Enhancing Drug Discovery and Development through the Integration of Medicinal Chemistry, Chemical Biology, and Academia-Industry Partnerships: Insights from Roche's Endocannabinoid System Projects.

The endocannabinoid system (ECS) is a critical regulatory network composed of endogenous cannabinoids (eCBs), their synthesizing and degrading enzymes, and associated receptors. It is integral to maintaining homeostasis and orchestrating key functions within the central nervous and immune systems. Given its therapeutic significance, we have launched a series of drug discovery endeavors aimed at ECS targets, including peroxisome proliferator-activated receptors (PPARs), cannabinoid receptors types 1 (CB1R) and 2 (CB2R), and monoacylglycerol lipase (MAGL), addressing a wide array of medical needs. The pursuit of new therapeutic agents has been enhanced by the creation of specialized labeled chemical probes, which aid in target localization, mechanistic studies, assay development, and the establishment of biomarkers for target engagement. By fusing medicinal chemistry with chemical biology in a comprehensive, translational end-to-end drug discovery strategy, we have expedited the development of novel therapeutics. Additionally, this strategy promises to foster highly productive partnerships between industry and academia, as will be illustrated through various examples.

CB1 Promotes Osteogenic Differentiation Potential of Periodontal Ligament Stem Cells by Enhancing Mitochondrial Transfer of Bone Marrow Mesenchymal Stem Cells.

OBJECTIVE: To reveal the role and mechanism of cannabinoid receptor 1 (CB1) and mitochondria in promoting osteogenic differentiation of periodontal ligament stem cells (PDLSCs) in the inflammatory microenvironment. METHODS: Bidirectional mitochondrial transfer was performed in bone mesenchymal stem cells (BMSCs) and PDLSCs. Laser confocal microscopy and quantitative flow cytometry were used to observe the mitochondrial transfer and quantitative mitochondrial transfer efficiency. Realtime reverse transcription polymerase chain reaction (RT-PCR) was employed to detect gene expression. Alkaline phosphatase (ALP) activity, alizarin red staining (ARS) and quantitative calcium ion analysis were used to evaluate the degree of osteogenic differentiation of PDLSCs. RESULTS: Bidirectional mitochondrial transfer was observed between BMSCs and PDLSCs. The indirect co-culture system could simulate intercellular mitochondrial transfer. Compared with the conditioned medium (CM) for BMSCs, that for HA-CB1 BMSCs could significantly enhance the mineralisation ability of PDLSCs. The mineralisation ability of PDLSCs could not be enhanced after removing the mitochondria in CM for HA-CB1 BMSCs. The expression level of HO-1, PGC-1α, NRF-1, ND1 and HK2 was significantly increased in HA-CB1 BMSCs. CONCLUSION: CM for HA-CB1 BMSCs could significantly enhance the damaged osteogenic differentiation ability of PDLSCs in the inflammatory microenvironment, and the mitochondria of CM played an important role. CB1 was related to the activation of the HO-1/PGC-1α/NRF-1 mitochondrial biogenesis pathway, and significantly increased the mitochondrial content in BMSCs.

Endocannabinoid signaling in stress, nausea, and vomiting.

BACKGROUND: Classical antiemetics that target the serotonin system may not be effective in treating certain nausea and vomiting conditions like cyclic vomiting syndrome (CVS) and cannabinoid hyperemesis syndrome (CHS). As a result, there is a need for better therapies to manage the symptoms of these disorders, including nausea, vomiting, and anxiety. Cannabis is often used for its purported antiemetic and anxiolytic effects, given regulation of these processes by the endocannabinoid system (ECS). However, there is considerable evidence that cannabinoids can also produce nausea and vomiting and increase anxiety in certain instances, especially at higher doses. This paradoxical effect of cannabinoids on nausea, vomiting, and anxiety may be due to the dysregulation of the ECS, altering how it maintains these processes and contributing to the pathophysiology of CVS or CHS. PURPOSE: The purpose of this review is to highlight the involvement of the ECS in the regulation of stress, nausea, and vomiting. We discuss how prolonged cannabis use, such as in the case of CHS or heightened stress, can dysregulate the ECS and affect its modulation of these functions. The review also examines the evidence for the roles of ECS and stress systems' dysfunction in CVS and CHS to better understand the underlying mechanisms of these conditions.

