Identification of Novel Cannabinoid CB2 Receptor Agonists from Botanical Compounds and Preliminary Evaluation of Their Anti-Osteoporotic Effects.

As cannabinoid CB2 receptors (CB2R) possess various pharmacological effects-including anti-epilepsy, analgesia, anti-inflammation, anti-fibrosis, and regulation of bone metabolism-without the psychoactive side effects induced by cannabinoid CB1R activation, they have become the focus of research and development of new target drugs in recent years. The present study was intended to (1) establish a double luciferase screening system for a CB2R modulator; (2) validate the agonistic activities of the screened compounds on CB2R by determining cAMP accumulation using HEK293 cells that are stably expressing CB2R; (3) predict the binding affinity between ligands and CB2 receptors and characterize the binding modes using molecular docking; (4) analyze the CB2 receptors-ligand complex stability, conformational behavior, and interaction using molecular dynamics; and (5) evaluate the regulatory effects of the screened compounds on bone metabolism in osteoblasts and osteoclasts. The results demonstrated that the screening system had good stability and was able to screen cannabinoid CB2R modulators from botanical compounds. Altogether, nine CB2R agonists were identified by screening from 69 botanical compounds, and these CB2R agonists exhibited remarkable inhibitory effects on cAMP accumulation and good affinity to CB2R, as evidenced by the molecular docking and molecular dynamics. Five of the nine CB2R agonists could stimulate osteoblastic bone formation and inhibit osteoclastic bone resorption. All these findings may provide useful clues for the development of novel anti-osteoporotic drugs and help elucidate the mechanism underlying the biological activities of CB2R agonists identified from the botanical materials.

Different Cytotoxic Effects of Vetiver Oil on Three Types of Cancer Cells, Mainly Targeting CNR2 on TNBC.

OBJECTIVE: To investigate vetiver oil (VO) selectivity effects on several cancer cell types and identify the ß-caryophyllene role and mechanisms to prevent cancer development. METHODS: Cytotoxic effects of VO on three types of cancer cells (WiDr, 4T1, T47D) were determined using MTT assay. VO's effects on the cell cycle and apoptosis were analyzed using flow cytometry. Intracellular Reactive Oxygen Species (ROS) of cells after treatment with VO was observed with DCFDA staining. Bioinformatics study and molecular docking were used to determine the molecular targets of VO. RESULTS: VO contained various essential oils in which ß-caryophyllene was the most abundant. 4T1 cells performed the lowest IC50 value. WiDr and 4T1 cells showed an arrest in the G2/M phase, while T47D showed an increase of sub G1 population after VO treatment. On the other hand, apoptosis was only observed in WiDr and T47D cells. ROS levels were increased significantly in WiDr and T47D cells but not in 4T1 cells. Cannabinoids CB2 receptor (CNR2) was highly expressed in 4T1 cells and commonly exhibited a low survival rate on Triple Negative Breast Cancer (TNBC) patients. CNR2 was the notable target of ß-caryophyllene and performed agonistic interaction, which might have contributed to its cytotoxic activity against 4T1 cells. CONCLUSION: The molecular interaction of VO cannabinoid agonists and the CNR2 receptor was the underlying cause of VO cytotoxicity, which is a VO distinction on TNBC. Therefore, VO is better suited for use as an anti-cancer agent in TNBC cells.

Antinociceptive and chondroprotective effects of prolonged ß-caryophyllene treatment in the animal model of osteoarthritis: Focus on tolerance development.

