inert after local hindpaw injection. Spinal co-administration of CB1 and CB2 antagonists inhibited WIN55,212-2’s anti-allodynic effects.By activating CB1 and CB2 receptors, cannabinoids inhibit the maintenance of vincristine-induced mechanical allodynia. The spinal cord is involved in the mediation of these anti-allodynic actions, at least partially.

Discovery of Potent and Selective CB2 Agonists Utilizing a Function-Based Computational Screening Protocol.

Nowadays, the identification of agonists and antagonists represents a great challenge in computer-aided drug design. In this work, we developed a computational protocol enabling us to design/screen novel chemicals that are likely to serve as selective CB2 agonists. The principle of this protocol is that by calculating the ligand-residue interaction profile (LRIP) of a ligand binding to a specific target, the agonist-antagonist function of a compound is then able to be determined after statistical analysis and free energy calculations. This computational protocol was successfully applied in CB2 agonist development starting from a lead compound, and a success rate of 70% was achieved. The functions of the synthesized derivatives were determined by in vitro functional assays. Moreover, the identified potent CB2 agonists and antagonists strongly interact with the key residues identified using the already known potent CB2 agonists/antagonists. The analysis of the interaction profile of compound 6, a potent agonist, showed strong interactions with F2.61, I186, and F2.64, while compound 39, a potent antagonist, showed strong interactions with L17, W6.48, V6.51, and C7.42. Still, some residues including V3.32, T3.33, S7.39, F183, W5.43, and I3.29 are hotspots for both CB2 agonists and antagonists. More significantly, we identified three hotspot residues in the loop, including I186 for agonists, L17 for antagonists, and F183 for both. These hotspot residues are typically not considered in CB1/CB2 rational ligand design. In conclusion, LRIP is a useful concept in rationally designing a compound to possess a certain function.

Wnt signaling is involved in crotalphine-induced analgesia in a rat model of neuropathic pain

The aberrant activation of Wnt/β-catenin and atypical Wnt/Ryk signaling pathways in the spinal cord is critical for the development and maintenance of neuropathic pain. Crotalphine is a structural analog to a peptide first identified in Crotalus durissus terrificus snake venom, which induces antinociception by activating kappa-opioid and CB2 cannabinoid receptors. Consistent with previous data, we showed that the protein levels of the canonical Wnt/β-catenin and the atypical Wnt/Ryk signaling pathways are increased in neuropathic rats. Importantly, the administration of crotalphine downregulates these protein levels, including its downstream cascades, such as TCF4 from the canonical pathway and NR2B glutamatergic receptor and Ca2+-dependent signals, via the Ryk receptor. The CB2 receptor antagonist, AM630, abolished the crotalphine-induced atypical Wnt/Ryk signaling pathway activation. However, the selective CB2 agonist affects both canonical and non-canonical Wnt signaling in the spinal cord. Next, we showed that crotalphine blocked hypersensitivity and significantly decreased the concentration of IL-1ɑ, IL-1β, IL-6, IL-10, IL-18, TNF-ɑ, MIP-1ɑ and MIP-2 induced by intrathecal injection of exogenous Wnt-3a agonist. Taken together, our findings show that crotalphine induces analgesia in a neuropathic pain model by down-regulating the canonical Wnt/β-catenin and the atypical Wnt/Ryk signaling pathways and, consequently controlling neuroinflammation. This effect is, at least in part, mediated by CB2 receptor activation. These results open a perspective for new approaches that can be used to target Wnt signaling in the context of chronic pain. PerspectiveOur work identified that crotalphine-induced activation of CB2 receptors plays a critical role in the impairment of Wnt signaling during neuropathic pain. This work suggests that drugs with opioid/cannabinoid activity may be a useful strategy to target Wnt signaling in the context of chronic pain.

