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Recent Advances on Type-2 Cannabinoid (CB2) Receptor Agonists and Their Therapeutic Potential
Abstract
In the last decade, selective modulators of type-2 cannabinoid receptor (CB2) have become a major focus to target endocannabinoid signaling in humans. Indeed, heterogeneously expressed within our body, CB2 actively regulates several physio-pathological processes, thus representing a promising target for developing specific and safe therapeutic drugs. If CB2 modulation has been extensively studied since the very beginning for the treatment of pain and inflammation, the more recent involvement of this receptor in other pathological conditions has further strengthened the pursuit of novel CB2 agonists in the last five years. Against this background, here we discuss the most recent evidence of the protective effects of CB2 against pathological conditions, emphasizing central nervous system disorders, bone and synovial diseases, and cancer. We also summarize the most recent advances in the development of CB2 agonists, focusing on the correlation between different chemical classes and diverse therapeutic applications. Data mining includes a review of the CB2 ligands disclosed in patents also released in the last five years. Finally, we discuss how the recent elucidation of CB2 tertiary structure has provided new details for the rational design of novel and more selective CB2 agonists, thus supporting innovative strategies to develop effective therapeutics. Our overview of the current knowledge on CB2 agonists provides pivotal information on the structure and function of different classes of molecules and opens possible avenues for future research.
... COVID-19 severity was associated with hyperinflammation, which is characterized by activation of the innate immune response, including the so-called cytokine storm-an excessive or uncontrolled release of proinflammatory cytokines [2]. For this reason, antiinflammatory interventions have been thought to block persistent inflammation, also due to the cytokine storm [3][4][5][6][7][8]. Moreover, an elevated diffuse inflammatory cytokine profile was associated with post-acute sequelae of SARS-CoV-2 (PASC), commonly known as long COVID. ...
... Chemical structures of the thromboxane receptor antagonist ramatroban (4), the pa caspase inhibitor emricasan(5), and the sodium-hydrogen antiporter 1 (NHE-1) inhibi rimeporide(6). ...
... Chemical structures of the thromboxane receptor antagonist ramatroban (4), the pan-caspase inhibitor emricasan(5), and the sodium-hydrogen antiporter 1 (NHE-1) inhibitor rimeporide(6). ...
People affected by COVID-19 are exposed to, among others, abnormal clotting and endothelial dysfunction, which may result in deep vein thrombosis, cerebrovascular disorders, and ischemic and non-ischemic heart diseases, to mention a few. Treatments for COVID-19 include antiplatelet (e.g., aspirin, clopidogrel) and anticoagulant agents, but their impact on morbidity and
mortality has not been proven. In addition, due to viremia-associated interconnected prothrombotic and proinflammatory events, anti-inflammatory drugs have also been investigated for their ability to mitigate against immune dysregulation due to the cytokine storm. By retrieving patent literature
published in the last two years, small molecules patented for long-COVID-related blood clotting and hematological complications are herein examined, along with supporting evidence from preclinical and clinical studies. An overview of the main features and therapeutic potentials of small
molecules is provided for the thromboxane receptor antagonist ramatroban, the pan-caspase inhibitor emricasan, and the sodium–hydrogen antiporter 1 (NHE-1) inhibitor rimeporide, as well as natural polyphenolic compounds.
... Two isoforms of CB 2 have been discovered in humans, namely CB 2 A and CB 2 B. CB 2 A is primarily found in the testis and is also expressed, albeit to a lesser extent, in certain regions of the brain, such as the amygdala, caudate, putamen, nucleus accumbens, cortex, hippocampus, and cerebellum. On the other hand, CB 2 B is the main isoform found in the spleen and leukocytes, but it is also present in other peripheral tissues [13,17]. ...
... Hence, subtype-selective probes are the most effective means of addressing these problems. CB 2 R agonists have shown vast potential, offering directions for future selective medication development [17,21,22]. Understanding ligand-protein interactions, target engagement, and the mechanism of action is crucial to the development of novel medications. ...
... The limited dependability of the antibodies used in immunochemistry investigations to study CB2R receptor expression underscores the need for further investigation to enhance our understanding of the participation of the CB2 receptor in the central nervous system and neuroinflammation. Numerous CB2 ligands have been synthesized, and their receptor function has been studied in detail ( Figure 2); however, drugs solely targeting CB2 have not yet made it to the clinic [16,17,22]. ...
The cannabinoid receptors CB1 and CB2 are class A G protein-coupled receptors (GPCRs) that are activated via endogenous lipids called endocannabinoids. The endocannabinoid system (ECS) plays a critical role in the regulation of several physiological states and a wide range of diseases. In recent years, drug discovery approaches targeting the cannabinoid type 2 receptor (CB2R) have gained prominence. Particular attention has been given to selective agonists targeting the CB2 receptors to circumvent the neuropsychotropic side effects associated with CB1 receptors. The pharmacological modulation of CB2R holds therapeutic promise for various diseases, such as inflammatory disorders and immunological conditions, as well as pain management and cancer treatment. Recently, the utilization of fluorescent probes has emerged as a valuable technique for investigating the interactions between ligands and proteins at an exceptional level of spatial and temporal precision. In this review, we aim to examine the progress made in the development of fluorescent probes targeting CB2 receptors and highlight their significance in facilitating the successful clinical translation of CB2R-based therapies.
... Thus, the entrance of ligands to the orthosteric site located within the 7TM domain occurs through the membrane bilayer [34][35][36]. This is the case of cannabinoid receptors, type 1 (CB 1 R) and type 2 (CB 2 R), that respond to endocannabinoids with long hydrophobic tails such as anandamide and 2-arachidonoyl glycerol, as well as phytocannabinoids like Δ⁹-tetrahydrocannabinol [37,38]. The pathway of ligand entry to CB 2 R, determined by MD simulations, defines two transient binding sites [39]: a membrane-facing pocket between TMs 1 and 7 [40] and a bundle-facing allosteric pocket located near the orthosteric site [41]. ...
... However, these findings also suggest that PM369 could offer new therapeutic strategies by controlling the signaling pathways mediated by CB 2 R. CB 2 R plays a crucial role in modulating immune responses and inflammation [66]. Its upregulation under pathological conditions and its association with anti-inflammatory effects highlight the therapeutic potential of targeting CB 2 R in various disease states such as autoimmune diseases, neuroinflammation, and tissue injury [37,38]. ...
... Since endocannabinoids (eCBs) are rapidly degraded, metabolically stable agonists that selectively target CB1Rs and CB2Rs, respectively, have proven useful tools to elucidate their physiological roles and to modulate the endocannabinoid system (ECS) 9,10 . Importantly, the tissue protective role of CB2Rs in pathophysiological processes related to inflammation and their lack of central effects have rendered them an attractive drug target 11 . Consequently, structurally diverse CB2R-selective agonists are being developed as drug candidates 12 for various diseases/pathological conditions ranging from chronic and inflammatory pain 13 , pruritus 14 , diabetic neuropathy 15 , liver cirrhosis 16 , and various types of ischemic-reperfusion injury [17][18][19] , to autoimmune, kidney and fibrotic diseases [4][5][6]15,[20][21][22][23][24][25][26] . ...
The cannabinoid CB2 receptor (CB2R) is a potential therapeutic target for distinct forms of tissue injury and inflammatory diseases. To thoroughly investigate the role of CB2R in pathophysiological conditions and for target validation in vivo, optimal pharmacological tool compounds are essential. Despite the sizable progress in the generation of potent and selective CB2R ligands, pharmacokinetic parameters are often neglected for in vivo studies. Here, we report the generation and characterization of a tetra-substituted pyrazole CB2R full agonist named RNB-61 with high potency (Ki 0.13 to 1.81 nM, depending on species) and a peripherally restricted action due to P-glycoprotein mediated efflux from the brain. 3H and 14C labelled RNB-61 showed apparent Kd values < 4 nM towards human CB2R in both cell and tissue experiments. The >6000-fold selectivity over CB1 receptors and negligible off-targets in vitro, combined with high oral bioavailability and suitable systemic pharmacokinetic (PK) properties, prompted the assessment of RNB-61 in a mouse ischemia-reperfusion model of acute kidney injury (AKI) and in a rat model of chronic kidney injury/inflammation and fibrosis (CKI) induced by unilateral ureteral obstruction. RNB-61 exerted dose-dependent nephroprotective and/or antifibrotic effects in the AKI/CKI models. Thus, RNB-61 is an optimal CB2R tool compound for preclinical in vivo studies with superior biophysical and PK properties over generally used CB2R ligands.
... The high concentration of cannabinoids in the flowers explains their preference, as these compounds modulate the body's homeostasis and mood by interacting with the peripheral and central nervous systems, especially with the cannabinoid-1 and cannabinoid-2 (CB1 and CB2) receptors. [33][34][35][36][37] However, some studies indicated different preferences for other plant parts. For example, Lozano et al. reported a significant inclination toward the seeds, which are rich in essential fatty acids and proteins. ...
Cannabis sativa (family Cannabaceae) is a plant of Asian origin, widely recognized for its psychoactive and therapeutic properties, which have been harnessed for recreational and medicinal purposes since ancient times. Currently, a number of scientific studies have investigated the medicinal value of Cannabis, while traditional knowledge regarding its therapeutic and cosmetic applications is steadily declining. This research project aimed to collect data on the traditional uses of Cannabis sativa in the provinces of Taounate and Al Hoceïma, located in Northern Morocco, by conducting an ethnobotanical survey. Eighty-five structured questionnaires were distributed to traditional practitioners, peasants, herbalists, and users of the plant over a period of four months. The questionnaires consisted of open-ended and closed-ended questions, covering topics such as the plant parts used, the modes of administration, the indications, the dosage, the frequency of use, and the adverse effects of Cannabis sativa L. Descriptive statistics was used to analyze the data and chi-square tests were used to determine the associations between the variables. The study revealed that Cannabis sativa is commonly utilized as a recreational or medicinal plant for several purposes, including the treatment of physical and mental health conditions, cosmetic application, and even as a building material. Furthermore, the findings indicated that various factors, such as demographic data and patterns of cannabis use, are associated with the specific plant parts used and the modes of administration.
... This result demonstrated the potential of the molecular-constrained optimization method in generating CBD analogs that exhibit enhanced interactions with the targets. Additionally, given the recognized significance of the endocannabinoid system in AD [48,49], we employed the CIRCE [50] to validate the interaction between CBD analogs and this system. This software predicts whether a query compound acts as a ligand in the endocannabinoid system by comparing its similarity to known ligands interacting with CB1, CB2, and GPRs. ...