Osteoarthritis (OA) is a chronic joint disease in which cartilage degeneration leads to chronic pain. The endocannabinoid system has attracted attention as an emerging drug target for OA. However, the therapeutic potential of cannabinoids is limited by psychoactive side-effects related to CB1 activation and tolerance development for analgesic effects. ß-Caryophyllene (BCP) is a low-efficacy natural agonist of CB2 and a common constituent of human diet with well-established anti-inflammatory properties. The results presented herein show the anti-nociceptive and chondroprotective potential of BCP in an animal model of OA induced by intra-articular injection of monoiodoacetate (MIA). Behavioural assessment included pressure application measurement and kinetic weight bearing tests. Histological assessment of cartilage degeneration was quantified using OARSI scoring. Experiments established the dose-response effects of BCP and pharmacological mechanisms of the antinociceptive action dependent on CB2 and opioid receptors. Chronic BCP treatment was able to hamper cartilage degeneration without producing tolerance for the analgesic effects. The data presented herein show that BCP is able to produce both acute and prolonged antinociceptive and chondroprotective effects. Together with the safety profile and legal status of BCP, these results indicate a novel and promising disease-modifying strategy for treating OA.

Cannabinoid 2 receptor agonist and L-arginine combination attenuates diabetic cardiomyopathy in rats via NF-Ä¸ß inhibition.

Beta-caryophyllene (BCP), a cannabinoid 2 (CB2) receptor agonist has recently been found to have cardioprotective activity as an anti-inflammatory and antioxidant molecule. L-arginine (LA), a nitric oxide (NO) donor, is a potential regulator of cardiovascular function. Considering the role of CB2 receptor activation and NO regulation in cardiovascular diseases, the combination of BCP with LA may be a possible treatment of diabetic cardiomyopathy (DCM). Hence, we investigated the efficacy of the novel combination of BCP with LA on cardiovascular inflammation and oxidative stress in diabetic rats. DCM was induced by streptozotocin (55 mg/kg) in Sprague-Dawley rats intraperitoneally. BCP, LA, and BCP with LA were administered to diabetic rats for 4 weeks. After completion of the study, hemodynamic parameters, biochemical parameters, and inflammatory cytokine levels were analyzed. Also, oxidative stress parameters, nuclear factor kappa beta (NF-ĸß) expression, and histopathology in cardiac tissues were estimated. The combination of BCP (200 mg/kg) with LA (200 mg/kg) significantly normalized the hemodynamic parameters and decreased the glucose, cardiac markers, interleukin-6, and tumor necrosis factor-alpha levels. Treatment of BCP and LA showed a significant decrease in oxidative stress and downregulated the cardiac expression of NF-ĸß. Thus, the combination of BCP with LA improves cardiac functions by attenuating inflammation through NF-Ä¸ß inhibition in DCM.

New Pyridone-Based Derivatives as Cannabinoid Receptor Type 2 Agonists.

The activation of the human cannabinoid receptor type II (CB2R) is known to mediate analgesic and anti-inflammatory processes without the central adverse effects related to cannabinoid receptor type I (CB1R). In this work we describe the synthesis and evaluation of a novel series of N-aryl-2-pyridone-3-carboxamide derivatives tested as human cannabinoid receptor type II (CB2R) agonists. Different cycloalkanes linked to the N-aryl pyridone by an amide group displayed CB2R agonist activity as determined by intracellular [cAMP] levels. The most promising compound 8d exhibited a non-toxic profile and similar potency (EC50 = 112 nM) to endogenous agonists Anandamide (AEA) and 2-Arachidonoylglycerol (2-AG) providing new information for the development of small molecules activating CB2R. Molecular docking studies showed a binding pose consistent with two structurally different agonists WIN-55212-2 and AM12033 and suggested structural requirements on the pyridone substituents that can satisfy the orthosteric pocket and induce an agonist response. Our results provide additional evidence to support the 2-pyridone ring as a suitable scaffold for the design of CB2R agonists and represent a starting point for further optimization and development of novel compounds for the treatment of pain and inflammation.

Pharmacological potential of JWH133, a cannabinoid type 2 receptor agonist in neurodegenerative, neurodevelopmental and neuropsychiatric diseases.

The pharmacological activation of cannabinoid type 2 receptors (CB2R) gained attention due to its ability to mitigate neuroinflammatory events without eliciting psychotropic actions, a limiting factor for the drugs targeting cannabinoid type 1 receptors (CB1R). Therefore, ligands activating CB2R are receiving enormous importance for therapeutic targeting in numerous neurological diseases including neurodegenerative, neuropsychiatric and neurodevelopmental disorders as well as traumatic injuries and neuropathic pain where neuroinflammation is a common accompaniment. Since the characterization of CB2R, many CB2R selective synthetic ligands have been developed with high selectivity and functional activity. Among numerous ligands, JWH133 has been found one of the compounds with high selectivity for CB2R. JWH133 has been reported to exhibit numerous pharmacological activities including antioxidant, anti-inflammatory, anticancer, cardioprotective, hepatoprotective, gastroprotective, nephroprotective, and immunomodulatory. Recent studies have shown that JWH133 possesses potent neuroprotective properties in several neurological disorders, including neuropathic pain, anxiety, epilepsy, depression, alcoholism, psychosis, stroke, and neurodegeneration. Additionally, JWH133 showed to protect neurons from oxidative damage and inflammation, promote neuronal survival and neurogenesis, and serve as an immunomodulatory agent. The present review comprehensively examined neuropharmacological activities of JWH133 in neurological disorders including neurodegenerative, neurodevelopmental and neuropsychiatric using synoptic tables and elucidated pharmacological mechanisms based on reported observations. Considering the cumulative data, JWH133 appears to be a promising CB2R agonist molecule for further evaluation and it can be a prototype agent in drug discovery and development for a unique class of agents in neurotherapeutics. Further, regulatory toxicology and pharmacokinetic studies are required to determine safety and proceed for clinical evaluation.

Plant-Derived Trans-ß-Caryophyllene Boosts Glucose Metabolism and ATP Synthesis in Skeletal Muscle Cells through Cannabinoid Type 2 Receptor Stimulation.

Skeletal muscle plays a pivotal role in whole-body glucose metabolism, accounting for the highest percentage of glucose uptake and utilization in healthy subjects. Impairment of these key functions occurs in several conditions including sedentary lifestyle and aging, driving toward hyperglycemia and metabolic chronic diseases. Therefore, strategies pointed to improve metabolic health by targeting skeletal muscle biochemical pathways are extremely attractive. Among them, we focused on the natural sesquiterpene and cannabinoid type 2 (CB2) receptor agonist Trans-ß-caryophyllene (BCP) by analyzing its role in enhancing glucose metabolism in skeletal muscle cells. Experiments were performed on C2C12 myotubes. CB2 receptor membrane localization in myotubes was assessed by immunofluorescence. Within glucose metabolism, we evaluated glucose uptake (by the fluorescent glucose analog 2-NBDG), key enzymes of both glycolytic and oxidative pathways (by spectrophotometric assays and metabolic radiolabeling) and ATP production (by chemiluminescence-based assays). In all experiments, CB2 receptor involvement was tested with the CB2 antagonists AM630 and SR144528. Our results show that in myotubes, BCP significantly enhances glucose uptake, glycolytic and oxidative pathways, and ATP synthesis through a CB2-dependent mechanism. Giving these outcomes, CB2 receptor stimulation by BCP could represent an appealing tool to improve skeletal muscle glucose metabolism, both in physiological and pathological conditions.

Modern approaches to the development of synthetic cannabinoid receptor probes.

The endocannabinoid system, which spans the central and peripheral nervous systems and regulates many biologic processes, is an important target for probe discovery and medications development. Whereas the earliest endocannabinoid receptor probes were derivatives of the non-selective phytocannabinoids isolated from Cannabis species, modern drug discovery techniques have expanded the definitions of what constitutes a CB1R or CB2R cannabinoid receptor ligand. This review highlights recent advances in synthetic cannabinoid receptor chemistry and pharmacology. We provide examples of new CB1R- and CB2R-selective probes, and discuss rational approaches to the design of peripherally-restricted agents. We also describe structural classes of positive- and negative allosteric modulators (PAMs and NAMs) of CB1R and CB2R. Finally, we introduce new opportunities for cannabinoid receptor probe development that have emerged in recent years, including biased agonists that may lead to medications lacking adverse effects.