Dietary limonene promotes gastrointestinal barrier function via upregulating tight/adherens junction proteins through cannabinoid receptor type-1 antagonistic mechanism and alters cellular metabolism in intestinal epithelial cells.

Limonene, a dietary monocyclic monoterpene commonly found in citrus fruits and various aromatic plants, has garnered increasing interest as a gastrointestinal protectant. This study aimed to assess the effects of limonene on intestinal epithelial barrier function and investigate the involvement of cannabinoid receptor type-1 (CB1R) in vitro. Additionally, the study focused on examining the metabolomic changes induced by limonene in the intestinal epithelial cells (Caco-2). Initial analysis of transepithelial electrical resistance (TEER) revealed that both l-limonene and d-limonene, isomers of limonene, led to a dose- and time-dependent increase in TEER in normal cells and those inflamed by pro-inflammatory cytokines mixture (CytoMix). Furthermore, both types of limonene reduced CytoMix-induced paracellular permeability, as demonstrated by a decrease in Lucifer yellow flux. Moreover, d-limonene and l-limonene treatment increased the expression of tight junction molecules (TJs) such as occludin, claudin-1, and ZO-1, at both the transcriptional and translational levels. d-Limonene upregulates E-cadherin, a molecule involved in adherens junctions (AJs). Mechanistic investigations demonstrated that d-limonene and l-limonene treatment significantly inhibited CB1R at the protein, while the mRNA level remained unchanged. Notably, the inhibitory effect of d-limonene on CB1R was remarkably similar to that of pharmacological CB1R antagonists, such as rimonabant and ORG27569. d-limonene also alters Caco-2 cell metabolites. A substantial reduction in ß-glucose and 2-succinamate was detected, suggesting limonene may impact intestinal epithelial cells' glucose uptake and glutamate metabolism. These findings suggest that d-limonene's CB1R antagonistic property could effectively aid in the recovery of intestinal barrier damage, marking it a promising gastrointestinal protectant.

Cardioprotective effect of CB1 receptor antagonist AM251 against ß receptor-stimulated myocardial infarction via modulation of NF-kB signaling pathway in diabetic mice.

We substantiated the effect of AM251, a cannabinoid receptor-1 (CB1R) antagonist, against ß-receptor stimulated myocardial infarction (MI) in streptozotocin (STZ)-induced diabetic mice via modulation- of the NF-kB signaling pathway. The different parameters were assessed such as ECG, hemodynamic, cardiac injury markers, oxidative stress parameters, pro-inflammatory cytokines, and histopathological abnormalities. Mice were fed a high-fat diet for 30 days. On day 7, to trigger diabetes, 150 mg/kg of STZ was injected intraperitoneally. On day 10, to determine whether diabetes developed, the blood level of glucose was monitored. From days 11-30, diabetic mice were injected with either CB1R agonist oleamide or antagonist AM251 or both, with concurrent administrations of ß-agonist isoproterenol on days 28 and 29 to induce MI. In comparison to normal, the myocardial infarcted diabetic animals demonstrated alterations in ECG, hemodynamic profiles, and diminished enzymatic activities (CK-MB, LDH, SOD, GSH, catalase), with concurrently increased MDA levels, which indicated increased oxidative stress in the myocardium. Additionally, higher concentrations of cytokines that signal myocardial inflammation, such as IL-1ß, IL-6, and TNF-α, were also noted. Furthermore, elevated myonecrosis, edema, and cell infiltration which is confirmed by histopathology of heart tissue. Treatment with AM251 significantly ameliorated myocardial redox status, reduced cytokines, and repaired enzymatic activities leading to subsequent recovery in cardiac function. AM251 effectively suppressed myonecrosis and edema. This study also showed that AM251 protects against myocardial inflammation and oxidative stress triggered by isoproterenol by blocking NF-kB signalling pathway. However, upregulation of the CB1R through oleamide showed significant cardiac toxicity. Conversely, the concurrent administration of oleamide and AM251 failed to induce cardiotoxic effects in isoproterenol-induced MI in diabetic mice which indicates downregulation of the CB1R might be associated with the cardioprotective effect.