Anandamide reduces the migration of lymphocytes to the intestine by CB2 activation and reduces TNF-α in the target organs, protecting mice from graft-versus-host disease

Graft-versus-host disease (GVHD) is a serious inflammatory illness that often occurs as a secondary complication of bone marrow transplantation. Current therapies have limited effectiveness and fail to achieve a balance between inflammation and the graft-versus-tumor effect. In this study, we investigate the effects of the endocannabinoid anandamide on the complex pathology of GVHD. We assess the effects of an irreversible inhibitor of fatty acid amine hydrolase or exogenous anandamide and find that they increase survival and reduce clinical signs in GVHD mice. In the intestine of GVHD mice, treatment with exogenous anandamide also leads to a reduction in the number of CD3+, CD3+CD4+, and CD3+CD8+ cells, which reduces the activation of CD3+CD4+ and CD3+CD8+ cells, as assessed by enhanced CD28 expression, a T cell co-stimulatory molecule. Exogenous AEA was also able to reduce TNF-α and increase IL-10 in the intestine of GVHD mice. In the liver, exogenous AEA reduces injury, TNF-α levels, and the number of CD3+CD8+ cells. Interestingly, anandamide reduces Mac-1α, which lowers the adhesion of transplanted cells in mesenteric veins. These effects are mimicked by JWH133—a CB2 selective agonist—and abolished by treatment with a CB2 antagonist. Furthermore, the effects caused by anandamide treatment on survival were related to the CB2 receptor, as the CB2 antagonist abolished it. This study shows the critical role of the CB2 receptor in the modulation of the inflammatory response of GVHD by treatment with anandamide, the most prominent endocannabinoid.

Prenatal exposure to CB2 receptors agonist differentially impacts male and female germ cells via histone modification

Cannabis use during pregnancy is increasing in the last few years potentially because of decreased perception of the risk of harm. Regardless, recent evidence demonstrated that prenatal cannabis exposure is associated with adverse outcomes. To date there is limited evidence of the impact of cannabis exposure during pregnancy on the reproductive health of the offspring. The biological effects of cannabis are mediated by two cannabinoid receptors, CB1 and CB2. We previously demonstrated that CB2 is highly expressed in mouse male and female fetal germ cells. In this study, we investigated the effects of prenatal exposure to a selective CB2 agonist, JWH-133, on the long-term reproductive health of male and female offspring and on the involved molecular epigenetic mechanisms. Notably, we focused on epigenetic histone modifications that can silence or activate gene expression, playing a pivotal role in cell differentiation. We reported that prenatal activation of CB2 has a sex-specific impact on germ cell development of the offspring. In male it determines a delay of germ cell differentiation coinciding with an enrichment of H3K27me3, while in female it causes a reduction of the follicles number through an increased apoptotic process not linked to modified H3K27me3 level.

P-600 cannabinoids exposure during pregnancy affects female offsprings reproduction

Abstract Study question Could in-utero exposure to cannabinoids alter the reproductive health of female offspring? Summary answer Prenatal exposure to cannabinoids causes a reduction of the ovarian reserve in F1 with compromised reproductive lifespan and low birth weight in F1/ F2 generations. What is known already Cannabis use during pregnancy is associated with adverse neonatal outcomes: preterm birth, intrauterine growth retardation, low birth weight, and neurological developmental changes. However, the impact on the germ line is still little known. Cannabis/cannabinoids exert their biological effect by activating two main cannabinoid receptors: CB1 and CB2. We previously demonstrated that fetal oocytes express CB2 receptors at a higher level than CB1. In vitro treatment of fetal oocytes with JWH-133, a selective CB2 agonist, induces an acceleration of meiosis and an increase in the percentage of γ-H2AX-positive cells and in TUNEL-positive cells (apoptotic cells). Study design, size, duration Five pregnant CD1 female mice were intraperitoneally injected with a single dose of JWH-133, at 1.5 mg/kg for 5 days between embryonic period (E) 12.5 to E16.5, while control pregnant females received a vehicle containing saline. At delivery, on post-natal day (PND) 2, 30 and 365, F1 pups were counted, weighted and tissues from females were collected. At adult age, F1 females were mated with control mice and F2 generation was morphologically analysed. Participants/materials, setting, methods Ovaries from control and in utero exposed mice were fixed in paraformaldehyde and stained with Haematoxylin and Eosin (H&E). Gonads were embedded in paraplast and sectioned at 5μm Leica-RM 2035 Microtome. Follicles in each ovary were counted serially in every third section through the entire ovary. Only healthy, non-atretic follicles with visible oocyte nuclei were scored. Main results and the role of chance We demonstrate that in vivo exposure to the cannabinoid JWH-133 during fetal life, through the administration of the drug to pregnant females, causes a significant reduction in the offspring body weight at birth (LBW). Histological analysis of the ovaries isolated from newborn females (PND2) shows a significant reduction of the primordial and primary follicles. This reduced ovarian reserve is observed also at PND30 and at PND365. At old adult age, these females, crossed with control males, show a drastic decrease in their litter size (F2) across the life course in comparison with control females. This suggests that the pool of primordial follicles becomes prematurely depleted in in-utero exposed F1 females, compromising their reproductive lifespan. Moreover, we find that F2 pups from prenatally exposed mothers show LBW but have an unaltered number of ovarian follicles in females. Since F2 pups have never been exposed to the drug, our results suggest that LBW is transmitted from the mother intergenerationally, while the reduced ovarian reserve is a consequence of direct exposure to the drug during fetal life of F1 females and it is not transmitted to the F2 generation. Limitations, reasons for caution The effects observed in mice with JWH-133 cannot be directly translated to cannabis effects since the active principle of cannabis (THC) activates both the cannabinoid receptors. Moreover, this study lacks molecular analysis of gene expression implicated in intergenerational transmission. Wider implications of the findings This is the first direct demonstration that prenatal exposure to cannabinoids could have long-term critical consequences for females’ reproductive health. In humans, diminished ovarian reserve is characterized by poor fertility outcomes and it represents a major challenge in reproductive medicine. Trial registration number not applicable