Cannabidiol (CBD), a major non-psychoactive component of the cannabis plant, has shown therapeutic potential in Alzheimer’s disease (AD). In this study, we identified potential CBD targets associated with AD using a drug-target binding affinity prediction model and generated CBD analogs using a genetic algorithm combined with a molecular docking system. As a result, we identified six targets associated with AD: Endothelial NOS (ENOS), Myeloperoxidase (MPO), Apolipoprotein E (APOE), Amyloid-beta precursor protein (APP), Disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), and Presenilin-1 (PSEN1). Furthermore, we generated CBD analogs for each target that optimize for all desired drug-likeness properties and physicochemical property filters, resulting in improved pIC50 values and docking scores compared to CBD. Molecular dynamics (MD) simulations were applied to analyze each target’s CBD and highest-scoring CBD analogs. The MD simulations revealed that the complexes of ENOS, MPO, and ADAM10 with CBD exhibited high conformational stability, and the APP and PSEN1 complexes with CBD analogs demonstrated even higher conformational stability and lower interaction energy compared to APP and PSEN1 complexes with CBD. These findings demonstrated the capable binding of the six identified targets with CBD and the enhanced binding stability achieved with the developed CBD analogs for each target.
... These findings have been substantiated by post-mortem data from diabetic human donors [8,46] or by measuring the thickness of retinal layers (OCT) in subjects [47] and in diabetic rats [8]. Numerous reports have shown the neuroprotective actions of CB2R agonists in the brain and their therapeutic potential in a plethora of neurological disorders [48]. In contrast, fewer studies have examined the role of CB2R activation in the retina. ...
Oxidative stress, neurodegeneration, neuroinflammation, and vascular leakage are believed to play a key role in the early stage of diabetic retinopathy (ESDR). The aim of this study was to investigate the blockade of cannabinoid receptor 1 (CB1R) and activation of cannabinoid receptor 2 (CB2R) as putative therapeutics for the treatment of the early toxic events in DR. Diabetic rats [streptozotocin (STZ)-induced] were treated topically (20 μL, 10 mg/mL), once daily for fourteen days (early stage DR model), with SR141716 (CB1R antagonist), AM1710 (CB2R agonist), and the dual treatment SR141716/AM1710. Immunohistochemical-histological, ELISA, and Evans-Blue analyses were performed to assess the neuroprotective and vasculoprotective properties of the pharmacological treatments on diabetes-induced retinal toxicity. Activation of CB2R or blockade of CB1R, as well as the dual treatment, attenuated the nitrative stress induced by diabetes. Both single treatments protected neural elements (e.g., RGC axons) and reduced vascular leakage. AM1710 alone reversed all toxic insults. These findings provide new knowledge regarding the differential efficacies of the cannabinoids, when administered topically, in the treatment of ESDR. Cannabinoid neuroprotection of the diabetic retina in ESDR may prove therapeutic in delaying the development of the advanced stage of the disease.
... Increased local eCB levels and CB₂R expression upon inflammation/tissue injury trigger fast signaling responses in immune and other cells leading mostly to a sequence of activities of a protective nature. Furthermore, eCBs and/or synthetic CB₂R activators have been reported to attenuate inflammation and associated tissue injury in a huge number of pathological conditions/ diseases, ranging from cardiovascular, gastrointestinal, liver, kidney, lung, neurodegenerative/neuroinflammatory, skin pathologies, rheumatoid arthritis, endometriosis, to eye diseases [7,8] (Figure 1). ...
Is CB₂R a hidden treasure trove for treating inflammatory diseases?
Expert scientists, Mauro Maccarrone and Uwe Grether, explain how CB₂R can be used to treat inflammatory diseases. Looking at GPCRs, G-protein-coupled receptors, they discuss their important role in multiple physio-pathological conditions throughout the body, including treating inflammatory diseases. Receptors are important protein structures that allow cells to interact with their environment or control activities within a cell. Within these GPCRs, Grether and Maccarrone analyse the role of type-2 cannabinoid receptors (CB₂R), which represent a promising target for developing novel therapeutics to treat a plethora of diseases linked to its deregulation, as CB₂R and the closely related type-1 cannabinoid receptor (CB₁R) are essential elements of the endocannabinoid system (ECS). Overall, the ECS plays a fundamental role for human health and disease state, as increased local eCB levels and CB₂R expression upon inflammation/tissue injury trigger fast signaling responses in immune and other cells leading mostly to a sequence of activities of a protective nature. This allows eCBs and/or synthetic CB₂R activators to attenuate inflammation and associated tissue injury in a huge number of pathological conditions/ diseases, ranging from cardiovascular, gastrointestinal, liver, kidney, lung, neurodegenerative/neuroinflammatory, skin pathologies, rheumatoid arthritis, endometriosis, to eye diseases.
Herein, we studied the expression of endocannabinoid receptor 2 (CB2R), a known inflammation mediator, in several lysosomal storage disorder (LSD) animal models and evaluated it as a potential biomarker and therapeutic target for these diseases. CB2R was highly elevated in the plasma of Farber disease and mucopolysaccharidosis (MPS) type IIIA mice, followed by Fabry disease and MPS type I mice. Mice with acid sphingomyelinase‐deficient Niemann‐Pick disease (ASMD) and rats with MPS type VI exhibited little or no plasma CB2R elevation. High‐level expression of CB2R was also observed in tissues of Farber and MPS IIIA mice. Treatment of MPS IIIIA patient cells with CB2R agonists led to a reduction of CB2R and monocyte chemoattractant protein‐1 (MCP‐1), a chemotactic factor that is elevated in this LSD. Treatment of MPS IIIA mice with one of these agonists (JWH133) led to a reduction of plasma and tissue CB2R and MCP‐1, a reduction of glial fibrillary acidic protein (GFAP) in the brain, and an improvement in hanging test performance. JWH133 treatment of Farber disease mice also led to a reduction of MCP‐1 in tissues and plasma, and treatment of these mice by enzyme replacement therapy (ERT) led to a reduction of plasma CB2R, indicating its potential to monitor treatment response. Overall, these findings suggest that CB2R should be further examined as a potential therapeutic target for the LSDs and may also be a useful biomarker to monitor the impact of therapies.
The endocannabinoid system, which includes cannabinoid receptor 1 and 2 subtypes (CB1R and CB2R, respectively), is responsible for the onset of various pathologies including neurodegeneration, cancer, neuropathic and inflammatory pain, obesity, and inflammatory bowel disease. Given the high similarity of CB1R and CB2R, generating subtype-selective ligands is still an open challenge. In this work, the Cannabinoid Iterative Revaluation for Classification and Explanation (CIRCE) compound prediction platform has been generated based on explainable machine learning to support the design of selective CB1R and CB2R ligands. Multilayer classifiers were combined with Shapley value analysis to facilitate explainable predictions. In test calculations, CIRCE predictions reached ∼80% accuracy and structural features determining ligand predictions were rationalized. CIRCE was designed as a web-based prediction platform that is made freely available as a part of our study.
The discovery of selective agonists of cannabinoid receptor 2 (CB2) is strongly pursued to successfully tuning endocannabinoid signaling for therapeutic purposes. However, the design of selective CB2 agonists is still challenging because of the high homology with the cannabinoid receptor 1 (CB1) and for the yet unclear molecular basis of the agonist/antagonist switch. Here, the 1,3-benzoxazine scaffold is presented as a versatile chemotype for the design of CB2 agonists from which 25 derivatives were synthesized. Among these, compound 7b5 (CB2 EC50 = 110 nM, CB1 EC50 > 10 μM) demonstrated to impair proliferation of triple negative breast cancer BT549 cells and to attenuate the release of pro-inflammatory cytokines in a CB2-dependent manner. Furthermore, 7b5 abrogated the activation of extracellular signal-regulated kinase (ERK) 1/2, a key pro-inflammatory and oncogenic enzyme. Finally, molecular dynamics studies suggested a new rationale for the in vitro measured selectivity and for the observed agonist behavior.
The cannabis derivative marijuana is the most widely used recreational drug in the Western world, that is consumed by an estimated 83 million individuals (~3% of the world population). In recent years, there has been a marked transformation in society regarding the risk perception of cannabis, driven by its legalization and medical use in many states in the USA and worldwide. Compelling research evidence and the FDA cannabis-derived cannabidiol approval for severe childhood epilepsy have confirmed the large therapeutic potential of cannabidiol itself, Δ9-tetrahydrocannabinol (THC) and other plant-derived cannabinoids (phytocannabinoids). Of note, our body has a complex endocannabinoid system (ECS) - made of receptors, metabolic enzymes and transporters - that is also regulated by phytocannabinoids. The first endocannabinoid to be discovered 30 years ago was anandamide (N-arachidonoyl-ethanolamine); since then, distinct elements of ECS have been the target of drug design programs aimed at curing (or at least slowing down) a number of human diseases, both in the central nervous system and at the periphery. Here, a critical review of our knowledge of the goods and bads of ECS as a therapeutic target are presented, in order to define the benefits of ECS-active phytocannabinoids and ECS-oriented synthetic drugs for human health. Significance Statement The endocannabinoid system plays important roles everywhere in our body and is either involved in mediating key processes of central and peripheral diseases or represents a therapeutic target for treatment. Understanding structure, function, and pharmacology of the components of this complex system, and in particular of key receptors (like CB1R and CB2R) and metabolic enzymes (like FAAH and MAGL), will advance our understanding of endocannabinoid signaling and activity at molecular, cellular, and system levels providing new opportunities to treat patients.
Simple Summary
Extracellular matrix is a three-dimensional network of macromolecules that provide structural and biochemical support to surrounding cells. Extracellular matrix plays a critical role in the development and progression of cancer. The extracellular matrix of the tumor is very different from the matrix of the normal tissue. Mainly fibroblasts produce and regulate matrix remodeling, but in cancer, the tumor matrix also originates from cancer cells. We describe the mechanisms of how the protein composition and structure of the extracellular matrix changes during cancer progression and how abnormal matrix deregulates the behavior of stromal cells and influences cancer progression.
Abstract
The extracellular matrix (ECM) is highly dynamic as it is constantly deposited, remodeled and degraded to maintain tissue homeostasis. ECM is a major structural component of the tumor microenvironment, and cancer development and progression require its extensive reorganization. Cancerized ECM is biochemically different in its composition and is stiffer compared to normal ECM. The abnormal ECM affects cancer progression by directly promoting cell proliferation, survival, migration and differentiation. The restructured extracellular matrix and its degradation fragments (matrikines) also modulate the signaling cascades mediated by the interaction with cell-surface receptors, deregulate the stromal cell behavior and lead to emergence of an oncogenic microenvironment. Here, we summarize the current state of understanding how the composition and structure of ECM changes during cancer progression. We also describe the functional role of key proteins, especially tenascin C and fibronectin, and signaling molecules involved in the formation of the tumor microenvironment, as well as the signaling pathways that they activate in cancer cells.
Cancer cells rewire their metabolic programs to favor biological processes that promote cell survival, proliferation, and dissemination. Among this relevant reprogramming, sphingolipid metabolism provides metabolites that can favor or oppose these hallmarks of cancer. The sphingolipid ceramide 1-phosphate (C1P) and the enzyme responsible for its biosynthesis, ceramide kinase (CERK), are well established regulators of cell growth and survival in normal, as well as malignant cells through stress-regulated signaling pathways. This metabolite also promotes cell survival, which has been associated with the feedback regulation of other antitumoral sphingolipids or second messengers. C1P also regulates cancer cell invasion and migration of different types of cancer, including lung, breast, pancreas, prostate, or leukemia cells. More recently, CERK and C1P have been implicated in the control of inflammatory responses. The present review provides an updated view on the important role of CERK/C1P in the regulation of cancer cell growth, survival, and dissemination.
Protein synthesis is essential for cells to perform life metabolic processes. Pathological alterations of protein content can lead to particular diseases. Cells have an intrinsic array of mechanisms and pathways that are activated when protein misfolding, accumulation, aggregation or mislocalization occur. Some of them (like the unfolded protein response) represent complex interactions between endoplasmic reticulum sensors and elongation factors that tend to increase expression of chaperone proteins and/or repress translation in order to restore protein homeostasis (also known as proteostasis). This is even more important in neurons, as they are very susceptible to harmful effects associated with protein overload and proteostatic mechanisms are less effective with age. Several neurodegenerative pathologies such as Alzheimer's, Parkinson's, and Huntington's diseases, amyotrophic lateral sclerosis and frontotemporal dementia exhibit a particular molecular signature of distinct, unbalanced protein overload. In amyotrophic lateral sclerosis and frontotemporal dementia, the majority of cases present intracellular inclusions of ubiquitinated transactive response DNA-binding protein of 43 kDa (TDP-43). TDP-43 is an RNA binding protein that participates in RNA metabolism, among other functions. Dysregulation of TDP-43 (e.g. aggregation and mislocalization) can dramatically affect neurons, and this has been linked to disease development. Expression of amyotrophic lateral sclerosis/frontotemporal dementia TDP-43-related mutations in cellular and animal models has been shown to recapitulate key features of the amyotrophic lateral sclerosis/frontotemporal dementia disease spectrum. These variants can be causative of degeneration onset and progression. Most neurodegenerative diseases (including amyotrophic lateral sclerosis and frontotemporal dementia) have no cure at the moment; however, modulating translation has recently emerged as an attractive approach that can be performed at several steps (i.e. regulating activation of initiation and elongation factors, inhibiting unfolded protein response activation or inducing chaperone expression and activity). This review focuses on the features of protein imbalance in neurodegenerative disorders and the relevance of developing therapeutical compounds aiming at restoring proteostasis. We strive to highlight the importance of research on drugs that, not only restore protein imbalance without compromising translational activity of cells, but are also as safe as possible for the patients.
Background
The cannabinoid CB 2 receptor (CB 2 R), which is a target to afford neuroprotection, and N-methyl-D-aspartate (NMDA) ionotropic glutamate receptors, which are key in mediating excitatory neurotransmission, are expressed in both neurons and glia. As NMDA receptors are the target of current medication in Alzheimer’s disease patients and with the aim of finding neuromodulators of their actions that could provide benefits in dementia, we hypothesized that cannabinoids could modulate NMDA function.
Methods
Immunocytochemistry was used to analyze the colocalization between CB 2 and NMDA receptors; bioluminescence resonance energy transfer was used to detect CB 2 -NMDA receptor complexes. Calcium and cAMP determination, mitogen-activated protein kinase (MAPK) pathway activation, and label-free assays were performed to characterize signaling in homologous and heterologous systems. Proximity ligation assays were used to quantify CB 2 -NMDA heteromer expression in mouse primary cultures and in the brain of APP Sw/Ind transgenic mice, an Alzheimer’s disease model expressing the Indiana and Swedish mutated version of the human amyloid precursor protein (APP).
Results
In a heterologous system, we identified CB 2 -NMDA complexes with a particular heteromer print consisting of impairment by cannabinoids of NMDA receptor function. The print was detected in activated primary microglia treated with lipopolysaccharide and interferon-γ. CB 2 R activation blunted NMDA receptor-mediated signaling in primary hippocampal neurons from APP Sw/Ind mice. Furthermore, imaging studies showed that in brain slices and in primary cells (microglia or neurons) from APP Sw/Ind mice, there was a marked overexpression of macromolecular CB 2 -NMDA receptor complexes thus becoming a tool to modulate excessive glutamate input by cannabinoids.
Conclusions
The results indicate a negative cross-talk in CB 2 -NMDA complexes signaling. The expression of the CB 2 -NMDA receptor heteromers increases in both microglia and neurons from the APP Sw/Ind transgenic mice, compared with levels in samples from age-matched control mice.
In glucocorticoid (GC)-induced osteonecrosis of the femoral head (ONFH), downregulated osteogenic ability and damaged blood supply are two key pathogenic mechanisms. Studies suggested that cannabinoid receptor 2 (CB2) is expressed in bone tissue and it plays a positive role in osteogenesis. However, whether CB2 could enhance bone formation and blood supply in GC-induced ONFH remains unknown. In this study, we focused on the effect of CB2 in GC-induced ONFH and possible mechanisms in vitro and in vivo. By using GC-induced ONFH rat model, rat-bone mesenchymal stem cells (BMSCs) and human umbilical vein endothelial cells (HUVECs) to address the interaction of CB2 in vitro and in vivo, we evaluate the osteogenic and angiogenic effect variation and possible mechanisms. Micro-CT, histological staining, angiography, calcein labeling, Alizarin red staining (ARS), alkaline phosphatase (ALP), tartrate-resistant acid phosphatase (TRAP) staining, TUNEL staining, migration assay, scratch assay, and tube formation were applied in this study. Our results showed that selective activation of CB2 alleviates GC-induced ONFH. The activation of CB2 strengthened the osteogenic activity of BMSCs under the influence of GCs by promotion of GSK-3β/β-catenin signaling pathway. Furthermore, CB2 promoted HUVECs migration and tube-forming capacities. Our findings indicated that CB2 may serve as a rational new treatment strategy against GC-induced ONFH by osteogenesis activation and maintenance of blood supply.
The cannabinoid 1 (CB1) receptor regulates appetite and body weight; however, unwanted central side effects of both agonists (in wasting disorders) or antagonists (in obesity and diabetes) have limited their therapeutic utility. At the peripheral level, CB1 receptor activation impacts the energy balance of mammals in a number of different ways: inhibiting satiety and emesis, increasing food intake, altering adipokine and satiety hormone levels, altering taste sensation, decreasing lipolysis (fat break down), and increasing lipogenesis (fat generation). The CB1 receptor also plays an important role in the gut–brain axis control of appetite and satiety. The combined effect of peripheral CB1 activation is to promote appetite, energy storage, and energy preservation (and the opposite is true for CB1 antagonists). Therefore, the next generation of CB1 receptor medicines (agonists and antagonists, and indirect modulators of the endocannabinoid system) have been peripherally restricted to mitigate these issues, and some of these are already in clinical stage development. These compounds also have demonstrated potential in other conditions such as alcoholic steatohepatitis and diabetic nephropathy (peripherally restricted CB1 antagonists) and pain conditions (peripherally restricted CB1 agonists and FAAH inhibitors). This review will discuss the mechanisms by which peripheral CB1 receptors regulate body weight, and the therapeutic utility of peripherally restricted drugs in the management of body weight and beyond.
The application of scaffolding materials is believed to hold enormous potential for tissue regeneration. Despite the widespread application and rapid advance of several tissue-engineered scaffolds such as natural and synthetic polymer-based scaffolds, they have limited repair capacity due to the difficulties in overcoming the immunogenicity, simulating in-vivo microenvironment, and performing mechanical or biochemical properties similar to native organs/tissues. Fortunately, the emergence of decellularized extracellular matrix (dECM) scaffolds provides an attractive way to overcome these hurdles, which mimic an optimal non-immune environment with native three-dimensional structures and various bioactive components. The consequent cell-seeded construct based on dECM scaffolds, especially stem cell-recellularized construct, is considered an ideal choice for regenerating functional organs/tissues. Herein, we review recent developments in dECM scaffolds and put forward perspectives accordingly, with particular focus on the concept and fabrication of decellularized scaffolds, as well as the application of decellularized scaffolds and their combinations with stem cells (recellularized scaffolds) in tissue engineering, including skin, bone, nerve, heart, along with lung, liver and kidney.
The tight regulation of microglia activity is key for precise responses to potential threats, while uncontrolled and exacerbated microglial activity is neurotoxic. Microglial toll‐like receptors (TLRs) are indispensable for sensing different types of assaults and triggering an innate immune response. Cannabinoid receptor 2 (CB2) signaling is a key pathway to control microglial homeostasis and activation, and its activation is connected to changes in microglial activity. We aimed to investigate how CB2 signaling impacts TLR‐mediated microglial activation. Here, we demonstrate that deletion of CB2 causes a dampened transcriptional response to prototypic TLR ligands in microglia. Loss of CB2 results in distinct microglial gene expression profiles, morphology, and activation. We show that the CB2‐mediated attenuation of TLR‐induced microglial activation is mainly p38 MAPK‐dependent. Taken together, we demonstrate that CB2 expression and signaling are necessary to fine‐tune TLR‐induced activation programs in microglia. CB2−/− microglia gene expression profile and changes in morphology after TLR stimulation are in line with a dampened microglial activation pattern. CB2 deletion alters TLR‐induced microglial activation via the p38 MAPK pathway.
The activation of the cannabinoid receptor type‐2 (CB2) afforded neuroprotection in amyotrophic lateral sclerosis (ALS) models. The objective of this study was to further investigate the relevance of the CB2 receptor through investigating the consequences of its inactivation. TDP‐43(A315T) transgenic mice were crossed with CB2 receptor knock‐out mice to generate double mutants. Temporal and qualitative aspects of the pathological phenotype of the double mutants were compared to TDP‐43 transgenic mice expressing the CB2 receptor. The double mutants exhibited significantly accelerated neurological decline, such that deteriorated rotarod performance was visible at 7 weeks, whereas rotarod performance was normal up to 11 weeks in transgenic mice with intact expression of the CB2 receptor. A morphological analysis of spinal cords confirmed an earlier death (visible at 65 days) of motor neurons labelled with Nissl staining and ChAT immunofluorescence in double mutants compared to TDP‐43 transgenic mice expressing the CB2 receptor. Evidence of glial reactivity, measured using GFAP and Iba‐1 immunostaining, was seen in double mutants at 65 days, but not in TDP‐43 transgenic mice expressing the CB2 receptor. However, at 90 days, both genotypes exhibited similar changes for all these markers, although surviving motor neurons of transgenic mice presented some morphological abnormalities in absence of the CB2 receptor that were not as evident in the presence of this receptor. This faster deterioration seen in double mutants led to premature mortality compared with TDP‐43 transgenic mice expressing the CB2 receptor. We also investigated the consequences of a pharmacological inactivation of the CB2 receptor using the selective antagonist AM630 in TDP‐43 transgenic mice, but results showed only subtle trends towards a greater deterioration. In summary, our results confirmed the potential of the CB2 receptor agonists as a neuroprotective therapy in ALS and strongly support the need to progress towards an evaluation of this potential in patients.
Colorectal cancer (CRC) is between the top three occurring cancers worldwide. The anticancer effects of Cannabinoid receptor 2 (CB2) agonist (GW833972A) in the presence and absence of its inverse agonist (SR144528) on Human colorectal adenocarcinoma cells (HT-29) was investigated. Following cell viability assays on HT-29 and HFF cells, the molecular mechanism(s) of cytotoxicity and apoptotic pathways of cell death were analyzed. The anticancer effects of CB2 agonist were measured with tumor cell migration and colony-forming assays. Real-time PCR and Western blotting techniques were used to examine any alterations in the expression of apoptotic genes. A concentration and time-dependent cytotoxicity of CB2 agonist with IC50 value of 24.92 ± 6.99 μM was obtained. The rate of lipid peroxidation was elevated, while the TNF-α concentration was declined, significantly (p < 0.05). CB2 agonist (50 μM) reduced the colony-forming capability by 83% and tumor cell migration by 50%. Apoptotic effects of CB2 agonist were revealed with the increase of apoptotic cells in Acridine orange/Ethidium bromide staining, clear DNA fragmentation, pro-apoptotic genes and proteins upregulation (Caspase-3 and p53), and significant downregulation of anti-apoptotic Bcl-2. All assessments demonstrated that CB2 agonist-induced effects were reversed by CB2 inverse agonist. These data suggest that CB2 agonists at micro-molar concentrations might be considered in the CRC treatment, and their effectiveness attributes to the apoptosis induction via upregulation of caspase-3 and p53 and downregulation of Bcl-2.
Graphic abstract
Abdominal pain is a key symptom of inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS), for which there are inadequate therapeutic options. We tested whether olorinab-a highly selective, full agonist of the cannabinoid receptor 2 (CB2)-reduced visceral hypersensitivity in models of colitis and chronic visceral hypersensitivity (CVH). In rodents, colitis was induced by intrarectal administration of nitrobenzene sulfonic acid derivatives. Control or colitis animals were administered vehicle or olorinab (3 or 30 mg/kg) twice daily by oral gavage for 5 days, starting 1 day before colitis induction. CVH mice were administered olorinab (1, 3, 10, or 30 mg/kg) twice daily by oral gavage for 5 days, starting 24 days after colitis induction. Visceral mechanosensitivity was assessed in vivo by quantifying visceromotor responses (VMR) to colorectal distension (CRD). Ex vivo afferent recordings determined colonic nociceptor firing evoked by mechanical stimuli. Colitis and CVH animals displayed significantly elevated VMRs to CRD and colonic nociceptor hypersensitivity. Olorinab treatment significantly reduced VMRs to control levels in colitis and CVH animals. Additionally, olorinab reduced nociceptor hypersensitivity in colitis and CVH states in a concentration- and CB2-dependent manner. In contrast, olorinab did not alter VMRs nor nociceptor responsiveness in control animals. CB2 mRNA was detected in colonic tissue, particularly within epithelial cells, and dorsal root ganglia, with no significant differences between healthy, colitis, and CVH states. These results demonstrate olorinab reduces visceral hypersensitivity via CB2 agonism in animal models, suggesting that olorinab may provide a novel therapy for IBD- and IBS-associated abdominal pain.
A molecular docking approach was employed to evaluate the binding affinity of six triterpenes, namely epifriedelanol, friedelin, α-amyrin, α-amyrin acetate, β-amyrin acetate, and bauerenyl acetate, towards the cannabinoid type 1 receptor (CB1). Molecular docking studies showed that friedelin, α-amyrin, and epifriedelanol had the strongest binding affinity towards CB1. Molecular dynamics simulation studies revealed that friedelin and α-amyrin engaged in stable non-bonding interactions by binding to a pocket close to the active site on the surface of the CB1 target protein. The studied triterpenes showed a good capacity to penetrate the blood–brain barrier. These results help to provide some evidence to justify, at least in part, the previously reported antinociceptive and sedative properties of Vernonia patula.
Simple Summary
This review examines the complex function of autophagy in malignancy and explores its regulation by cannabinoids in different cancers. Autophagy is an important process in the maintenance of cellular homeostasis, through the degradation and recycling of cytoplasmic constituents. The action of autophagy is highly dependent on tumour stage and type and the receptors with which ligands interact. Cannabinoids are growingly being acknowledged for their anticancer activities and are known to stimulate several mechanisms such as apoptosis and autophagy. Better understanding the mechanism of action behind autophagy and its regulation by cannabinoids will allow the development of novel cancer therapeutics.
Abstract
Autophagy is a “self-degradation” process whereby malfunctioned cytoplasmic constituents and protein aggregates are engulfed by a vesicle called the autophagosome, and subsequently degraded by the lysosome. Autophagy plays a crucial role in sustaining protein homeostasis and can be an alternative source of energy under detrimental circumstances. Studies have demonstrated a paradoxical function for autophagy in cancer, displaying both tumour suppressive and tumour promotive roles. In early phases of tumour development autophagy promotes cancer cell death. In later phases, autophagy enables cancer cells to survive and withstand therapy. Cannabinoids, which are derivatives of the Cannabis sativa L. plant, have shown to be associated with autophagy induction in cells. There is an emerging interest in studying the signalling pathways involved in cannabinoid-induced autophagy and their potential application in anticancer therapies. In this review, the molecular mechanisms involved in the autophagy degradation process will be discussed. This review also highlights a role for autophagy in cancer progression, with cannabinoid-induced autophagy presenting a novel strategy for anticancer therapy.
Alginate hydrogels have been widely used as excellent scaffold materials for implantation in biological systems because of their good biocompatibility. However, it is difficult to repair bone defects with these materials because of their poor mechanical properties. The aim of the present study was to fabricate a novel degradable alginate/palygorskite (PAL) composite hydrogel with good mechanical properties and investigate its potential for application in bone defect repair. The modified alginate-based hydrogel with increasing PAL content exhibited better mechanical properties than the original alginate hydrogel. In addition, the resulting composite hydrogel was thoroughly characterized by scanning electron microscopy (SEM). With increasing PAL content, the swelling ratio of the hydrogel increased in PBS (pH = 7.4). In vitro cytocompatibility was evaluated using bone marrow-derived mesenchymal stem cells (BMSCs) to confirm that the developed composite hydrogel was cytocompatible after 1, 3, and 7 days. All these results suggest that the developed composite hydrogel has great potential for bone tissue engineering applications. JWH133 is a selective agonist of cannabinoid receptor type 2 (CB2), which exerts dual anti-inflammatory and anti-osteoclastogenic effects. We co-cultured BMSCs with composite hydrogels loaded with JWH133, and analysis of proliferation and osteogenic differentiation indicated that the composite hydrogel loaded with JWH133 may enhance the osteogenic differentiation of rat BMSCs. Furthermore, we found that the composite hydrogel loaded with JWH133 inhibited osteoclast formation and the mRNA expression of osteoclast-specific markers. In summary, the developed composite hydrogel has a high drug-loading capacity, good biocompatibility, and strong potential as a drug carrier for treating osteoporosis by promoting osteoblast and inhibiting osteoclast formation and function.
In this Seminar, we highlight the main developments in the field of Alzheimer's disease. The most recent data indicate that, by 2050, the prevalence of dementia will double in Europe and triple worldwide, and that estimate is 3 times higher when based on a biological (rather than clinical) definition of Alzheimer's disease. The earliest phase of Alzheimer's disease (cellular phase) happens in parallel with accumulating amyloid β, inducing the spread of tau pathology. The risk of Alzheimer's disease is 60–80% dependent on heritable factors, with more than 40 Alzheimer's disease-associated genetic risk loci already identified, of which the APOE alleles have the strongest association with the disease. Novel biomarkers include PET scans and plasma assays for amyloid β and phosphorylated tau, which show great promise for clinical and research use. Multidomain lifestyle-based prevention trials suggest cognitive benefits in participants with increased risk of dementia. Lifestyle factors do not directly affect Alzheimer's disease pathology, but can still contribute to a positive outcome in individuals with Alzheimer's disease. Promising pharmacological treatments are poised at advanced stages of clinical trials and include anti-amyloid β, anti-tau, and anti-inflammatory strategies.
A number of age-associated neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD) and Amyotrophic Lateral Sclerosis (ALS), possess a shared characteristic of region-specific neurodegeneration. However, the mechanisms which determine why particular regions within the nervous system are selectively vulnerable to neurodegeneration, whilst others remain relatively unaffected throughout disease progression, remain elusive. Here, we review how regional susceptibility to the ubiquitous physiological phenomenon of normal ageing might underlie the vulnerability of these same regions to neurodegeneration, highlighting three regions archetypally associated with AD, PD and ALS (the hippocampus, substantia nigra pars compacta and ventral spinal cord, respectively), as especially prone to age-related alterations. Placing particular emphasis on these three regions, we comprehensively explore differential regional susceptibility to nervous system tissue, cellular and molecular level ageing to provide an integrated perspective on why age-related neurodegenerative diseases exhibit region-selective vulnerability. Combining these principles with increasingly recognised differences between chronological and biological ageing (termed differential or ‘delta’ ageing) might ultimately guide therapeutic approaches for these devastating neurodegenerative diseases, for which a paucity of disease modifying and/or life promoting treatments currently exist.
The aim of the present study was to explore the potential mechanism underlying the involvement of CB2 in osteoporosis. Micro‐CT was utilized to examine femur bone architecture. Also, real‐time PCR and Western blot analysis were utilized to detect the effect of 2‐AG on the expression of CB2 and Notch, or the interaction between CB2 and Notch 2. 2‐AG treatment up‐regulated BMD, Tb.Sp and SMI in OVX mice, whereas proportion of bone volume in total volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and bone mineral density (BMD) were decreased in 2‐AG‐treated OVX mice. Accordingly, 2‐AG administration up‐regulated Notch 1 expression in OVX mice but had no effect on CB2 and Notch 2 expression. Meanwhile, 2‐AG administration promoted the differentiation of hBMSCs in OVX mice, while exhibiting no effect on the proliferation of hBMSCs. Furthermore, in the cellular models, 2‐AG treatment also up‐regulated Notch 1 expression but had no effect on CB2 and Notch 2 expression, while Notch 1 shRNA had no effect on CB2 and Notch 2 expression. 2‐AG promoted cell proliferation and differentiation, which were inhibited by Notch 1 shRNA. NICD had no effect on CB2 level but increased Notch 1 expression, and CB2 shRNA decreased CB2 and Notch 1 expression. Finally, CB2 shRNA inhibited cell proliferation and differentiation, whereas NICD promoted proliferation and differentiation of hBMSCs. Our results provided further evidence for the association of CB2 gene with BMD and osteoporosis, and identified CB2 as a promising target for the treatment of osteoporosis.
Background and purpose
The endocannabinoid system became a promising target for osteoarthritis (OA) treatment. Functional selectivity of cannabinoids may increase their beneficial properties while reducing side effects. The aim of the present study was to evaluate the analgesic potential of two functionally biased CB2 agonists in different treatment regimens to propose the best pharmacological approach for OA management.
Experimental approach
Two functionally selective CB2 agonists were administered i.p. – JWH133 (cAMP biased) and GW833972A (β-arrestin biased), in a chemically induced model of OA in rats. The drugs were tested in acute and chronic treatment regimens. Analgesic effects were assessed by pressure application measurement and kinetic weight bearing. X-ray microtomography was used for the morphometric analysis of the femur’s subchondral bone tissue. Underlying biochemical changes were analysed via RT-qPCR.
Key results
Dose-response studies established the effective dose for both JWH133 and GW833972A. In chronic treatment paradigms, JWH133 was able to elicit analgesia throughout the course of the experiment, whereas GW833972A lost its efficacy after 2 days of treatment. Later studies revealed improvement in subchondral bone architecture and decrement of matrix metalloproteinases and proinflammatory factors expression following JWH133 chronic treatment.
Conclusion and implications
Data presents analgesic and disease-modifying potential of CB2 agonists in OA treatment. Moreover, the study revealed more pronounced tolerance development for analgesic effects of the β-arrestin biased CB2 agonist GW833972A. These results provide a better understanding of the molecular underpinnings of the anti-nociceptive potential of CB2 agonists and may improve drug development processes for any cannabinoid-based chronic pain therapy.
Patients suffering from malignant diseases receive very often highly emetogenic chemotherapy as part of their treatment.
With the aim of assessing the efficacy of cannabinoids in treating chemotherapy-induced nausea and vomiting (CINV), we searched the literature published until April 2020 in Medline/PubMed, Embase, the Cochrane Controlled Trials Register, and in specific web pages. Randomized clinical trials comparing cannabinoids efficacy in managing CINV with that of placebo reported absence of vomit-ing (3 trials, 168 patients) and absence of nausea and vomiting (3 trials, 288 participants). In comparison with patients receiving other antiemetics, patients receiving cannabinoids reported no nausea (5 trials, 258 participants), no vomiting (4 trials, 209 participants), and absence of both (4 trials, 414 patients). Across all trials, cannabinoids were more effective in relieving the symptoms of nausea and vomiting induced by cytotoxic therapy than placebo was and slightly better than conventional antiemetics. A retrospective review com-paring nabilone, dronabinol, delta-9-THC, and delta 8-THC with other antiemetics used to manage CINV in pediatric patients showed that these drugs could also be used as adjuvant antiemetics. Cancer patients on highly emetogenic chemotherapy but with insufficiently effective standard antiemetic therapy can be given cannabis preparations containing similar amounts of tetrahydrocannabinol and can-nabidiol, which should be received in strict compliance with the professional guidelines for the treatment of CINV.
The Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB), the US data center for the global PDB archive and a founding member of the Worldwide Protein Data Bank partnership, serves tens of thousands of data depositors in the Americas and Oceania and makes 3D macromolecular structure data available at no charge and without restrictions to millions of RCSB.org users around the world, including >660 000 educators, students and members of the curious public using PDB101.RCSB.org. PDB data depositors include structural biologists using macromolecular crystallography, nuclear magnetic resonance spectroscopy, 3D electron microscopy and micro-electron diffraction. PDB data consumers accessing our web portals include researchers, educators and students studying fundamental biology, biomedicine, biotechnology, bioengineering and energy sciences. During the past 2 years, the research-focused RCSB PDB web portal (RCSB.org) has undergone a complete redesign, enabling improved searching with full Boolean operator logic and more facile access to PDB data integrated with >40 external biodata resources. New features and resources are described in detail using examples that showcase recently released structures of SARS-CoV-2 proteins and host cell proteins relevant to understanding and addressing the COVID-19 global pandemic.
Abstract Cannabinoid 1 receptor (CB1R) expression is upregulated in the liver with viral hepatitis, cirrhosis, and both alcoholic and non-alcoholic fatty liver disease (FLD), whereas its expression is muted under usual physiological conditions. Inhibiting CB1R has been shown to be beneficial in preserving hepatic function in FLD but it is unclear if inhibiting CB1R during an inflammatory response to an acute hepatic injury, such as toxin-induced injury, would also be beneficial. We found that intrinsic CB1R in hepatocytes regulated liver inflammation-related gene transcription. We tested if nullification of hepatocyte-specific CB1R (hCNR1−/−) in mice protects against concanavalin A (Con A)-induced liver injury. We looked for evidence of liver damage and markers of inflammation in response to Con A by measuring liver enzyme levels and proinflammatory cytokines (e.g., TNF-α, IL-1β, IL-6, IL-17) in serum collected from hCNR1−/− and control mice. We observed a shift to the right in the dose-response curve for liver injury and inflammation in hCNR1−/− mice. We also found less inflammatory cell infiltration and focal necrosis in livers of hCNR1−/− mice compared to controls, resulting from downregulated apoptotic markers. This anti-apoptotic mechanism results from increased activation of nuclear factor kappa B (NF-κB), especially cAMP-dependent cannabinoid signaling and membrane-bound TNF-α, via downregulated TNF-α receptor 2 (TNFR2) transcription levels. Collectively, these findings provide insight into involvement of CB1R in the pathogenesis of acute liver injury.
Cannabis smoking is the dominant route of delivery, with the airway epithelium functioning as the site of first contact. The endocannabinoid system is responsible for mediating the physiological effects of inhaled phytocannabinoids. The expression of the endocannabinoid system in the airway epithelium and contribution to normal physiological responses remains to be defined.
To begin to address this knowledge gap, a curated dataset of 1090 unique human bronchial brushing gene expression profiles was created. The dataset included 616 healthy subjects, 136 subjects with asthma, and 338 subjects with COPD. A 32-gene endocannabinoid signature was analysed across all samples with sex and disease specific-analyses performed. Immunohistochemistry and immunoblots were performed to probe in situ and in vitro protein expression.
CB 1 , CB 2 , and TRPV1 protein signal is detectable in human airway epithelial cells in situ and in vitro , justifying examining the downstream endocannabinoid pathway. Sex status was associated with differential expression of 7/32 genes. In contrast, disease status was associated with differential expression of 21/32 genes in asthmatics and 26/32 genes in COPD subjects. We confirm at the protein level that TRPV1, the most differentially expressed candidate in our analyses, was up-regulated in airway epithelial cells from asthmatics relative to healthy subjects.
Our data demonstrate that endocannabinoid system is expressed in human airway epithelial cells with expression impacted by disease status and minimally by sex. The data suggest that cannabis consumers may have differential physiological responses in the respiratory mucosa.
Bone tissue renewal can be outlined as a complicated mechanism centered on the interaction between osteogenic and angiogenic events capable of leading to bone formation and tissue renovation. The achievement or debacle of bone regeneration is focused on the primary role of vascularization occurrence; in particular, the turning point is the opportunity to vascularize the bulk scaffolds, in order to deliver enough nutrients, growth factors, minerals and oxygen for tissue restoration. The optimal scaffolds should ensure the development of vascular networks to warrant a positive suitable microenvironment for tissue engineering and renewal. Vascular Endothelial Growth Factor (VEGF), a main player in angiogenesis, is capable of provoking the migration and proliferation of endothelial cells and indirectly stimulating osteogenesis, through the regulation of the osteogenic growth factors released and through paracrine signaling. For this reason, we concentrated our attention on two principal groups involved in the renewal of bone tissue defects: the cells and the scaffold that should guarantee an effective vascularization process. The application of Mesenchymal Stem Cells (MSCs), an excellent cell source for tissue restoration, evidences a crucial role in tissue engineering and bone development strategies. This review aims to provide an overview of the intimate connection between blood vessels and bone formation that appear during bone regeneration when MSCs, their secretome—Extracellular Vesicles (EVs) and microRNAs (miRNAs) —and bone substitutes are used in combination.
Objective
Nuclear factor-erythroid 2-related factor 2 (Nrf2) is shown to as a negative-regulatory cause in osteoclasts differentiation. Cannabinoid receptor type 2 (CB2) is verified to regulate osteoclast differentiation, though with diversed results.
Methods
In current research, we studied the Nrf2 role on osteoclast differentiation regulation with the CB2-selective agonists, AM1241, or CB2-selective antagonist, AM630, in RAW 264.7 macrophages. The nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation activator was confirmed by tartrate-resistant acid phosphatase (TRAP) staining as well as the TRAP activity analysis. In addition, Nrf2 siRNA was used to characterize the function of Nrf2 during osteoclast differentiation. We analyzed HO-1 and Nrf2 proteins levels with western blotting.
Results
The results showed that AM1241 promoted, while AM630 suppressed, osteoclast differentiation in RAW 264.7 cells. Both AM1241 and AM630 increased the expressions of HO-1 and Nrf2. Nrf2 silencing promoted osteoclast differentiation and abolished the function of AM630 to inhibit osteoclast differentiation.
Conclusions
Our results suggested that Nrf2 was required for inhibiting osteoclast differentiation induced by RANKL of RAW 264.7 cells by AM630, which may provide the insights of a novel method to treat osteoclastogenic bone disease.
Neuropathic pain conditions including neuropathic orofacial pain (NOP) are difficult to treat. Contemporary therapeutic agents for neuropathic pain are often ineffective in relieving pain and are associated with various adverse effects. Finding new options for treating neuropathic pain is a major priority in pain-related research. Cannabinoid-based therapeutic strategies have emerged as promising new options. Cannabinoids mainly act on cannabinoid 1 (CB1) and 2 (CB2) receptors, and the former is widely distributed in the brain. The therapeutic significance of cannabinoids is masked by their adverse effects including sedation, motor impairment, addiction and cognitive impairment, which are thought to be mediated by CB1 receptors in the brain. Alternative approaches have been developed to overcome this problem by selectively targeting CB2 receptors, peripherally restricted CB1 receptors and endocannabinoids that may be locally synthesized on demand at sites where their actions are pertinent. Many preclinical studies have reported that these strategies are effective for treating neuropathic pain and produce no or minimal side effects. Recently, we observed that inhibition of degradation of a major endocannabinoid, 2-arachydonoylglycerol, can attenuate NOP following trigeminal nerve injury in mice. This review will discuss the above-mentioned alternative approaches that show potential for treating neuropathic pain including NOP.
Abnormal cannabidiol (abn-CBD) exerts neuroprotective effects in vivo and in vitro. In the present study, we investigated the impact of abn-CBD on the glial production of proinflammatory mediators and scar formation within in vitro models. Primary astrocytic-microglial cocultures and astrocytic cultures from neonatal C57BL/6 mice and CB2 receptor knockout mice were stimulated with lipopolysaccharide (LPS), and the concentrations of tumor necrosis factor α (TNFα), interleukin-6 (IL-6) and nitrite were determined. Furthermore, we performed a live cell microscopy-based scratch-wound assay. After LPS stimulation, TNFα, IL-6 and nitrite production was more strongly increased in cocultures than in isolated astrocytes. Abn-CBD treatment attenuated the LPS-induced production of TNFα and nitrite in cocultures, while IL-6 production remained unaltered. In isolated astrocytes, only LPS-induced TNFα production was reduced by abn-CBD. Similar effects were observed after abn-CBD application in cocultures of CB2 knockout mice. Interestingly, LPS-induced TNFα and nitrite levels were far lower in CB2 knockout cultures compared to wildtypes, while IL-6 levels did not differ. In the scratch-wound assay, treatment with abn-CBD decelerated wound closure when microglial cells were present. Our data shows a differential role of abn-CBD for modulation of glial inflammation and astrocytic scar formation. These findings provide new explanations for mechanisms behind the neuroprotective potential of abn-CBD.
Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) are the most frequent primary liver cancers, accounting for approximately 80% and 15%, respectively. HCC carcinogenesis occurs mostly in cirrhosis and is a complex multi-step process, from precancerous lesions (low-grade and high-grade dysplastic nodules) to progressed HCC. During the different stages of liver carcinogenesis, there is an accumulation of pathological, genetic and epigenetic changes leading to initiation, malignant transformation and finally tumor progression. In contrast, a small subset of HCC occurs in normal liver from the transformation of hepatocellular adenoma (HCA), a benign hepatocellular tumor. The recent molecular classification enables to stratify HCAs according to their risk of complication, in particular malignant transformation, associated with mutations in exon 3 of the catenin beta 1 (CTNNB1) gene. Cholangiocarcinoma (CCA) derives from the multistep malignant transformation of preneoplastic lesions, like biliary intraepithelial neoplasia (BilIN) and intraductal papillary neoplasm of the bile duct (IPNB), for which a pre-operative diagnosis remains difficult. Different genetic alterations are involved in BilIN and IPNB progression, leading to the development of tubular or intestinal adenocarcinoma.The aims of this review are to describe the main clinical and molecular features of preneoplastic lesions leading to the development of HCC and CCA, their implications in clinical practice and the perspectives for future research.
Significance
Analgesic tolerance can result upon chronic use of opioid drugs necessitating dose escalation to treat pain. The biased mu opioid receptor agonist SR-17018 maintains antinociceptive efficacy without evidence of tolerance when given chronically to mice. Here, we show that SR-17018 and related compounds are noncompetitive agonists that stabilize the receptor in an active state that can still be blocked by orthosteric antagonists. We propose that this mode of activation may contribute to the biased agonists’ apparent ability to preferentially induce G protein signaling in cellular assays and may underlie the enduring efficacy observed for SR-17018 in vivo.
Evolution of tumor hallmarks is a result of accommodation of tumor cells with their nearby milieu called tumor microenvironment (TME). Accommodation or adaptive responses is highly important for a cell to survive, without which no cell is allowed to take any further steps in tumorigenesis. Metabolism of cancer cells is largely depended on stroma. Composition and plasticity of cells within the stroma is highly affected from inflammatory setting of TME. Hypoxia which is a common event in many solid cancers, is known as one of the key hallmarks of chronic inflammation and the master regulator of metastasis. Transforming growth factor (TGF)-β is produced in the chronic inflammatory and chronic hypoxic settings, and it is considered as a cardinal factor for induction of all tumor hallmarks. Aging, obesity and smoking are the main predisposing factors of cancer, acting mainly through modulation of TME.
Background
Cannabis sativa has been attributed to different pharmacological properties. A number of secondary metabolites such as tetrahydrocannabinol (THC), cannabinol (CBD), and different analogs, with highly promising biological activity on CB1 and CB2 receptors, have been identified.
Methods
Thus, this study aimed was to evaluate the activity of THC, CBD, and their analogs using molecular docking and molecular dynamics simulations (MD) methods. Initially, the molecules (ligands) were selected by bioinformatics searches in databases. Subsequently, CB1 and CB2 receptors were retrieved from the protein data bank database. Afterward, each receptor and its ligands were optimized to perform molecular docking. Then, MD Simulation was performed with the most stable ligand-receptor complexes. Finally, the Molecular Mechanics-Generalized Born Surface Area (MM-PBSA) method was applied to analyze the binding free energy between ligands and cannabinoid receptors.
Results
The results obtained showed that ligand LS-61176 presented the best affinity in the molecular docking analysis. Also, this analog could be a CB1 negative allosteric modulator like CBD and probably an agonist in CB2 like THC and CBD according to their dynamic behavior in silico. The possibility of having a THC and a CBD analogue (LS-61176) as a promising molecule for experimental evaluation since it could have no central side-effects on CB1 and have effects of CB2 useful in pain, inflammation, and some immunological disorders. Docking results were validate using ROC curve for both cannabinoids receptor where AUC for CB1 receptor was 0.894 ± 0.024, and for CB2 receptor AUC was 0.832 ± 0,032, indicating good affinity prediction.
Background
Obesity and type 2 diabetes are two interrelated metabolic disorders characterized by insulin resistance and a mild chronic inflammatory state. We previously observed that leptin (ob/ob) and leptin receptor (db/db) knockout mice display a distinct inflammatory tone in the liver and adipose tissue. The present study aimed at investigating whether alterations in these tissues of the molecules belonging to the endocannabinoidome (eCBome), an extension of the endocannabinoid (eCB) signaling system, whose functions are important in the context of metabolic disorders and inflammation, could reflect their different inflammatory phenotypes.
Results
The basal eCBome lipid and gene expression profiles, measured by targeted lipidomics and qPCR transcriptomics, respectively, in the liver and subcutaneous or visceral adipose tissues, highlighted a differentially altered eCBome tone, which may explain the impaired hepatic function and more pronounced liver inflammation remarked in the ob/ob mice, as well as the more pronounced inflammatory state observed in the subcutaneous adipose tissue of db/db mice. In particular, the levels of linoleic acid-derived endocannabinoid-like molecules, of one of their 12-lipoxygenase metabolites and of Trpv2 expression, were always altered in tissues exhibiting the highest inflammation. Correlation studies suggested the possible interactions with some gut microbiota bacterial taxa, whose respective absolute abundances were significantly different between ob/ob and the db/db mice.
Conclusions
The present findings emphasize the possibility that bioactive lipids and the respective receptors and enzymes belonging to the eCBome may sustain the tissue-dependent inflammatory state that characterize obesity and diabetes, possibly in relation with gut microbiome alterations.
Background
Lenabasum is a synthetic cannabinoid receptor type-2 (CB2) agonist able to exert potent anti-inflammatory effects, but its role on T cells remains unknown.
Objectives
The present study was undertaken to investigate anti-inflammatory mechanisms of lenabasum in T lymphocyte subsets and its in vivo therapeutic efficacy in experimental autoimmune encephalomyelitis (EAE).
Methods
Mononuclear cells from 17 healthy subjects (HS) and 25 relapsing-remitting multiple sclerosis (RRMS) patients were activated in presence or absence of lenabasum and analysed by flow cytometry and qRT-PCR. EAE mice were treated with lenabasum and clinical score and neuroinflammation were evaluated.
Results
Lenabasum significantly reduced TNF-a production from CD4+ T cells and CD8+ T cells in a dose-dependent manner in both HS and RRMS patients. In MS patients, lenabasum also reduced activation marker CD25 and inhibited IL-2 production from both T cell subsets and IFN-γ and IL-17 from committed Th1 and Th17 cells, respectively. These effects were blocked by the pre-treatment with selective CB2 inverse agonist SR144528. In vivo treatment of EAE mice with lenabasum significantly ameliorated disease severity, reduced neuroinflammation and demyelination in spinal cord.
Conclusion
Lenabasum exerts potent T cell-mediated immunomodulatory effects, suggesting CB2 as a promising pharmacological target to counteract neuroinflammation in MS.
Objective
Joint injury-induced perturbations to the endocannabinoid system (ECS), a regulator of both inflammation and nociception, remain largely uncharacterized. We employed a mouse model of ACL rupture to assess alterations to nociception, inflammation, and the ECS while using in vitro models to determine whether CB2 agonism can mitigate inflammatory signaling in macrophages and fibroblast-like synoviocytes (FLS).
Design
Mice underwent noninvasive ACL rupture (ACLR) via tibial compression-based loading. Nociception was measured longitudinally using mechanical allodynia and knee hyperalgesia testing. Synovitis was assessed using histological scoring and histomorphometry. Gene and protein markers of inflammation were characterized in whole joints and synovium. Immunohistochemistry assessed injury-induced alterations to CB1+, CB2+, and F4/80+ cells in synovium. To assess whether CB2 agonism can inhibit pro-inflammatory macrophage polarization, murine bone marrow-derived macrophages (mBMDM) were stimulated with IL-1β or conditioned medium from IL-1β-treated FLS and treated with vehicle (DMSO), the CB2 agonist HU308, or cannabidiol (CBD). Macrophage polarization was assessed as the ratio of M1-associated (IL1b, MMP1b, and IL6) to M2-associated (IL10, IL4, and CD206) gene expression. Human FLS (hFLS) isolated from synovial tissue of OA patients were treated with vehicle (DMSO) or HU308 following TNF-α or IL-1β stimulation to assess inhibition of catabolic/inflammatory gene expression.
Results
ACLR induces synovitis, progressively-worsening PTOA severity, and an immediate and sustained increase in both mechanical allodynia and knee hyperalgesia, which persist beyond the resolution of molecular inflammation. Enrichment of CB2, but not CB1, was observed in ACLR synovium at 3d, 14d, and 28d, and CB2 was found to be associated with F4/80 (+) cells, which are increased in number in ACLR synovium at all time points. The CB2 agonist HU308 strongly inhibited mBMDM M1-type polarization following stimulation with either IL-1β or conditioned medium from IL-1β-treated mFLS, which was characterized by reductions in Il1b, Mmp1b, and Il6 and increases in Cd206 gene expression. Cannabidiol similarly inhibited IL-1β-induced mBMDM M1 polarization via a reduction in Il1b and an increase in Cd206 and Il4 gene expression. Lastly, in OA hFLS, HU308 treatment inhibited IL-1β-induced CCL2, MMP1, MMP3, and IL6 expression and further inhibited TNF-α-induced CCL2, MMP1, and GMCSF expression, demonstrating human OA-relevant anti-inflammatory effects by targeting CB2.
Conclusions
Joint injury perturbs the intra-articular ECS, characterized by an increase in synovial F4/80(+) cells, which express CB2, but not CB1. Targeting CB2 in murine macrophages and human FLS induced potent anti-inflammatory and anti-catabolic effects, which indicates that the CB2 receptor plays a key role in regulating inflammatory signaling in the two primary effector cells in the synovium. The intraarticular ECS is therefore a potential therapeutic target for blocking pathological inflammation in future disease-modifying PTOA treatments.
G-protein-coupled receptors (GPCRs) represent the largest family of drug targets. Upon activation, GPCRs signal primarily via a diverse set of heterotrimeric G proteins. Most GPCRs can couple to several different G protein subtypes. However, how drugs act at GPCRs contributing to the selectivity of G protein recognition is poorly understood. Here, we examined the G protein selectivity profile of the dopamine D2 receptor (D2), a GPCR targeted by antipsychotic drugs. We show that D2 discriminates between six individual members of the Gi/o family, and its profile of functional selectivity is remarkably different across its ligands, which all engaged D2 with a distinct G protein coupling pattern. Using structural modeling, receptor mutagenesis, and pharmacological evaluation, we identified residues in the D2 binding pocket that shape these ligand-directed biases. We further provide pharmacogenomic evidence that natural variants in D2 differentially affect its G protein biases in response to different ligands.
Vascular smooth muscle proliferation and migration triggered by inflammatory stimuli are involved in the development and progression of atherosclerosis and restenosis. Cannabinoids may modulate cell proliferation in various cell types through cannabinoid 2 (CB2) receptors. Herein, we have investigated the effects of CB2 receptor agonists on TNF‐alpha‐induced proliferation, migration and signal transduction in human coronary artery smooth muscle cells (HCASMCs). HCASMCs were stimulated with TNF‐alpha and smooth muscle cell proliferation was determined by the extent of BrdU incorporation and the migration was assayed. CB2 and/or CB1 receptor expressions were determined by immunoflourescence staining, western, RT‐PCR, real‐time PCR and flow cytometry. Moderate levels of CB2 and CB1 receptors were detectable in HCASMCs compared to the very high levels of CB2 receptors expressed in THP‐1 monocytes. TNF‐alpha triggered up to ∼2.5 fold increase in CB2 receptor mRNA expression and/or protein expression in HCASMCs. Further, TNF‐alpha induced Ras, p38 MAPK, ERK ½, Akt and SAPK/JNK activation, with concordant increase in the proliferation and migration of smooth muscle cells. The CB2 agonists, JWH133 and HU308, dose‐dependently attenuated these effects of TNF‐alpha. Since the above‐mentioned TNF‐alpha‐induced phenotypic changes are critical in the initiation and progression of atherosclerosis and restenosis our findings suggest that CB2 agonists may offer a novel approach in the treatment of these pathologies by decreasing vascular smooth muscle proliferation and migration.
The G-protein-coupled cannabinoid receptor type 2 (CB 2 R) is a key element of the endocannabinoid (EC) system. EC/CB 2 R signaling has significant therapeutic potential in major pathologies affecting humans such as allergies, neurodegenerative disorders, inflammation or ocular diseases. CB 2 R agonism exerts anti-inflammatory and tissue protective effects in preclinical animal models of cardiovascular, gastrointestinal, liver, kidney, lung and neurodegenerative disorders. Existing ligands can be subdivided into endocannabinoids, cannabinoid-like and synthetic CB 2 R ligands that possess various degrees of potency on and selectivity against the cannabinoid receptor type 1. This review is an account of granted CB 2 R ligand patents from 2010 up to the present, which were surveyed using Derwent Innovation ® .
The type 1 and type 2 cannabinoid receptors are G protein-coupled receptors implicated in a variety of physiological processes and diseases. Synthetic cannabinoid receptor agonists (SCRAs) were originally developed to explore the therapeutic benefits of cannabinoid receptor activation, although more recently, these compounds have been diverted to the recreational drug market and are increasingly associated with incidences of toxicity. A prominent concept in contemporary pharmacology is functional selectivity or biased agonism, which describes the ability of ligands to elicit differential activation of signalling pathways through stabilisation of distinct receptor conformations. Biased agonists may maximise drug effectiveness by reducing on-target adverse effects if they are mediated by signalling pathways distinct from those that drive the therapeutic effects. For the cannabinoid receptors, it remains unclear as to which signalling pathways mediate desirable and adverse effects. However, given their structural diversity and potential to induce a plethora of signalling effects, SCRAs provide the most promising prospect for detecting and studying bias at the cannabinoid receptors. This review summarises the emerging evidence of SCRA bias at the cannabinoid receptors.
The G-protein coupled cannabinoid type 2 (CB2) receptor is a key element of the endocannabinoid system (ECS). EC/CB2 receptor signaling has tremendous therapeutic potential in some of the major pathologies affecting humans. CB2 receptor agonism exerts anti-inflammatory and tissue-protective effects in preclinical animal models of cardiovascular, gastrointestinal, liver, kidney lung and neurodegenerative disorders. Existing molecules targeting CB2R can be sub-divided into endocannabinoids and related fatty acid derivatives, phytocannabinoids and synthetic CB2 receptor ligands that possess various degrees of potency on CB2R and selectivity against the cannabinoid type 1 receptor (CB1R). While early CB2R ligands suffer from high lipophilicity, low solubility and tight plasma protein binding resulting in high in vivo clearance, medicinal chemists overcame these initial issues and provided highly potent, selective, drug-like and developable assets from structurally diverse chemotypes, which is reflected by a number of ongoing promising clinical trials. These achievements are complemented by the discovery of allosteric ligands and various high-quality chemical probes that will enable a better understanding of CB2R expression and mechanism of action, thereby unlocking the receptor's full therapeutic potential. In this review we describe the current knowledge on CB2R ligands by highlighting representative key molecules from different chemotypes and relevant results from pharmacological and clinical studies.
Multiple sclerosis is a chronic inflammatory demyelinating disorder of the central nervous system that eventually leads to progressive neurodegeneration and disability. Recent findings highlighted the emerging role of each target of the endocannabinoid system in controlling the symptoms and disease progression of multiple sclerosis. Therefore, multi-target modulators of the endocannabinoid system could provide a more effective pharmacological strategy as compared to the single target modulation. In this work, N-cycloheptyl-1,2-dihydro-5-bromo-1-(4-fluorobenzyl)-6-methyl-2-oxo-pyridine-3-carboxamide (B2) was identified as the most promising compound with dual agonism at cannabinoid receptors type-1 and cannabinoid receptors type-2 and good drug-like properties. In in vitro assays, B2 reduced glutamate release from rat synaptosomes through interaction with cannabinoid receptors type-1 and modulated the production of the pro- and anti-inflammatory cytokines (interleukins IL-1β and IL-6 and interleukin IL-10 respectively) via cannabinoid receptors type-2 activation. Furthermore, B2 demonstrated antinociceptive effects in an animal model of neuropathic pain and efficacy in an experimental autoimmune encephalomyelitis model of multiple sclerosis.
Pharmacological modulation of cannabinoid type 2 receptor (CB2R) holds promise for the treatment of numerous con-ditions, including inflammatory diseases, autoimmune disorders, pain, and cancer. Despite the significance of this re-ceptor, researchers lack reliable tools to address questions concerning the expression and complex mechanism of CB2R signaling, especially in cell-type and tissue-dependent context. Herein, we report for the first time a versatile ligand platform for the modular design of a collection of highly specific CB2R fluorescent probes, used successfully across ap-plications, species and cell types. These include flow cytometry of endogenously expressing cells, real-time confocal microscopy of mouse splenocytes and human macrophages, as well as FRET-based kinetic and equilibrium binding assays. High CB2R specificity was demonstrated by competition experiments in living cells expressing CB2R at native levels. The probes were effectively applied to FACS analysis of microglial cells derived from a mouse model relevant to Alzheimer's disease.
Artificial intelligence and multi-objective optimization represent promising solutions to bridge chemical and biological landscape by addressing the automated de novo design of compounds as a result of a human-like creative process. In the present study, we conceived a novel pair based multi-objective approach implemented in an adapted SMILES generative algorithm based on Recurrent Neural Networks for the automated de novo design of new molecules whose overall features are optimized by finding the best trade-offs among relevant physicochemical properties (MW, logP, HBA, HBD) and additional similarity-based constraints biasing specific biological targets. In this respect, we carried out the de novo design of chemical libraries targeting Neuraminidase, Acetylcholinesterase and the main protease of Severe Acute Respiratory Syndrome Coronavirus 2. Several quality metrics were employed to assess drug-likeness, chemical feasibility, diversity content and validity. Molecular docking was finally carried out to better evaluate the scoring and posing of the de novo generated molecules with respect to X-ray cognate ligands of the corresponding molecular counterparts. Our results indicate that artificial intelligence and multi-objective optimization allow to capture the latent links joining chemical and biological aspects, thus providing easy-to-use options for customizable design strategies, which are especially effective for both lead generation and lead optimization. The algorithm is freely downloadable at https://github.com/alberdom88/moo-denovo and all the data are available as Supporting Information.
Objectives:
In this study we investigated the role of the Cannabinoid Receptor type 2 (CB2) in the bone loss associated with Celiac Disease (CD) evaluating the effect of its pharmacological modulation on osteoclast activity. We previously demonstrated a significant association between the CB2 Q63R variant and CD, suggesting it as a possible disease biomarker. Moreover, CB2 stimulation is beneficial for reducing osteoclast activity in several bone pathologic conditions.
Methods:
in vitro osteoclasts (OCs) were differentiated from peripheral blood mononuclear cells of healthy donors, CD children at diagnosis and after one year of gluten free diet (GFD) and characterized by Real Time PCR and Western Blot for the expression of CB2 and specific osteoclastic markers, TRAP and Cathepsin K. TRAP assay and Bone Resorption assay were performed to evaluate osteoclast activity before and after 48 h exposure to CB2 selective drugs (JWH-133 and AM630) and Vitamin D.
Results:
We found in CD patients an osteoclast hyper-activation and low levels of CB2. CB2 stimulation with JWH-133 agonist is more effective than Vitamin D in reducing osteoclast activity while CB2 blockade with AM630 increases osteoclast activation. The anti-osteoporotic effect of JWH-133 decreases when used in co-treatment with vitamin D. GFD reduces osteoclast activity without restore CB2 expression.
Conclusions:
CB2 could be a molecular marker to predict the risk of bone alterations in CD and a pharmacological target to reduce bone mass loss in patients who need a direct intervention on bone metabolism, in addition to the GFD.
Evidence from several novel opioid agonists and knockout animals suggests that improved opioid therapeutic window, notably for analgesia versus respiratory depression, is a result of ligand bias downstream of activation of the m-opioid receptor (MOR) toward G protein signaling and away from other pathways, such as arrestin recruitment. Here, we argue that published claims of opioid bias based on application of the operational model of agonism are frequently confounded by failure to consider the assumptions of themodel. These include failure to account for intrinsic efficacy and ceiling effects in different pathways, distortions introduced by analysis of amplified (G protein) versus linear (arrestin) signaling mechanisms, and nonequilibrium effects in a dynamic signaling cascade. We show on both theoretical and experimental grounds that reduced intrinsic efficacy that is unbiased across different downstream pathways, when analyzed without due considerations, does produce apparent but erroneous MOR ligand bias toward G protein signaling, and the weaker the G protein partial agonism is the greater the apparent bias. Experimentally, such apparently G protein-biased opioids have been shown to exhibit low intrinsic efficacy for G protein signaling when ceiling effects are properly accounted for. Nevertheless, such agonists do display an improved therapeutic window for analgesia versus respiratory depression. Reduced intrinsic efficacy for G proteins rather than any supposed G protein bias provides a more plausible, sufficient explanation for the improved safety. Moreover, genetic models of G protein-biased opioid receptors and replication of previous knockout experiments suggest that reduced or abolished arrestin recruitment does not improve therapeutic window for MORinduced analgesia versus respiratory depression. © 2020 by The American Society for Pharmacology and Experimental Therapeutics.
Alzheimer’s disease (AD) is a dementing, neurodegenerative disorder characterized by increased accumulation of beta-amyloid peptides (Aβ), degeneration of hippocampal neurons and the gradual development of learning and memory deficits. Therapeutically, there are still no ideal medicines available and this represents an urgent need for the development of new strategies to treat AD. Emerging lines of evidence suggest that modulation of the cannabinoid system exhibits neuroprotective effects in various neurological diseases, including AD. However, a consensus is yet to emerge as to the impact of hippocampal cannabinoid receptor 2 (CB2R) in protection of hippocampal neurons against Aβ induced neuronal toxicity. Here, we report that chronic treatment of primary hippocampal neuronal cultures with 100 nM Aβ1-42 oligomers for 7 days results in neurotoxicity, which includes increases in lactate dehydrogenase (LDH) levels, suggesting an Aβ1-42 –induced neuron apoptosis. Further, chronic Aβ1-42 reduces the ratio of phosphorylated Akt (pAkt)/Akt, in turn decreases neuronal Bcl-2/Bax ratio, and leads to an increase of caspase-3, which likely underlines the signal pathway of chronic Aβ1-42–induced neuron apoptosis. Interestingly, pre-treatments of CB2R agonist (JWH133, 10 µM) with Aβ1-42 prevents Aβ1-42-induced the decrease of pAkt/Akt ratio, the decrease of Bcl-2/Bax ratio, and the increase of caspase-3, and protects hippocampal neurons against Aβ1-42-induced apoptosis. All neuroprotective effects of JWH133 are abolished by a selective CB2R antagonist, AM630. Taken together, the activation of hippocampal CB2Rs protects neurons against Aβ1-42 toxicity, and the CB2R-mediated enhancement of the pAkt signaling is likely involved in the protection of hippocampal neurons against Aβ1-42-induced neuronal toxicity.
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 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).
Cannabinoids are a group of chemical compounds that have been used for thousands of years due to their psychoactive function and systemic physiological effects. There are at least two types of cannabinoid receptors, CB1 and CB2, which belong to the G protein-coupled receptor superfamily, can trigger different signaling pathways to exert their physiological functions. In this study, several representative agonists and antagonists of both CB1 and CB2 were systematically studied to predict their binding affinities and selectivity against both cannabinoid receptors using a set of hierarchical molecular modeling and simulation techniques, including homology modeling, molecular docking, molecular dynamics (MD) simulations and endpoint binding free energy calculations using the MM-PBSA-WSAS method, and MM-GBSA free energy decomposition. Encouragingly, the calculated binding free energies correlated very well with the experimental values and the correlation coefficient square, 0.60, was much higher than that of an efficient but less accurate docking scoring function (R2=0.37). The hotspot residues for CB1 and CB2 in both active and inactive conformations were identified via MM-GBSA free energy decomposition analysis. The comparisons of binding free energies, ligand-receptor interaction patterns and hotspot residues among the four systems, namely, agonist-bound CB1, agonist-bound CB2, antagonist-bound CB1 and antagonist-bound CB2, enabled us to investigate and identify distinct binding features of these four systems with which one can rationally design potent, selective and function-specific modulators for the cannabinoid receptors.
Drugs selectively targeting CB2 hold promise for treating neurodegenerative disorders, inflammation, and pain while avoiding psychotropic side effects mediated by CB1. The mechanisms underlying CB2 activation and signaling are poorly understood but critical for drug design. Here we report the cryo-EM structure of the human CB2-Gi signaling complex bound to the agonist WIN 55,212-2. The 3D structure reveals the binding mode of WIN 55,212-2 and structural determinants for distinguishing CB2 agonists from antagonists, which are supported by a pair of rationally designed agonist and antagonist. Further structural analyses with computational docking results uncover the differences between CB2 and CB1 in receptor activation, ligand recognition, and Gi coupling. These findings are expected to facilitate rational structure-based discovery of drugs targeting the cannabinoid system.
Human endocannabinoid systems modulate multiple physiological processes mainly through the activation of cannabinoid receptors CB1 and CB2. Their high sequence similarity, low agonist selectivity, and lack of activation and G protein-coupling knowledge have hindered the development of therapeutic applications. Importantly, missing structural information has significantly held back the development of promising CB2-selective agonist drugs for treating inflammatory and neuropathic pain without the psychoactivity of CB1. Here, we report the cryoelectron microscopy structures of synthetic cannabinoid-bound CB2 and CB1 in complex with G i , as well as agonist-bound CB2 crystal structure. Of important scientific and therapeutic benefit, our results reveal a diverse activation and signaling mechanism, the structural basis of CB2-selective agonists design, and the unexpected interaction of cholesterol with CB1, suggestive of its endogenous allosteric moduating role.
Activation of cannabinoid receptor type II (CB2R) by AM1241 has been demonstrated to protect dopaminergic neurons in Parkinson's disease (PD) animals. However, the specific mechanisms of the action of the CB2R agonist AM1241 for PD treatment have not been characterized. Wild‐type (WT), CB1R knockout (CB1‐KO), and CB2R knockout (CB2‐KO) mice were exposed to 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) for 1 week to obtain a PD mouse model. The therapeutic effects of AM1241 were evaluated in each group. Behavioral tests, analysis of neurotransmitters, and immunofluorescence results demonstrated that AM1241 ameliorated PD in WT animals and CB1‐KO animals. However, AM1241 did not ameliorate PD symptoms in CB2‐KO mice. RNA‐seq analysis identified the lncRNA Xist as an important regulator of the protective actions of AM1241. Specifically, AM1241 allowed WT and CB1‐KO animals treated with MPTP to maintain normal expression of Xist, which affected the expression of miR‐133b‐3p and Pitx3. In vitro, overexpression of Xist or AM1241 protected neuronal cells from death induced by 6‐hydroxydopamine and increased Pitx3 expression. The CB2 receptor agonist AM1241 alleviated PD via regulation of the Xist/miR‐133b‐3p/Pitx3 axis, and revealed a new approach for PD treatment. The CB2 receptor agonist, AM1241, alleviated Parkinson's disease (PD) via regulation of the Xist/miR‐133b‐3p/Pitx3 axis, and revealed a new approach for PD treatment.