Activation of CB2 Receptors by (-)-Cannabichromene but Not (+)-Cannabichromene.

Introduction: Cannabichromene (CBC) is a minor constituent of cannabis that is a selective cannabinoid CB2 receptor agonist and activator of TRPA1. To date, it has not been shown whether (-)-CBC, (+)-CBC, or both can mediate these effects. In this study, we investigate the activity of the CBC enantiomers at CB1, CB2, and Transient receptor potential ankyrin 1 (TRPA1) receptors in vitro. Materials and Methods: CBC enantiomers were purified from synthetic CBC by chiral chromatography, and their optical activity was confirmed by spectroscopy. Human CB1 and CB2 receptor activity was measured using a fluorescent assay of membrane potential in stably transfected AtT20 cells. TRPA1 activation was measured using a fluorescent assay of intracellular calcium in stably transfected HEK293 cells. Results: The (-)-CBC activated CB2 with an EC50 of 1.5 µM, to a maximum of 60% of (-)CP55940. (+)-CBC did not activate CB2 at concentrations up to 30 µM. Only 30 µM (-)-CBC produced detectable activation of CB1, (+)-CBC was inactive. Both (-)-CBC and (+)-CBC activated TRPA1; at 30 µM (-)-CBC produced an activation 50% of that of the reference agonist cinnamaldehyde (300 µM), 30 µM (+)-CBC activated TRPA1 to 38% of the cinnamaldehyde maximum. Discussion: It is unclear whether (-)-CBC is the sole or even the predominant enantiomer of CBC enzymatically synthesized in cannabis. This study shows that (-)-CBC is the active isomer at CB2 receptors, while both isomers activate TRPA1. The results suggest that medicinal preparations of CBC that target cannabinoid receptors would be most effective when (-)-CBC is the dominant isomer.

Interpreting mono- and poly-SCRA intoxications from an activity-based point of view: JWH-018 equivalents in serum as a comparative measure.

Synthetic cannabinoid receptor agonists (SCRAs) are a class of synthetic drugs that mimic and greatly surpass the effect of recreational cannabis. Acute SCRA intoxications are in general difficult to assess due to the large number of compounds involved, differing widely in both chemical structure and pharmacological properties. The rapid pace of emergence of unknown SCRAs hampers on one hand the timely availability of methods for identification and quantification to confirm and estimate the extent of the SCRA intoxication. On the other hand, lack of knowledge about the harm potential of emerging SCRAs hampers adequate interpretation of serum concentrations in intoxication cases. In the present study, a novel comparative measure for SCRA intoxications was evaluated, focusing on the cannabinoid activity (versus serum concentrations), which can be measured in serum extracts with an untargeted bioassay assessing ex vivo CB1 activity. Application of this principle to a series of SCRA intoxication cases (n = 48) allowed for the determination of activity equivalents, practically entailing a conversion from different SCRA serum concentrations to a JWH-018 equivalent. This allowed for the interpretation of both mono- (n = 34) and poly-SCRA (n = 14) intoxications, based on the intrinsic potential of the present serum levels to exert cannabinoid activity (cf. pharmacological/toxicological properties). A non-distinctive toxidrome was confirmed, showing no relation to CB1 activity. The JWH-018 equivalent was partly related to the poison severity score (PSS) and causality of the clinical intoxication elicited by the SCRA. Altogether, this equivalent concept allows to comparatively and timely interpret (poly-)SCRA intoxications based on CB1 activity.

Acute rimonabant treatment prevents anhedonia and memory loss in rats submitted to mild restraint stress.

Stress-related disorders are becoming increasingly common and are often associated with cognitive impairments. Within this context, the endocannabinoid (ECB) system, particularly the type 1 cannabinoid (CB1) receptor, seems to play a decisive role in restoring body homeostasis. There is consistent evidence in the literature that disrupted CB1-mediated neurotransmission can ultimately contribute to stress-related diseases. Therefore, the present study aimed to evaluate the participation of CB1 receptors in the integrity of stress-induced peripheral and behavioral responses. For this purpose, male adult Wistar rats underwent physical restraint (1 h/day, for 7 days), followed by a single administration of rimonabant (CB1 receptor antagonist, 3 mg/Kg, intraperitonial) at the end of stress protocol. Animals were then subjected to evaluation of neuroendocrine responses, behavioral tests and quantification of Iba-1 (microglial) immunoreactivity in the parvocellular subdivisions of the paraventricular nucleus of the hypothalamus (PVN). No effects of restraint stress or rimonabant administration were detected on body mass variation. However, stress significantly increased adrenal relative mass and corticosterone secretion, and reduced thymus relative size. The stress effects on adrenal size and corticosterone plasma levels were absent in rimonabant-treated rats, but the thymus size was further reduced in the restraint-rimonabant group. Restraint stress also induced anhedonia, a depression-like behavior, and reduced object recognition index, indicating memory recovery impairment. Treatment with the CB1 antagonist significantly reversed stress-induced anhedonia and memory deficit. In the PVN, restraint stress reduced the number of Iba-1 positive cells in the medial parvocellular region of vehicle- but not rimonabant-treated animals. Taken together, these results indicate that the acute inhibition of the CB1-mediated endogenous pathway restores stress-induced depression-like behaviors and memory loss, suggesting a role for endocannabinoids in the neuro-immune-endocrine interplay at both peripheral and hypothalamic levels.

A Comprehensive Exploration of the Multifaceted Neuroprotective Role of Cannabinoids in Alzheimer's Disease across a Decade of Research.

Alzheimer's disease (AD), a progressive neurodegenerative disorder, manifests through dysregulation of brain function and subsequent loss of bodily control, attributed to ß-amyloid plaque deposition and TAU protein hyperphosphorylation and aggregation, leading to neuronal death. Concurrently, similar cannabinoids to the ones derived from Cannabis sativa are present in the endocannabinoid system, acting through receptors CB1R and CB2R and other related receptors such as Trpv-1 and GPR-55, and are being extensively investigated for AD therapy. Given the limited efficacy and adverse effects of current available treatments, alternative approaches are crucial. Therefore, this review aims to identify effective natural and synthetic cannabinoids and elucidate their beneficial actions for AD treatment. PubMed and Scopus databases were queried (2014-2024) using keywords such as "Alzheimer's disease" and "cannabinoids". The majority of natural (Δ9-THC, CBD, AEA, etc.) and synthetic (JWH-133, WIN55,212-2, CP55-940, etc.) cannabinoids included showed promise in improving memory, cognition, and behavioral symptoms, potentially via pathways involving antioxidant effects of selective CB1R agonists (such as the BDNF/TrkB/Akt pathway) and immunomodulatory effects of selective CB2R agonists (TLR4/NF-κB p65 pathway). Combining anticholinesterase properties with a cannabinoid moiety may enhance therapeutic responses, addressing cholinergic deficits of AD brains. Thus, the positive outcomes of the vast majority of studies discussed support further advancing cannabinoids in clinical trials for AD treatment.

Hypothalamic cannabinoid signaling: Consequences for eating behavior.

In parallel to the legalization of cannabis for both medicinal and recreational purposes, cannabinoid use has steadily increased over the last decade in the United States. Cannabinoids, such as tetrahydrocannabinol and anandamide, bind to the central cannabinoid-1 (CB1) receptor to impact several physiological processes relevant for body weight regulation, including appetite and energy expenditure. The hypothalamus integrates peripheral signals related to energy balance, houses several nuclei that orchestrate eating, and expresses the CB1 receptor. Herein we review literature to date concerning cannabinergic action in the hypothalamus with a specific focus on eating behaviors. We highlight hypothalamic areas wherein researchers have focused their attention, including the lateral, arcuate, paraventricular, and ventromedial hypothalamic nuclei, and interactions with the hormone leptin. This review serves as a comprehensive analysis of what is known about cannabinoid signaling in the hypothalamus, highlights gaps in the literature, and suggests future directions.

Licofelone, a Dual COX/LOX Inhibitor, Ameliorates Paclitaxel-Induced Mechanical Allodynia in Rats in a Cannabinoid Receptor-Dependent Manner.

The use of paclitaxel as a chemotherapeutic drug is limited by the development of dose-dependent paclitaxel-induced neuropathic pain (PINP). Recently, we observed that the combination of indomethacin plus minocycline (IPM) attenuates PINP in a mouse model in a cannabinoid (CB) receptor-dependent manner. Indomethacin inhibits cyclooxygenase (COX) activity, and minocycline inhibits 5-lipoxygenase (5-LOX) activity. Male Sprague Dawley rats with paclitaxel-induced mechanical allodynia were treated with indomethacin, minocycline, IPM combination, licofelone (a dual COX/LOX inhibitor), or their vehicles. AM251, a CB1 receptor antagonist, and AM630, a CB2 receptor antagonist, were administered before the IPM combination or licofelone. Mechanical allodynia was measured using a dynamic plantar aesthesiometer. Molecular docking was performed using CB-Dock2. Licofelone and IPM combination had antiallodynic effects, which were significantly higher than either indomethacin or minocycline alone. AM251 and AM630 blocked the antiallodynic effects of IPM combination and licofelone. Molecular docking showed that licofelone binds to both CB1 and CB2 receptors with a high affinity similar to the phytocannabinoid 1-trans-delta-9-tetrahydrocannabinol and the synthetic cannabinoid WIN 55,212-2. Licofelone inhibits COX and LOX and/or directly interacts with CB receptors to produce antiallodynic effects in a rat model of PINP. The findings further suggest that licofelone could be a therapeutic agent for managing PINP.

Local activation of CB1 receptors by synthetic and endogenous cannabinoids dampens burst firing mode of reticular thalamic nucleus neurons in rats under ketamine anesthesia.

The reticular thalamic nucleus (RTN) is a thin shell that covers the dorsal thalamus and controls the overall information flow from the thalamus to the cerebral cortex through GABAergic projections that contact thalamo-cortical neurons (TC). RTN neurons receive glutamatergic afferents fibers from neurons of the sixth layer of the cerebral cortex and from TC collaterals. The firing mode of RTN neurons facilitates the generation of sleep-wake cycles; a tonic mode or desynchronized mode occurs during wake and REM sleep and a burst-firing mode or synchronized mode is associated with deep sleep. Despite the presence of cannabinoid receptors CB1 (CB1Rs) and mRNA that encodes these receptors in RTN neurons, there are few works that have analyzed the participation of endocannabinoid-mediated transmission on the electrical activity of RTN. Here, we locally blocked or activated CB1Rs in ketamine anesthetized rats to analyze the spontaneous extracellular spiking activity of RTN neurons. Our results show the presence of a tonic endocannabinoid input, since local infusion of AM 251, an antagonist/inverse agonist, modifies RTN neurons electrical activity; furthermore, local activation of CB1Rs by anandamide or WIN 55212-2 produces heterogeneous effects in the basal spontaneous spiking activity, where the main effect is an increase in the spiking rate accompanied by a decrease in bursting activity in a dose-dependent manner; this effect is inhibited by AM 251. In addition, previous activation of GABA-A receptors suppresses the effects of CB1Rs on reticular neurons. Our results show that local activation of CB1Rs primarily diminishes the burst firing mode of RTn neurons.

Activity-based detection of synthetic cannabinoid receptor agonists in plant materials.

BACKGROUND: Since late 2019, fortification of 'regular' cannabis plant material with synthetic cannabinoid receptor agonists (SCRAs) has become a notable phenomenon on the drug market. As many SCRAs pose a higher health risk than genuine cannabis, recognizing SCRA-adulterated cannabis is important from a harm reduction perspective. However, this is not always an easy task as adulterated cannabis may only be distinguished from genuine cannabis by dedicated, often expensive and time-consuming analytical techniques. In addition, the dynamic nature of the SCRA market renders identification of fortified samples a challenging task. Therefore, we established and applied an in vitro cannabinoid receptor 1 (CB1) activity-based procedure to screen plant material for the presence of SCRAs. METHODS: The assay principle relies on the functional complementation of a split-nanoluciferase following recruitment of ß-arrestin 2 to activated CB1. A straightforward sample preparation, encompassing methanolic extraction and dilution, was optimized for plant matrices, including cannabis, spiked with 5 µg/mg of the SCRA CP55,940. RESULTS: The bioassay successfully detected all samples of a set (n = 24) of analytically confirmed authentic Spice products, additionally providing relevant information on the 'strength' of a preparation and whether different samples may have originated from separate batches or possibly the same production batch. Finally, the methodology was applied to assess the occurrence of SCRA adulteration in a large set (n = 252) of herbal materials collected at an international dance festival. This did not reveal any positives, i.e. there were no samples that yielded a relevant CB1 activation. CONCLUSION: In summary, we established SCRA screening of herbal materials as a new application for the activity-based CB1 bioassay. The simplicity of the sample preparation, the rapid results and the universal character of the bioassay render it an effective and future-proof tool for evaluating herbal materials for the presence of SCRAs, which is relevant in the context of harm reduction.

Long-term effects on cardiorespiratory and behavioral responses in male and female rats prenatally exposed to cannabinoid.

Development of the respiratory system can be affected by the use of drugs during pregnancy, as the prenatal phase is highly sensitive to pharmacological interventions, resulting in long-term consequences. The deleterious effects of external cannabinoids during gestation may be related to negative interference in central nervous system formation, cardiorespiratory system function, and behavioral disorders. Nevertheless, the impact of external cannabinoids on cardiorespiratory network development, chemosensitivity, and its future consequences in adulthood is still unclear. We evaluated the effects of prenatal exposure to a synthetic cannabinoid (WIN 55,212-2, 0.5 mg·kg-1·day-1) on the cardiorespiratory control and panic-like behavior of male and female rats in adulthood. Exogenous cannabinoid exposure during pregnancy resulted in a sex-dependent difference in breathing control. Specifically, males showed increased chemosensitivity to CO2 and O2, whereas females exhibited decreased sensitivity. Altered cardiovascular control was evident, with prenatally treated males and females being more susceptible to hypertension and tachycardia under adverse environmental conditions. Moreover, WIN-treated males exhibited higher fragmentation of sleep episodes, whereas females displayed anxiolytic and panicolytic behavioral responses to CO2. However, no changes were observed in the mechanical component of the respiratory system, and there were no neuroanatomical alterations, such as changes in the expression of CB1 receptors in the brainstem or in the quantification of catecholaminergic and serotonergic neurons. These findings highlight that external interference in cannabinoid signaling during fetal development causes sex-specific, long-lasting effects for the cardiorespiratory system and behavioral responses in adulthood.NEW & NOTEWORTHY The surge in recreational cannabis use and cannabinoid-based medication prescription among pregnant women has been notable in recent years, fueled by the misconception that natural products are inherently safe. Significant gaps persist regarding the potential risks of maternal consumption of cannabinoids and the long-term effects on the cardiorespiratory system of their offspring, which may be determined by sex. Accordingly, this research aims to diminish this lack of information and raise a note of caution.

Negative allosteric modulation of CB1 cannabinoid receptor signaling suppresses opioid-mediated tolerance and withdrawal without blocking opioid antinociception.

The direct blockade of CB1 cannabinoid receptors produces therapeutic effects as well as adverse side-effects that limit their clinical potential. CB1 negative allosteric modulators (NAMs) represent an indirect approach to decrease the affinity and/or efficacy of orthosteric cannabinoid ligands or endocannabinoids at CB1. We recently reported that GAT358, a CB1-NAM, blocked opioid-induced mesocorticolimbic dopamine release and reward via a CB1-allosteric mechanism of action. Whether a CB1-NAM dampens opioid-mediated therapeutic effects such as analgesia or alters other unwanted opioid side-effects remain unknown. Here, we characterized the effects of GAT358 on nociceptive behaviors in the presence and absence of morphine in male rats. We examined the impact of GAT358 on formalin-evoked pain behavior and Fos protein expression, a marker of neuronal activation, in the lumbar spinal cord. We also assessed the impact of GAT358 on morphine-induced slowing of colonic transit, tolerance, and withdrawal behaviors in male mice. GAT358 attenuated morphine antinociceptive tolerance without blocking acute antinociception and reduced morphine-induced slowing of colonic motility without impacting fecal boli production. GAT358 also produced antinociception in the presence and absence of morphine in the formalin model of inflammatory nociception and reduced the number of formalin-evoked Fos protein-like immunoreactive cells in the lumbar spinal cord. Finally, GAT358 mitigated the somatic signs of naloxone-precipitated, but not spontaneous, opioid withdrawal following chronic morphine dosing. Our results support the therapeutic potential of CB1-NAMs as novel drug candidates aimed at preserving opioid-mediated analgesia while preventing their unwanted side-effects. Our studies also uncover previously unrecognized antinociceptive properties associated with an arrestin-biased CB1-NAM.

Gut Microbiota-Mediated Alterations of Hippocampal CB1R Regulating the Diurnal Variation of Cognitive Impairment Induced by Hepatic Ischemia-Reperfusion Injury in Mice.

Patients suffering from hepatic ischemia-reperfusion injury (HIRI) frequently exhibit postoperative cognitive deficits. Our previous observations have emphasized the diurnal variation in hepatic ischemia-reperfusion injury-induced cognitive impairment, in which gut microbiota-associated hippocampal lipid metabolism plays an important role. Herein, we further investigated the molecular mechanisms involved in the process. Hepatic ischemia-reperfusion surgery was performed under morning (ZT0, 08:00) and evening (ZT12, 20:00). Fecal microbiota transplantation was used to associate HIRI model with pseudo-germ-free mice. The novel object recognition test and Y-maze test were used to assess cognitive function. 16S rRNA gene sequencing and analysis were used for microbial analysis. Western blotting was used for hippocampal protein analysis. Compared with the ZT0-HIRI group, ZT12-HIRI mice showed learning and short term memory impairment, accompanied by down-regulated expression of hippocampal CB1R, but not CB2R. Both gut microbiota composition and microbiota metabolites were significantly different in ZT12-HIRI mice compared with ZT0-HIRI. Fecal microbiota transplantation from the ZT12-HIRI was demonstrated to induce cognitive impairment behavior and down-regulated hippocampal CB1R and ß-arrestin1. Intraperitoneal administration of CB1R inhibitor AM251 (1 mg/kg) down-regulated hippocampal CB1R and caused cognitive impairment in ZT0-HIRI mice. And intraperitoneal administration of CB1R agonist WIN 55,212-2 (1 mg/kg) up-regulated hippocampal CB1R and improved cognitive impairment in ZT12-HIRI mice. In summary, the results suggest that gut microbiota may regulate the diurnal variation of HIRI-induced cognitive function by interfering with hippocampal CB1R.

Additive antinociceptive action of intrathecal anandamide reuptake inhibitor and morphine in the management of post-incisional pain in rats.

Spinal opioids have mixed efficacy and their adverse effects force treatment cessation of postoperative pain. Consequently, there is an ongoing search for new therapeutic strategies. Here, we evaluated the analgesic efficacy of intrathecal UCM707, an anandamide reuptake inhibitor, and morphine combination. Firstly, we assessed the effects of morphine (1, 5 and 10 µg), UCM707 (75 µg) and its combination in the hot plate. Then, morphine + UCM707 at sub-effective doses was evaluated in a rat post-incisional pain model. In addition, µ-, CB1r-, CB2r- and TRPV1-antagonists were pre-administered before the combination. Activation of µ-opioid and CB1r, and Cnr1, Cnr2, Oprm1 and TRPV1 expressions were evaluated in the lumbar sacra and periaqueductal grey by [35 S]-GTPγS binding autoradiography and qPCR studies. In the hot plate, morphine (1 µg) and UCM707 (75 µg) induced a more robust analgesic effect than each drug alone. Morphine plus UCM707 did not modify µ-opioid nor CB1 receptor function in the PAG or LS. Cnr1 and TRPV1 expression increased in the lumbar sacra (LS). Morphine plus UCM707 significantly reduced post-incisional pain at 1 and 4 days after surgery. Cnr1, Cnr2 and TRPV1 expressions increased in the LS. Blockade of µ-opioid receptor reduced combination effects on days 1 and 4. CB1r- and CB2r-antagonism reduced morphine + UCM707 effects on days 1 and 4, respectively. CB1r and TRPV1-antagonism improved their antinociceptive effects on day 4. These results revealed a synergistic/additive analgesic effect of UCM707 and morphine combination controlling postincisional pain. CB1r, CB2r and TRPV1 contribute differently as central sensitization occurs.

Pharmaceutical targeting of the cannabinoid type 1 receptor impacts the crosstalk between immune cells and islets to reduce insulitis in humans.

AIMS/HYPOTHESIS: Insulitis, a hallmark of inflammation preceding autoimmune type 1 diabetes, leads to the eventual loss of functional beta cells. However, functional beta cells can persist even in the face of continuous insulitis. Despite advances in immunosuppressive treatments, maintaining functional beta cells to prevent insulitis progression and hyperglycaemia remains a challenge. The cannabinoid type 1 receptor (CB1R), present in immune cells and beta cells, regulates inflammation and beta cell function. Here, we pioneer an ex vivo model mirroring human insulitis to investigate the role of CB1R in this process. METHODS: CD4+ T lymphocytes were isolated from peripheral blood mononuclear cells (PBMCs) from male and female individuals at the onset of type 1 diabetes and from non-diabetic individuals, RNA was extracted and mRNA expression was analysed by real-time PCR. Single beta cell expression from donors with type 1 diabetes was obtained from data mining. Patient-derived human islets from male and female cadaveric donors were 3D-cultured in solubilised extracellular matrix gel in co-culture with the same donor PBMCs, and incubated with cytokines (IL-1ß, TNF-α, IFN-γ) for 24-48 h in the presence of vehicle or increasing concentrations of the CB1R blocker JD-5037. Expression of CNR1 (encoding for CB1R) was ablated using CRISPR/Cas9 technology. Viability, intracellular stress and signalling were assayed by live-cell probing and real-time PCR. The islet function measured as glucose-stimulated insulin secretion was determined in a perifusion system. Infiltration of immune cells into the islets was monitored by microscopy. Non-obese diabetic mice aged 7 weeks were treated for 1 week with JD-5037, then euthanised. Profiling of immune cells infiltrated in the islets was performed by flow cytometry. RESULTS: CNR1 expression was upregulated in circulating CD4+ T cells from individuals at type 1 diabetes onset (6.9-fold higher vs healthy individuals) and in sorted islet beta cells from donors with type 1 diabetes (3.6-fold higher vs healthy counterparts). The peripherally restricted CB1R inverse agonist JD-5037 arrested the initiation of insulitis in humans and mice. Mechanistically, CB1R blockade prevented islet NO production and ameliorated the ATF6 arm of the unfolded protein response. Consequently, cyto/chemokine expression decreased in human islets, leading to sustained islet cell viability and function. CONCLUSIONS/INTERPRETATION: These results suggest that CB1R could be an interesting target for type 1 diabetes while highlighting the regulatory mechanisms of insulitis. