CB1 Activation Research Articles

N-linoleyltyrosine resisted the growth of non-small cell lung cancer cells via the regulation of CB1 and CB2 involvement of PI3K and ERK pathways.

Background: N-linoleyltyrosine (NITyr), one of the anandamide analogs, exerts activity via the endocannabinoid receptors (CB1 and CB2), which showed anti-tumor effects in various tumors. Therefore, we speculated that NITyr might show anti-non-small cell lung cancer (NSCLC) effects via the CB1 or CB2 receptor. The purpose of the investigation was to reveal the anti-tumor ability of NITyr on A549 cells and its mechanisms. Methods: The viability of A549 cells was measured by MTT assay, and the cell cycle and apoptosis were both examined by flow cytometry; in addition, cell migration was tested by wound healing assay. Apoptosis-related markers were measured by immunofluorescence. The downstream signaling pathways (PI3K, ERK, and JNK) of CB1 or CB2 were examined through Western blotting. The expressions of CB1 and CB2 were detected by immunofluorescence. Finally, the AutoDock software was used to validate the binding affinity between the targets, such as CB1 and CB2, with NITyr. Results: We found that NITyr inhibited cell viability, hindered the cell cycle, resulted in apoptosis, and inhibited migration. The CB1 inhibitor, AM251, and the CB2 inhibitor, AM630, weakened the aforementioned phenomenon. The immunofluorescence assay suggested that NITyr upregulated the expression of CB1 and CB2. Western blot analysis indicated that NITyr upregulated the expression of p-ERK, downregulated the expression of p-PI3K, and did not affect p-JNK expression. In conclusion, NITyr showed a role in inhibiting NSCLC through the activation of CB1 and CB2 receptors involved in PI3K and ERK pathways.

Distinct activation mechanisms regulate subtype selectivity of Cannabinoid receptors

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

Combined non-psychoactive Cannabis components cannabidiol and β-caryophyllene reduce chronic pain via CB1 interaction in a rat spinal cord injury model.

The most frequently reported use of medical marijuana is for pain relief. However, its psychoactive component Δ9-tetrahydrocannabinol (THC) causes significant side effects. Cannabidiol (CBD) and β-caryophyllene (BCP), two other cannabis constituents, possess more benign side effect profiles and are also reported to reduce neuropathic and inflammatory pain. We evaluated the analgesic potential of CBD and BCP individually and in combination in a rat spinal cord injury (SCI) clip compression chronic pain model. Individually, both phytocannabinoids produced dose-dependent reduction in tactile and cold hypersensitivity in male and female rats with SCI. When co-administered at fixed ratios based on individual A50s, CBD and BCP produced enhanced dose-dependent reduction in allodynic responses with synergistic effects observed for cold hypersensitivity in both sexes and additive effects for tactile hypersensitivity in males. Antinociceptive effects of both individual and combined treatment were generally less robust in females than males. CBD:BCP co-administration also partially reduced morphine-seeking behavior in a conditioned place preference (CPP) test. Minimal cannabinoidergic side effects were observed with high doses of the combination. The antinociceptive effects of the CBD:BCP co-administration were not altered by either CB2 or μ-opioid receptor antagonist pretreatment but, were nearly completely blocked by CB1 antagonist AM251. Since neither CBD or BCP are thought to mediate antinociception via CB1 activity, these findings suggest a novel CB1 interactive mechanism between these two phytocannabinoids in the SCI pain state. Together, these findings suggest that CBD:BCP co-administration may provide a safe and effective treatment option for the management of chronic SCI pain.