The Selective CB2 Agonist COR167 Reduced Symptoms in a Mice Model of Trauma-Induced Peripheral Neuropathy through HDAC-1 Inhibition.

Neuropathic pain is a chronic disabling condition with a 7-10% of prevalence in the general population that is largely undertreated. Available analgesic therapies are poorly effective and are often accompanied by numerous side effects. Growing evidence indicates cannabinoids are a valuable treatment opportunity for neuropathic pain. The endocannabinoid system is an important regulator of pain perception through the CB1 receptors, but CB1 agonists, while largely effective, are not always satisfactory pain-relieving agents in clinics because of their serious adverse effects. Recently, several CB2 agonists have shown analgesic, anti-hyperalgesic, and anti-allodynic activity in the absence of CB1-induced psychostimulant effects, offering promise in neuropathic pain management. The aim of this study was to evaluate the anti-neuropathic activity of a novel selective CB2 agonist, COR167, in a preclinical model of peripheral neuropathy, the spared nerve injury (SNI). Oral COR167, in a dose-dependent manner, attenuated mechanical allodynia and thermal hyperalgesia after acute and repeated administration, showing the absence of tolerance induction. At anti-neuropathic doses, COR167 did not show any alteration in the locomotor behavior. SNI mice showed increased microglial levels of HDAC1 protein in the ipsilateral side of the spinal cord, along with NF-kB activation. COR167 treatment prevented the HDAC1 overexpression and the NF-kB activation and increased the levels of the anti-inflammatory cytokine IL-10 through a CB2-mediated mechanism. Oral administration of COR167 shows promising therapeutic potential in the management of neuropathic pain conditions.

Distinct activation mechanisms regulate subtype selectivity of Cannabinoid receptors

Design of cannabinergic subtype selective ligands is challenging because of high sequence and structural similarities of cannabinoid receptors (CB1 and CB2). We hypothesize that the subtype selectivity of designed selective ligands can be explained by the ligand binding to the conformationally distinct states between cannabinoid receptors. Analysis of ~ 700 μs of unbiased simulations using Markov state models and VAMPnets identifies the similarities and distinctions between the activation mechanism of both receptors. Structural and dynamic comparisons of metastable intermediate states allow us to observe the distinction in the binding pocket volume change during CB1 and CB2 activation. Docking analysis reveals that only a few of the intermediate metastable states of CB1 show high affinity towards CB2 selective agonists. In contrast, all the CB2 metastable states show a similar affinity for these agonists. These results mechanistically explain the subtype selectivity of these agonists by deciphering the activation mechanism of cannabinoid receptors.

Novel Cannabinoid Receptor 2 (CB2) Low Lipophilicity Agonists Produce Distinct cAMP and Arrestin Signalling Kinetics without Bias

Cannabinoid Receptor 2 (CB2) is a promising target for treating inflammatory diseases. We designed derivatives of 3-carbamoyl-2-pyridone and 1,8-naphthyridin-2(1H)-one-3-carboxamide CB2-selective agonists with reduced lipophilicity. The new compounds were measured for their affinity (radioligand binding) and ability to elicit cyclic adenosine monophosphate (cAMP) signalling and β-arrestin-2 translocation with temporal resolution (BRET-based biosensors). For the 3-carbamoyl-2-pyridone derivatives, we found that modifying the previously reported compound UOSS77 (also known as S-777469) by appending a PEG2-alcohol via a 3-carbomylcyclohexyl carboxamide (UOSS75) lowered lipophilicity, and preserved binding affinity and signalling profile. The 1,8-naphthyridin-2(1H)-one-3-carboxamide UOMM18, containing a cis configuration at the 3-carboxamide cyclohexyl and with an alcohol on the 4-position of the cyclohexyl, had lower lipophilicity but similar CB2 affinity and biological activity to previously reported compounds of this class. Relative to CP55,940, the new compounds acted as partial agonists and did not exhibit signalling bias. Interestingly, while all compounds shared similar temporal trajectories for maximal efficacy, differing temporal trajectories for potency were observed. Consequently, when applied at sub-maximal concentrations, CP55,940 tended to elicit sustained (cAMP) or increasing (arrestin) responses, whereas responses to the new compounds tended to be transient (cAMP) or sustained (arrestin). In future studies, the compounds characterised here may be useful in elucidating the consequences of differential temporal signalling profiles on CB2-mediated physiological responses.

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.

Structure-Based Virtual Screening and Molecular Dynamics Simulation Assessments of Depsidones as Possible Selective Cannabinoid Receptor Type 2 Agonists.

The discovery of natural drug metabolites is a leading contributor to fulfilling the sustainable development goal of finding solutions to global health challenges. Depsidones are a class of polyketides that have been separated from lichens, fungi, sponges, and plants and possess various bioactivities, including cytotoxic, antimicrobial, antimalarial, antituberculosis, acetylcholinesterase and α-glucosidase inhibition, and anti-inflammatory effects. Endocannabinoid receptors (CB1 and CB2) are G-protein-coupled receptors (GPCRs), and their activation mediates many physiological processes. CB1 is the dominant subtype in the central nervous system, while CB2 is mainly expressed in the immune system. The two receptors exhibit high heterogeneity, making developing selective ligands a great challenge. Attempts to develop CB2 selective agonists for treating inflammatory diseases and neuropathic pain have not been successful due to the high homology of the binding sites of the CB receptors. In this work, 235 depsidones from various sources were investigated for the possibility of identifying CB2-selective agonists by performing multiple docking studies, including induced fit docking and Prime/molecular mechanics-generalized Born surface area (MM-GBSA) calculations to predict the binding mode and free energy. Simplicildone J (10), lobaric acid (110), mollicellin Q (101), garcinisidone E (215), mollicellin P (100), paucinervin Q (149), and boremexin C (161) had the highest binding scores (-12.134 kcal/mol, -11.944 kcal/mol, -11.479 kcal/mol, -11.394 kcal/mol, -11.322 kcal/mol, -11.305 kcal/mol, and -11.254 kcal/mol, respectively) when screened against the CB2 receptor (PDB ID: 6KPF). The molecular dynamic simulation was performed on the compounds with the highest binding scores. The computational outcomes show that garcinisidone E (215) and paucinervin Q (149) could be substantial candidates for CB2 receptor activation and warrant further in vivo and in vitro investigations.

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.

Cannabinoid CB1 receptors regulate salivation

Saliva serves multiple important functions within the body that we typically take for granted, such as helping prepare food for swallowing and defense against oral pathogens. Dry mouth is a primary symptom of Sjӧgren’s syndrome and is a side effect of many drug treatments. Cannabis users frequently report dry mouth, but the basis for this is still unknown. If the effects occur via the endogenous cannabinoid signaling system, then this may represent a novel mechanism for the regulation of salivation. We examined expression of cannabinoid CB1 receptors in submandibular salivary gland using immunohistochemistry and tested regulation of salivation by THC and cannabinoid-related ligands. We now report that CB1 receptors are expressed in the axons of cholinergic neurons innervating the submandibular gland. No staining is seen in submandibular gland epithelial cells (acinar and ductal), or myoepithelial cells (MECs). Treatment with THC (4 mg/kg, IP) or the cannabinoid receptor agonist CP55940 (0.5 mg/kg) reduced salivation in both male and female mice 1 h after treatment. CBD had no effect on its own but reversed the effect of THC in a concentration-dependent manner. Neither the CB1 receptor antagonist SR141716 (4 mg/kg) nor the CB2-selective agonist JWH133 (4 mg/kg) had an effect on salivation. We also found that fatty acid amide hydrolase (FAAH), the enzyme that metabolizes the endocannabinoid anandamide and related lipids, regulates salivation. Salivation was reduced in FAAH knockout mice as well as mice treated with the FAAH blocker URB597 (4 mg/kg). URB597 had no effect in CB1 knockout mice. FAAH protein is detected intracellularly in acinar but not ductal epithelial cells. In lipidomics experiments, we found that FAAH knockout mice chiefly had elevated levels of acylethanolamines, including anandamide, and reduced levels of acyglycines. Our results are consistent with a model wherein endocannabinoids activate CB1 receptors on cholinergic axons innervating the submandibular gland. THC likely acts by plugging into this system, activating CB1 receptors to reduce salivation, thus offering a mechanism underlying the dry mouth reported by cannabis users.

Cannabinoid Receptors and Glial Response Following a Basal Forebrain Cholinergic Lesion.

The endocannabinoid system modulates learning, memory, and neuroinflammatory processes, playing a key role in neurodegeneration, including Alzheimer's disease (AD). Previous results in a rat lesion model of AD showed modulation of endocannabinoid receptor activity in the basalo-cortical pathway following a specific lesion of basal forebrain cholinergic neurons (BFCNs), indicating that the glial neuroinflammatory response accompanying the lesion is related to endocannabinoid signaling. In this study, 7 days after the lesion, decreased astrocyte and increased microglia immunoreactivities (GFAP and Iba-1) were observed, indicating microglia-mediated neuroinflammation. Using autoradiographic studies, the density and functional coupling to G-proteins of endocannabinoid receptor subtypes were studied in tissue sections from different brain areas where microglia density increased, using CB1 and CB2 selective agonists and antagonists. In the presence of the specific CB1 receptor antagonist, SR141716A, [3H]CP55,940 binding (receptor density) was completely blocked in a dose-dependent manner, while the selective CB2 receptor antagonist, SR144528, inhibited binding to 25%, at best. [35S]GTPγS autoradiography (receptor coupling to Gi/0-proteins) evoked by CP55,940 (CB1/CB2 agonist) and HU308 (more selective for CB2) was abolished by SR141716A in all areas, while SR144528 blocked up to 51.8% of the coupling to Gi/0-proteins evoked by CP55,940 restricted to the nucleus basalis magnocellularis. Together, these results demonstrate that there are increased microglia and decreased astrocyte immunoreactivities 1 week after a specific deletion of BFCNs, which projects to cortical areas, where the CB1 receptor coupling to Gi/0-proteins is upregulated. However, at the lesion site, the area with the highest neuroinflammatory response, there is also a limited contribution of CB2.

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.

A Cannabinoid 2-Selective Agonist Inhibits Allogeneic Skin Graft Rejection In Vivo.

Previous work from our laboratory showed that a CB2 selective agonist, O-1966, blocked the proliferative response of C57BL/6 mouse spleen cells exposed to spleen cells of C3HeB/FeJ mice in vitro in the mixed lymphocyte reaction (MLR). The MLR is widely accepted as an in vitro correlate of in vivo grant rejection. Mechanisms of the immunosuppression induced by the cannabinoid were explored, and it was shown that O-1966 in this in vitro assay induced CD25+Foxp3+ Treg cells and IL-10, as well as down-regulated mRNA for CD40 and the nuclear form of the transcription factors NF-κB and NFAT in T-cells. The current studies tested the efficacy of O-1966 in prolonging skin grafts in vivo. Full thickness flank skin patches (1-cm2) from C3HeB/FeJ mice were grafted by suturing onto the back of C57BL/6 mice. O-1966 or vehicle was injected intraperitoneally into treated or control groups of animals beginning 1 h pre-op, and then every other day until 14 days post-op. Graft survival was scored based on necrosis and rejection. Treatment with 5 mg/kg of O-1966 prolonged mean graft survival time from 9 to 11 days. Spleens harvested from O-1966 treated mice were significantly smaller than those of vehicle control animals based on weight. Flow cytometry analysis of CD4+ spleen cells showed that O-1966 treated animals had almost a 3-fold increase in CD25+Foxp3+ Treg cells compared to controls. When dissociated spleen cells were placed in culture ex vivo and stimulated with C3HeB/FeJ cells in an MLR, the cells from the O-1966 treated mice were significantly suppressed in their proliferative response to the allogeneic cells. These results support CB2 selective agonists as a new class of compounds to prolong graft survival in transplant patients.