Endocannabinoid System as a Promising Therapeutic Target in Inflammatory Bowel Disease - A Systematic Review.

Inflammatory bowel disease (IBD) is a general term used to describe a group of chronic inflammatory conditions of the gastrointestinal tract of unknown etiology, including two primary forms: Crohn's disease (CD) and ulcerative colitis (UC). The endocannabinoid system (ECS) plays an important role in modulating many physiological processes including intestinal homeostasis, modulation of gastrointestinal motility, visceral sensation, or immunomodulation of inflammation in IBD. It consists of cannabinoid receptors (CB1 and CB2), transporters for cellular uptake of endocannabinoid ligands, endogenous bioactive lipids (Anandamide and 2-arachidonoylglycerol), and the enzymes responsible for their synthesis and degradation (fatty acid amide hydrolase and monoacylglycerol lipase), the manipulation of which through agonists and antagonists of the system, shows a potential therapeutic role for ECS in inflammatory bowel disease. This review summarizes the role of ECS components on intestinal inflammation, suggesting the advantages of cannabinoid-based therapies in inflammatory bowel disease.

Effect of acute and subchronic administration of (R)-WIN55,212-2 induced neuroprotection and anti inflammatory actions in rat retina: CB1 and CB2 receptor involvement.

Cannabinoids have been shown to protect the retina from ischemic/excitotoxic insults. The aim of the present study was to investigate the neuroprotective and anti-inflammatory properties of the synthetic cannabinoid (R)-WIN55,212-2 (CB1/CB2 receptor agonist) when administered acutely or subchronically in control and AMPA treated retinas. Sprague-Dawley rats were intravitreally administered (acutely) with vehicle or AMPA, in the absence or presence of (R)-WIN55,212-2 (10-7-10-4M) alone or in combination with AM251 [CB1 receptor antagonist/inverse agonist,10-4M] and AM630 (CB2 receptor antagonist,10-4M). In addition, AMPA was co-administered with the racemic (R,S)-WIN55,212 (10-4Μ). (R)-WIN55,212-2 was also administered subchronically (25,100 µg/kg,i.p.,4d) in control and AMPA treated rats. Immunohistochemical studies were performed using antibodies against the CB1R, and retinal markers for retinal neurons (brain nitric oxide synthetase, bNOS) and microglia (ionized calcium binding adaptor molecule 1, Iba1). ELISA assay was employed to assess TNFα levels in AMPA treated retinas. Intravitreal administration of (R)-WIN55,212-2 reversed the AMPA induced loss of bNOS expressing amacrine cells, an effect that was blocked by both AM251 and AM630. (R,S)WIN55,212 had no effect. (R)-WIN55,212-2 also reduced a) the AMPA induced activation of microglia, by activating CB2 receptors that were shown to be colocalized with Iba1+ reactive microglial cells, and b) TNFα levels in retina. (R)-WIN55,212-2 administered subchronically led to the downregulation of CB1 receptors at the high dose of 100 µg/kg(i.p.), and to the attenuation of the WIN55,212-2 induced neuroprotection of amacrine cells. At the same dose, (R)-WIN55,212-2 did not attenuate the AMPA induced increase in the number of reactive microglia cells, suggesting CB2 receptor downregulation under subchronic conditions. This study provides new findings regarding the role of CB1 and CB2 receptor activation by the synthetic cannabinoid (R)-WIN55,212-2, administered acutely or sub-chronically, on neuron viability and microglia activation in healthy and diseased retina.

From a Designer Drug to the Discovery of Selective Cannabinoid Type 2 Receptor Agonists with Favorable Pharmacokinetic Profiles for the Treatment of Systemic Sclerosis.

Synthetic cannabinoids, as exemplified by SDB-001 (1), bind to both CB1 and CB2 receptors and exert cannabimimetic effects similar to (-)-trans-Δ9-tetrahydrocannabinol, the main psychoactive component present in the cannabis plant. As CB1 receptor ligands were found to have severe adverse psychiatric effects, increased attention was turned to exploiting the potential therapeutic value of the CB2 receptor. In our efforts to discover novel and selective CB2 receptor agonists, 1 was selected as a starting point for hit molecule identification and a class of 1H-pyrazole-3-carboxamide derivatives were thus designed, synthesized, and biologically evaluated. Systematic structure-activity relationship investigations resulted in the identification of the most promising compound 66 as a selective CB2 receptor agonist with favorable pharmacokinetic profiles. Especially, 66 treatment significantly attenuated dermal inflammation and fibrosis in a bleomycin-induced mouse model of systemic sclerosis, supporting that CB2 receptor agonists might serve as potential therapeutics for treating systemic sclerosis.

In vitro and in vivo pharmacological activity of minor cannabinoids isolated from Cannabis sativa.

The Cannabis sativa plant contains more than 120 cannabinoids. With the exceptions of ∆9-tetrahydrocannabinol (∆9-THC) and cannabidiol (CBD), comparatively little is known about the pharmacology of the less-abundant plant-derived (phyto) cannabinoids. The best-studied transducers of cannabinoid-dependent effects are type 1 and type 2 cannabinoid receptors (CB1R, CB2R). Partial agonism of CB1R by ∆9-THC is known to bring about the 'high' associated with Cannabis use, as well as the pain-, appetite-, and anxiety-modulating effects that are potentially therapeutic. CB2R activation by certain cannabinoids has been associated with anti-inflammatory activities. We assessed the activity of 8 phytocannabinoids at human CB1R, and CB2R in Chinese hamster ovary (CHO) cells stably expressing these receptors and in C57BL/6 mice in an attempt to better understand their pharmacodynamics. Specifically, ∆9-THC, ∆9-tetrahydrocannabinolic acid (∆9-THCa), ∆9-tetrahydrocannabivarin (THCV), CBD, cannabidiolic acid (CBDa), cannabidivarin (CBDV), cannabigerol (CBG), and cannabichromene (CBC) were evaluated. Compounds were assessed for their affinity to receptors, ability to inhibit cAMP accumulation, ßarrestin2 recruitment, receptor selectivity, and ligand bias in cell culture; and cataleptic, hypothermic, anti-nociceptive, hypolocomotive, and anxiolytic effects in mice. Our data reveal partial agonist activity for many phytocannabinoids tested at CB1R and/or CB2R, as well as in vivo responses often associated with activation of CB1R. These data build on the growing body of literature showing cannabinoid receptor-dependent pharmacology for these less-abundant phytocannabinoids and are critical in understanding the complex and interactive pharmacology of Cannabis-derived molecules.

Oleamide Induces Cell Death in Glioblastoma RG2 Cells by a Cannabinoid Receptor-Independent Mechanism.

The endocannabinoid system has been associated with antiproliferative effects in several types of tumors through cannabinoid receptor-mediated cell death mechanisms. Oleamide (ODA) is a CB1/CB2 agonist associated with cell growth and migration by adhesion and/or ionic signals associated with Gap junctions. Antiproliferative mechanisms related to ODA remain unknown. In this work, we evaluated the effects of ODA on cell viability and morphological changes in a rat RG2 glioblastoma cell line and compared these effects with primary astrocyte cultures from 8-day postnatal rats. RG2 and primary astrocyte cultures were treated with ODA at increasing concentrations (25, 50, 100, and 200 µM) for different periods of time (12, 24, and 48 h). Changes in RG2 cell viability and morphology induced by ODA were assessed by viability/mitochondrial activity test and phase contrast microscopy, respectively. The ratios of necrotic and apoptotic cell death, and cell cycle alterations, were evaluated by flow cytometry. The roles of CB1 and CB2 receptors on ODA-induced changes were explored with specific receptor antagonists. ODA (100 µM) induced somatic damage, detachment of somatic bodies, cytoplasmic polarization, and somatic shrinkage in RG2 cells at 24 and 48 h. In contrast, primary astrocytes treated at the same ODA concentrations exhibited cell aggregation but not cell damage. ODA (100 µM) increased apoptotic cell death and cell arrest in the G1 phase at 24 h in the RG2 line. The effects induced by ODA on cell viability of RG2 cells were independent of CB1 and CB2 receptors or changes in intracellular calcium transient. Results of this novel study suggest that ODA exerts specific antiproliferative effects on RG2 glioblastoma cells through unconventional apoptotic mechanisms not involving canonical signals.

Bioactive Chemical Composition of Cannabis Extracts and Cannabinoid Receptors.

Cannabis is widely used as a therapeutic drug, especially by patients suffering from psychiatric and neurodegenerative diseases. However, the complex interplay between phytocannabinoids and their targets in the human receptome remains largely a mystery, and there have been few investigations into the relationship between the chemical composition of medical cannabis and the corresponding biological activity. In this study, we investigated 59 cannabis samples used by patients for medical reasons. The samples were subjected to extraction (microwave and supercritical carbon dioxide) and chemical analyses, and the resulting extracts were assayed in vitro using the CB1 and CB2 receptors. Using a partial least squares regression analysis, the chemical compositions of the extracts were then correlated to their corresponding cannabinoid receptor activities, thus generating predictive models that describe the receptor potency as a function of major phytocannabinoid content. Using the current dataset, meaningful models for CB1 and CB2 receptor agonism were obtained, and these reveal the insignificant relationships between the major phytocannabinoid content and receptor affinity for CB1 but good correlations between the two at CB2 receptors. These results also explain the anomalies between the receptor activities of pure phytocannabinoids and cannabis extracts. Furthermore, the models for CB1 and CB2 agonism in cannabis extracts predict the cannabinoid receptor activities of individual phytocannabinoids with reasonable accuracy. Here for the first time, we disclose a method to predict the relationship between the chemical composition, including phytocannabinoids, of cannabis extracts and cannabinoid receptor responses.

Xie2-64, a novel CB2 receptor inverse agonist, reduces cocaine abuse-related behaviors in rodents.

Cocaine abuse remains a public health threat around the world. There are no pharmacological treatments approved for cocaine use disorder. Cannabis has received growing attention as a treatment for many conditions, including addiction. Most cannabis-based medication development has focused on cannabinoid CB1 receptor (CB1R) antagonists (and also inverse agonists) such as rimonabant, but clinical trials with rimonabant have failed due to its significant side-effects. Here we sought to determine whether a novel and selective CB2R inverse agonist, Xie2-64, has similar therapeutic potential for cocaine use disorder. Computational modeling indicated that Xie2-64 binds to CB2R in a way similar to SR144528, another well-characterized but less selective CB2R antagonist/inverse agonist, suggesting that Xie2-64 may also have CB2R antagonist profiles. Unexpectedly, systemic administration of Xie2-64 or SR144528 dose-dependently inhibited intravenous cocaine self-administration and shifted cocaine dose-response curves downward in rats and wild-type, but not in CB2R-knockout, mice. Xie2-64 also dose-dependently attenuated cocaine-enhanced brain-stimulation reward maintained by optical stimulation of ventral tegmental area dopamine (DA) neurons in DAT-Cre mice, while Xie2-64 or SR144528 alone inhibited optical brain-stimulation reward. In vivo microdialysis revealed that systemic or local administration of Xie2-64 into the nucleus accumbens reduced extracellular dopamine levels in a dose-dependent manner in rats. Together, these results suggest that Xie2-64 has significant anti-cocaine reward effects likely through a dopamine-dependent mechanism, and therefore, deserves further study as a new pharmacotherapy for cocaine use disorder.

Design, Synthesis, and Physicochemical and Pharmacological Profiling of 7-Hydroxy-5-oxopyrazolo[4,3-b]pyridine-6-carboxamide Derivatives with Antiosteoarthritic Activity In Vivo.

The hallmark of joint diseases, such as osteoarthritis (OA), is pain, originating from both inflammatory and neuropathic components, and compounds able to modulate the signal transduction pathways of the cannabinoid type-2 receptor (CB2R) can represent a helpful option in the treatment of OA. In this perspective, a set of 18 cannabinoid type-2 receptor (CB2R) ligands was developed based on an unprecedented structure. With the aim of improving the physicochemical properties of previously reported 4-hydroxy-2-quinolone-3-carboxamides, a structural optimization program led to the discovery of isosteric 7-hydroxy-5-oxopyrazolo[4,3-b]pyridine-6-carboxamide derivatives. These new compounds are endowed with high affinity for the CB2R and moderate to good selectivity over the cannabinoid type-1 receptor (CB1R), associated with good physicochemical characteristics. As to the functional activity at the CB2R, compounds able to act either as agonists or as inverse agonists/antagonists were discovered. Among them, compound 51 emerged as a potent CB2R agonist able to reduce pain in rats carrying OA induced by injection of monoiodoacetic acid (MIA).

Cannabinoid receptor 2 agonist promotes parameters implicated in mucosal healing in patients with inflammatory bowel disease.

BACKGROUND: Cannabis benefits patients with inflammatory bowel disease (IBD). Cannabinoid receptors are expressed in gut immune cells and in epithelial cells of inflamed guts. Mucosal healing (MH) requires epithelial layer restoration. OBJECTIVE: To analyze the effects of CB2 agonist on parameters implicated in gut inflammation and MH. METHODS: Mucosal samples from areas of inflamed/uninflamed colon from 16 patients with IBD were cultured without/with cannabinoid receptor 2 (CB2) agonist (JWH-133, 10 µM, 6 hours (hr)), and analyzed for epithelial/stromal cell proliferation, apoptosis (secretome matrix metalloproteinase 9 (MMP9) activity, which impairs epithelial permeability) and interleukin-8 (IL-8) levels (n = 5-9). In addition, Caco-2 (colon carcinoma epithelial cells) were cultured with biopsy secretomes (48 hr), and analyzed for phenotype and protein markers of proliferation (proliferating cell nuclear antigen), autophagy (LC3IIB) and permeability (Zonula occludens-1) (n = 4-6). RESULTS: Uninflamed tissue had higher epithelial proliferation (Ki67: 50%↑, p < 0.05), and reduced secretome MMP9 activity and IL-8 levels (>50%↓, p < 0.05) compared to inflamed tissue. Treatment with CB2 agonist had no effect on epithelial apoptosis, but increased epithelial Ki67 expression (25%), and reduced secretome MMP9 and IL-8 levels in inflamed biopsies. Secretomes of CB2-treated biopsies increased Caco-2 number, migration, proliferating cell nuclear antigen and LC3IIB expression (all, p < 0.05), but had no effect on ZO-1. CONCLUSION: Using ex vivo and in vitro human models, we demonstrated that manipulating the cannabinoid system affects colon cells and secretome characteristics that facilitate MH in IBD.

Celastrol is a novel selective agonist of cannabinoid receptor 2 with anti-inflammatory and anti-fibrotic activity in a mouse model of systemic sclerosis.

BACKGROUND: Increasing evidence indicated that the cannabinoid receptors were involved in the pathogenesis of organ fibrogenesis. PURPOSE: The purpose of this study was to discover novel cannabinoid receptor 2 (CB2) agonist and assess the potential of CB2 activation in treating systemic sclerosis. METHODS: A gaussia princeps luciferase-based split luciferase complementation assay (SLCA) was developed for detection of the interaction between CB2 and ß-arrestin2. A library of 366 natural products was then screened as potential CB2 agonist using SLCA approach. Several GPCR functional assays, including HTRF-based cAMP assay and calcium mobilization were also utilized to evaluated CB2 activation. Bleomycin-induced experimental systemic sclerosis was used to assess the in vivo anti-fibrotic effects. Dermal thickness and collagen content were evaluated via H&E and sirius red staining. RESULTS: Celastrol was identified as a new agonist of CB2 by using SLCA. Furthermore, celastrol triggers several CB2-mediated downstream signaling pathways, including calcium mobilization, inhibition of cAMP accumulation, and receptor desensitization in a dose-dependent manner, and it has a moderate selectivity on CB1. In addition, celastrol exhibited the anti-inflammatory properties on lipopolysaccharide (LPS) treated murine Raw 264.7 macrophages and primary macrophages. Finally, we found that celastrol exerts anti-fibrotic effects in the bleomycin-induced systemic sclerosis mouse model accompanied by reduced inflammatory conditions. CONCLUSION: Taken together, celastrol is identified a novel selective CB2 agonist using a new developed arrestin-based SLCA, and CB2 activation by celastrol reduces the inflammatory response, and prevents the development of dermal fibrosis in bleomycin-induced systemic sclerosis mouse model.

Soothing Effect of Pogostemon cablin Extract (Patchouli), via Cannabinoid Receptor 2 Modulation in Human Skin.

Skin functions as a neuro-immuno-endocrine tissue with well-defined neuronal networks and functions. The endocannabinoid system has been proven to be an important, homeostatic regulator for homeostatic and inflammatory events. The system comprises endogenous or exogenous ligands and receptors (CB1 and CB2). In the present study, we evaluated the soothing properties of a Pogostemon cablin (patchouli) extract. Agonist AM1241 and antagonist AM630 were used for CB2 receptor activation/inhibition. Expression of CB2 receptor and ß-endorphin was monitored by immunohistochemistry. Skin inflammation was induced with ultraviolet B (UVB) or lipopolysaccharide (LPS), and the following markers were used to highlight the anti-inflammatory properties of the extract: transient receptor potential vanilloid 1 (TRPV1), interleukin receptors 1 (IL1R1), and the interleukin 6 signal transducer (IL6ST). Our results demonstrated the implication of the CB2 receptor in the skin inflammation process. The expression of CB2 receptor and ß-endorphin was increased 48 hours after application of the extract. Furthermore, patchouli extract application helped to reduce IL1R1, IL6ST, and TRPV1 expression, in skin exposed to UVB or LPS. In conclusion, the application of the patchouli extract helps maintain skin integrity and reduce skin discomfort via modulation of CB2 receptor stimulation and the subsequent ß-endorphin release.

The therapeutic role of cannabinoid receptors and its agonists or antagonists in Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disease and its characteristic is the progressive degeneration of dopaminergic neurons within the substantia nigra (SN) of the midbrain. There is hardly any clinically proven efficient therapeutics for its cure in several recent preclinical advances proposed to treat PD. Recent studies have found that the endocannabinoid signaling system in particular the comprised two receptors, CB1 and CB2 receptors, has a significant regulatory function in basal ganglia and is involved in the pathogenesis of PD. Therefore, adding new insights into the biochemical interactions between cannabinoids and other signaling pathways may help develop new pharmacological strategies. Factors of the endocannabinoid system (ECS) are abundantly expressed in the neural circuits of basal ganglia, where they interact interactively with glutamatergic, γ-aminobutyric acid-ergic (GABAergic), and dopaminergic signaling systems. Although preclinical studies on PD are promising, the use of cannabinoids at the clinical level has not been thoroughly studied. In this review, we evaluated the available evidence and reviewed the involvement of ECS in etiologies, symptoms and treatments related to PD. Since CB1 and CB2 receptors are the two main receptors of endocannabinoids, we primarily put the focus on the therapeutic role of CB1 and CB2 receptors in PD. We will try to determine future research clues that will help understand the potential therapeutic benefits of the ECS in the treatment of PD, aiming to open up new strategies and ideas for the treatment of PD.