Endocannabinoids, anandamide and 2-AG, regulate mechanosensitivity of mucosal afferents in the Guinea pig bladder

Bladder afferents play a crucial role in urine storage and voiding, and conscious sensations from the bladder. Endocannabinoids, anandamide (AEA) and 2-arachidonolylglycerol (2-AG), are endogenous ligands of G-protein coupled cannabinoid receptors 1 and 2 (CB1 and CB2) found in the CNS and peripheral organs. They also have off-target effects on some ligand- and voltage-gated channels. The aim of this study is to determine the role of AEA and 2-AG in regulation of mechanosensitivity of probable nociceptive neurons innervating the bladder - capsaicin-sensitive mucosal afferents. The activity of these afferents was determined by ex vivo single unit extracellular recordings in the guinea pig bladder. A stable analogue of anandamide, methanandamide (mAEA) evoked initial excitatory response of mucosal afferents followed by potentiation of their responses to mechanical stimulation. In the presence of TRPV1 antagonist (AMG9810), mAEA's effect on mechanosensitivity switched from excitatory to inhibitory. The inhibitory effect of mAEA is due to activation of both CB1 and CB2 cannabinoid receptors since it was abolished by combined application of selective CB1 (NESS0327) and CB2 (SR144528) antagonists. 2-AG application evoked a brief excitation of mucosal afferents, without potentiation of their mechanosensitivity, followed by the inhibition of their responses to mechanical stimulation. CB2 receptor antagonist, SR144528 abolished the inhibitory effect of 2-AG. Our data indicated that anandamide and 2-AG have opposite effects on mechanosensitivity of mucosal capsaicin-sensitive afferents in the guinea pig bladder; mAEA potentiated while 2-AG inhibited responses of mucosal afferents to mechanical stimulation. These findings are important for understanding of the role of endocannabinoids in regulating bladder sensation and function.

The Anti-Tumorigenic Role of Cannabinoid Receptor 2 in Colon Cancer: A Study in Mice and Humans.

The endocannabinoid system, particularly cannabinoid receptor 2 (CB2 in mice and CNR2 in humans), has controversial pathophysiological implications in colon cancer. Here, we investigate the role of CB2 in potentiating the immune response in colon cancer in mice and determine the influence of CNR2 variants in humans. Comparing wild-type (WT) mice to CB2 knockout (CB2-/-) mice, we performed a spontaneous cancer study in aging mice and subsequently used the AOM/DSS model of colitis-associated colorectal cancer and a model for hereditary colon cancer (ApcMin/+). Additionally, we analyzed genomic data in a large human population to determine the relationship between CNR2 variants and colon cancer incidence. Aging CB2-/- mice exhibited a higher incidence of spontaneous precancerous lesions in the colon compared to WT controls. The AOM/DSS-treated CB2-/- and ApcMin/+CB2-/- mice experienced aggravated tumorigenesis and enhanced splenic populations of immunosuppressive myeloid-derived suppressor cells along with abated anti-tumor CD8+ T cells. Importantly, corroborative genomic data reveal a significant association between non-synonymous variants of CNR2 and the incidence of colon cancer in humans. Taken together, the results suggest that endogenous CB2 activation suppresses colon tumorigenesis by shifting the balance towards anti-tumor immune cells in mice and thus portray the prognostic value of CNR2 variants for colon cancer patients.

Comprehensive Characterization of a Systematic Library of Alkyl and Alicyclic Synthetic Cannabinoids Related to CUMYL-PICA, CUMYL-BUTICA, CUMYL-CBMICA, and CUMYL-PINACA.

Over 200 synthetic cannabinoid receptor agonists (SCRAs) have been identified as new psychoactive substances. Effective monitoring and characterization of SCRAs are hindered by the rapid pace of structural evolution. Ahead of possible appearance on the illicit drug market, new SCRAs were synthesized to complete a systematic library of cumyl-indole- (e.g., CUMYL-CPrMICA, CUMYL-CPMICA) and cumyl-indazole-carboxamides (e.g., CUMYL-CPrMINACA, CUMYL-CPMINACA), encompassing butyl, pentyl, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, and cyclohexylmethyl tails. Comprehensive pharmacological characterization was performed with three assay formats, monitoring the recruitment of either wild-type or C-terminally truncated (βarr2d366) β-arrestin2 to the activated cannabinoid 1 receptor (CB1) or monitoring Gβγ-mediated membrane hyperpolarization. Altered compound characterization was observed when comparing derived potency (EC50) and efficacy (Emax) values from both assays monitoring the same or a different signaling event, whereas ranges and ranking orders were similar. Structure-activity relationships (SAR) were assessed in threefold, resulting in the identification of the pendant tail as a critical pharmacophore, with the optimal chain length for CB1 activation approximating an n-pentyl (e.g., cyclopentylmethyl or cyclohexylmethyl tail). The activity of the SCRAs encompassing cyclic tails decreased with decreasing number of carbons forming the cyclic moiety, with CUMYL-CPrMICA showing the least CB1 activity in all assay formats. The SARs were rationalized via molecular docking, demonstrating the importance of the optimal steric contribution of the hydrophobic tail. While SAR conclusions remained largely unchanged, the differential compound characterization by both similar and different assay designs emphasizes the importance of detailing specific assay characteristics to allow adequate interpretation of potencies and efficacies.

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

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

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

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

Identification of CB1 Ligands among Drugs, Phytochemicals and Natural-Like Compounds: Virtual Screening and In Vitro Verification.

Cannabinoid receptor type 1 (CB1) is an important modulator of many key physiological functions and thus a compelling molecular target. However, safe CB1 targeting is a non-trivial task. In recent years, there has been a surge of data indicating that drugs successfully used in the clinic for years (e.g. paracetamol) show CB1 activity. Moreover, there is a lot of promise in finding CB1 ligands in plants other than Cannabis sativa. In this study, we searched for possible CB1 activity among already existing drugs, their metabolites, phytochemicals, and natural-like molecules. We conducted two iterations of virtual screening, verifying the results with in vitro binding and functional assays. The in silico procedure consisted of a wide range of structure- and ligand-based methods, including docking, molecular dynamics, and quantitative structure–activity relationship (QSAR). As a result, we identified travoprost and ginkgetin as CB1 ligands, which provides a starting point for future research on the impact of their metabolites or preparations on the endocannabinoid system. Moreover, we found five natural-like compounds with submicromolar or low micromolar affinity to CB1, including one mixed partial agonist/antagonist viable for hit-to-lead phase. Finally, the computational procedure established in this work will be of use for future screening campaigns for novel CB1 ligands.

The potential of cannabinoids in the treatment of lung cancer

Introduction: Lung cancer is the number-one cause of death due to neoplasms worldwide. The 5-year overall survival rate is only 22%. In advanced stages, the therapeutic options are limited to chemotherapy, radiotherapy, molecularly targeted therapy and immunotherapy. Phytocannabinoids, the components of Cannabis sativa, their synthetic derivatives and endogenous cannabinoids have demonstrated anticancer activity in various common cancers - breast, prostate, colorectal and lung cancers, among others. The aim of this review was to assess the potential value of cannabinoids in the treatment of lung cancer.
 State of knowledge: The majority of preclinical studies demonstrates that cannabinoids inhibit lung cancer cell viability both in vitro and in vivo. The main mechanism of anticancer activity is the induction of apoptosis, triggered by activation of CB1, CB2 and TRPV1 receptors or independently via other pathways. Cannabinoids influence the components of the tumour microenvironment - cancer associated fibroblasts, macrophages and lymphokine-activated-killer cells. Cannabinoids alter leukocyte infiltration into anti-cancer proportions, inhibit expression of EGFR and PAI-1 and increase the expression of TIMP-1. As a result they induce cytotoxicity, decrease proliferation, migration and invasive potential of lung cancer cells, suppress angiogenesis and metastasis forming. Patients with advanced lung cancer may also benefit from analgesic, antiemetic and appetite improving properties of cannabinoids.
 Summary: Cannabinoids can be a supplementary agent in systemic anticancer therapeutic regimen in the future. The exact mechanisms of action, specific doses in anticancer treatment, routes of administration and interactions with other anticancer drugs has yet to be determined. Thus the clinical studies on cannabinoids in lung cancer should be performed in the future.

CB1 activity of three classes of synthetic cannabinoids and their metabolites – The tail needs to know what the head is doing

Synthetic cannabinoids (SCs) are amongst the most prevalent of the new psychoactive substances. They encompass various structural classes with a wide range of CB1 activity. Most SCs are extensively bio-transformed in vivo and in vitro. The available data on the CB1 activity of SC metabolites are limited but such knowledge is needed to evaluate SC toxicology, duration of effects and to develop bioassays for screening purposes. To evaluate any structure-activity relationships, we determined the human CB1 receptor efficacy and potency of three SC classes and their metabolites. An activity assay incorporating AequoScreen recombinant CHO-K1 cells expressing the human CB1 receptor was used to study SCs and their metabolites. Dose-response curves were prepared in triplicate in three independent experiments and GraphPad Prism was used to calculate substance efficacy and potency. Eight concentrations, 29 nM–60 μM, were used for each drug and JWH-018 was used as a reference. The three SC classes investigated were (i) JWH-018, AM2201, THJ-018 and THJ-2201 and their corresponding 4-OH-pentyl and 5OH-pentyl metabolites; (ii) MMB-4en-PICA (MMB022) and MDMB-4en-PINACA and their corresponding ester-hydrolysis and dihydrodiol metabolites; and (iii) 5F-MDMB-PINACA, 5F-MDMB-PICA, 4F-MDMB-BINACA, and 4F-MDMB-BICA and their oxidative-defluorination and ester-hydrolysis metabolites. Parent SC potency values ranged from 7-200 nM. The 4-OH-pentyl metabolites of JWH-018, AM2201, THJ-018 and THJ-2201 had similar potency to their respective parent SC. The 5-OH-pentyl-JWH-018 and 5-OH-pentyl-THJ-018 metabolites were 5–10 times less potent than their parent SCs. No CB1 activity was observed for MMB-4en-PICA ester-hydrolysis and dihydrodiol metabolites, possibly due to the lower activity of the parent and a subsequent large loss of activity, whilst the dihydrodiol and ester-hydrolysis MDMB-4en-PINACA metabolites were 53 times and 130 times less potent respectively than MDMB-4en-PINACA. 5F-MDMB-PINACA and 5F-MDMB-PICA oxidative-defluorinated metabolites were 7 and 27 times less potent than the parent SCs. The 4OH-butyl metabolites of 4F-MDMB-BINACA, and 4F-MDMB-BICA were 6 and 11 times less potent than the parent drug, respectively, and their ester-hydrolysis metabolites were 21-47 times less potent. Where hydroxylations occur at the non-terminal (4) position of the pentyl tail of JWH-018 and THJ -2201 there is no loss of CB1 activity. The CB1 potency of the JWH-018 and THJ -2201 5OH-pentyl metabolites is reduced but in the same potency range as those reported for other parent SCs. Dihydrodiol metabolites of 4en-compounds show a large reduction in CB1 activity (> 53 times) compared to the parent, as do their ester hydrolysis metabolites (> 130-times). The oxidative defluorinated metabolites of the 4F-MDMB and 5F-MDMB compounds retain some CB1 activity (7–27 times reduction) but a more significant loss of activity is observed when ester hydrolysis occurs in the head group (21–417 times reduction). The head and core moieties of SCs are most influential in determining CB1 potency. The tail, when fulfilling certain steric requirements, acts as an anchor point in the CB1 receptor, influencing the overall position and rigidity of the SC in the CB1 receptor. From a urinary bioassay perspective, where metabolites can be 10 times more abundant than parent SCs, the activity of the metabolites might be important for SC detection. We have established a structure-activity relationship for three SC classes and their metabolites. The relative change in CB1 activity caused by a biotransformation on the tail of the SC depends on the head structure present and biotransformation of the head group leads to a greater reduction in potency than single hydroxylations on the tail. So, “the tail of the SC metabolite needs to know what the head is doing” when activating the CB1 receptor. The study is part of Eurostars-2 Joint Programme NPS-REFORM.

Towards better understanding SCRAs and metabolites in recreational drug intoxications associated with 5F-MDMB-PICA use

Synthetic cannabinoid receptor agonists (SCRAs) pose a danger to public health. Historically, SCRA use has been associated with a multitude of adverse events, including neurotoxicity and cardiotoxicity. Numerous case reports are published on observed SCRA toxicity, however clear studies on the toxicity of SCRAs in humans are lacking because of obvious ethical reasons. This study focused on a unique cohort of patients experiencing recreational drug toxicity, who had used 5F-MDMB-PICA, to better understand the effects of SCRAs and their metabolites in humans. All patients were evaluated regarding vital signs, Glasgow Coma Scale (GCS) and clinical features. Liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS) confirmed and quantified the presence of 5F-MDMB-PICA (and/or metabolites) as the only SCRA present in the serum of 71 patients (Janssens et al., Clinical Chemistry , 2022, in press, https://doi.org/10.1093/clinchem/hvac027 ). Cannabinoid activity was assessed in extracts from all serum samples by a cannabinoid receptor (CB1) bioassay, allowing to assess the relationship between serum concentrations and ex vivo human CB1 activation potential. Furthermore, a link with the clinical presentation was appraised. 5F-MDMB-PICA and metabolites were pharmacologically profiled in vitro to evaluate the theoretically possible contribution of active metabolites to overall cannabinoid activity. This revealed the presence of active in vivo metabolites M2 (oxidative defluorination) and M7 (ester hydrolysis) in serum. Serum levels of 5F-MDMB-PICA were correlated to the ex vivo cannabinoid activity, revealing a sigmoidal relationship. The latter could also be predicted based on the pharmacological characterization and an in-depth investigation of the bioassay outcome. Regarding the clinical presentation, the GCS showed a significant trend (decrease) upon increasing ex vivo cannabinoid activity. Trends for the occurrence of symptoms or an altered heart rate, blood pressure or body temperature were non-significant. Although high concentrations of 5F-MDMB-PICA metabolites (M2 and M7) were present in serum, a negligible contribution of these active metabolites to the overall cannabinoid activity in serum was anticipated, based on their in vitro CB1 receptor activation potential. Prediction of ex vivo cannabinoid activity was made possible by generating a mathematical formula based on the pharmacological characterization of reference standards of all compounds that were detected in serum. Including correction factors within this formula to account for incomplete recovery and matrix effects within the bioassay was necessary as – in contrast to the analytical method – no internal standard could be used due to retained CB1 activation potential of SCRA internal standards. The clinical presentation of 5F-MDMB-PICA-intoxicated patients was not unequivocally correlated to the ex vivo cannabinoid activity in their serum. On the one hand, the level of consciousness significantly correlated to the ex vivo cannabinoid activity that could be measured within extracts of their serum. This is in line with previous reports of SCRA misuse resulting in drowsiness or ‘zombie-like’ effects. The insignificance of other clinical trends, on the other hand, suggest that the clinical presentation regarding SCRA abuse cannot be deemed as clear-cut. This is the first study to evaluate in a large patient cohort the correlation between possible toxic effects of 5F-MDMB-PICA misuse and ex vivo cannabinoid activity. Pharmacological profiling and in-depth bioassay analysis allowed a better understanding of the contribution of 5F-MDMB-PICA and active metabolites to the total CB1 activity present in serum, suggesting a negligible contribution by metabolites of this specific SCRA despite the high concentrations in serum.

Linking in vitro and ex vivo CB1 activity with serum concentrations and clinical features in 5F-MDMB-PICA users to better understand SCRAs and their metabolites.

Synthetic cannabinoid receptor agonists (SCRAs) pose a danger to public health. This study focused on individuals experiencing recreational drug toxicity who had used 5F-MDMB-PICA.Patient records were evaluated regarding vital signs, Glasgow Coma Scale (GCS) and clinical features. Liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) confirmed and quantified the presence of 5F-MDMB-PICA (and/or metabolites) as the only SCRA present in the serum of 71 patients. Cannabinoid activity was evaluated by a cannabinoid receptor (CB1) bioassay, to assess the relationship between serum concentrations and ex vivo human CB1 activation potential. Furthermore, a link with the clinical presentation was appraised.5F-MDMB-PICA and five metabolites were pharmacologically profiled in vitro, revealing theoretically possible contributions of two active in vivo metabolites to overall cannabinoid activity. Serum concentrations of 5F-MDMB-PICA were correlated to the ex vivo cannabinoid activity, revealing a sigmoidal relationship. The latter could also be predicted based on pharmacological characterization of 5F-MDMB-PICA and its metabolites and an in-depth investigation of the bioassay outcome. Clinically, the GCS showed a significant trend (decrease) with increasing ex vivo cannabinoid activity.This is the first study to evaluate possible toxic effects of 5F-MDMB-PICA in a unique large patient cohort. It allows a better understanding of 5F-MDMB-PICA and metabolites in humans, suggesting a negligible contribution by 5F-MDMB-PICA metabolites to the overall cannabinoid activity in serum. Additionally, this work shows that in vitro pharmacological characterization allows close prediction of an individual's ex vivo CB1 activity, the latter showing a relationship with the level of consciousness.

Hear the sounds: the role of G protein-coupled receptors in the cochlea.

Sound is converted by hair cells in the cochlea into electrical signals, which are transmitted by spiral ganglion neurons (SGNs) and heard by the auditory cortex. G protein-coupled receptors (GPCRs) are crucial receptors that regulate a wide range of physiological functions in different organ and tissues. The research of GPCRs in the cochlea is essential for the understanding of the cochlea development, hearing disorders, and the treatment for hearing loss. Recently, several GPCRs have been found to play important roles in the cochlea. Frizzleds and Lgrs are dominant GPCRs that regulate stem cell self-renew abilities. Moreover, Frizzleds and Celsrs have been demonstrated to play core roles in the modulation of cochlear planar cell polarity (PCP). In addition, hearing loss can be caused by mutations of certain GPCRs, such as Vlgr1, Gpr156, S1P2, and Gpr126. And A1, A2A, and CB2 activation by agonists has protective functions on noise- or drug-induced hearing loss. Here, we review the key findings of GPCR in the cochlea and discuss the role of GPCR in the cochlea, such as stem cell fate, PCP, hearing loss, and hearing protection.

Visual-area-specific tonic modulation of GABA release by endocannabinoids sets the activity and coordination of neocortical principal neurons.

SummaryPerisomatic inhibition of pyramidal neurons (PNs) coordinates cortical network activity during sensory processing, and this role is mainly attributed to parvalbumin-expressing basket cells (BCs). However, cannabinoid receptor type 1 (CB1)-expressing interneurons are also BCs, but the connectivity and function of these elusive but prominent neocortical inhibitory neurons are unclear. We find that their connectivity pattern is visual area specific. Persistently active CB1 signaling suppresses GABA release from CB1 BCs in the medial secondary visual cortex (V2M), but not in the primary visual cortex (V1). Accordingly, in vivo, tonic CB1 signaling is responsible for higher but less coordinated PN activity in the V2M than in the V1. These differential firing dynamics in the V1 and V2M can be captured by a computational network model that incorporates visual-area-specific properties. Our results indicate a differential CB1-mediated mechanism controlling PN activity, suggesting an alternative connectivity scheme of a specific GABAergic circuit in different cortical areas.

2-Arachidonoylglycerol Reduces the Production of Interferon-Gamma in T Lymphocytes from Patients with Systemic Lupus Erythematosus.

Background: the endocannabinoid 2-arachidonoylglycerol (2-AG) plays a pivotal role in immune cells regulation. The plasma levels of 2-AG are increased in patients with systemic lupus erythematosus (SLE) and correlate with disease activity. Moreover, in plasmacytoid dendritic cells from SLE patients, 2-AG is able to control the production of type 1 interferon (IFN) through CB2 activation. The aim of this study was to evaluate the potential role of 2-AG on T lymphocytes from SLE patients. Methods: peripheral blood mononuclear cells (PBMCs) from SLE participants and age- and sex-matched healthy donors (HD) were isolated by Ficoll–Hypaque density-gradient centrifugation. The PBMCs were treated with increasing concentrations of 2-AG, and AM251 and AM630 were used to antagonize CB1 and CB2, respectively. Flow cytometry was used to assess the expression of CD3, CD4, CD8, CD25, IFN-ɣ, IL-4, and IL-17A. Results: 2-AG (1 μM) decreased IFN-ɣ expression (p = 0.0005) in the Th1 lymphocytes of SLE patients. 2-AG did not modulate the cytokine expression of any other T lymphocyte population from either SLE or HD. Treatment with both 2-AG and AM630 increased the IFN-ɣ expression in Th1 lymphocytes of SLE patients (p = 0.03). Discussion: 2-AG is able to modulate type 2 IFN production from CD4+ T lymphocytes from SLE patients through CB2 activation.

The Combination of Δ9-Tetrahydrocannabinol and Cannabidiol Suppresses Mitochondrial Respiration of Human Glioblastoma Cells via Downregulation of Specific Respiratory Chain Proteins.

Simple SummaryCannabidiol (CBD) is a phytocannabinoid from Cannabis sativa L. that exhibits no psychoactivity and, like the psychoactive cannabinoid Δ9-tetrahydrocannabinol (THC), shows anticancer effects in preclinical cell and animal models. Previous studies have indicated a stronger cancer-targeting effect when THC and CBD are combined. Here, we investigated how the combination of THC and CBD in a 1:1 ratio affects glioblastoma cell survival. The compounds were found to synergistically enhance cell death, which was attributed to mitochondrial damage and disruption of energy metabolism. A detailed look at the mitochondrial electron transfer chain showed that THC/CBD selectively decreased certain subunits of complexes I and IV. These data highlight the fundamental changes in cellular energy metabolism when cancer cells are exposed to a mixture of cannabinoids and underscore the potential of combining cannabinoids in cancer treatment.Phytocannabinoids represent a promising approach in glioblastoma therapy. Previous work has shown that a combined treatment of glioblastoma cells with submaximal effective concentrations of psychoactive Δ9-tetrahydrocannabinol (THC) and non-psychoactive cannabidiol (CBD) greatly increases cell death. In the present work, the glioblastoma cell lines U251MG and U138MG were used to investigate whether the combination of THC and CBD in a 1:1 ratio is associated with a disruption of cellular energy metabolism, and whether this is caused by affecting mitochondrial respiration. Here, the combined administration of THC and CBD (2.5 µM each) led to an inhibition of oxygen consumption rate and energy metabolism. These effects were accompanied by morphological changes to the mitochondria, a release of mitochondrial cytochrome c into the cytosol and a marked reduction in subunits of electron transport chain complexes I (NDUFA9, NDUFB8) and IV (COX2, COX4). Experiments with receptor antagonists and inhibitors showed that the degradation of NDUFA9 occurred independently of the activation of the cannabinoid receptors CB1, CB2 and TRPV1 and of usual degradation processes mediated via autophagy or the proteasomal system. In summary, the results describe a previously unknown mitochondria-targeting mechanism behind the toxic effect of THC and CBD on glioblastoma cells that should be considered in future cancer therapy, especially in combination strategies with other chemotherapeutics.

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

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

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

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

Functional analysis of key residues involved in CB1 and CB2 activation

Human endocannabinoid signaling is primarily mediated by the G‐protein coupled receptors CB1 and CB2. These receptors have been linked to a variety of physiological processes and are being pursued as prospective drug candidates because of their potential to treat pain and inflammation. Due to their high sequence homology, as well as similarity in activation and signaling mechanism, investigating the physiological role of these receptors with selective ligands has been problematic. Ligands that selectively bind to CB2 may provide therapeutic benefits without the psychoactive effects associated with the CB1 receptor. Recent structural determination of the two receptors has allowed us to identify potential key residues involved in ligand binding. In the studies presented here, single point‐mutations at the CB2 and CB1 receptor were developed and the receptor‐mediated signaling of these mutants were evaluated. Using cannabinoid agonists AM841 and AM12033, we compared each mutation and observed that certain changes did not affect the activity of the receptor while others prevented or severely attenuated signaling. These results may provide a pathway for developing novel CB2 specific compounds with improved therapeutic potential.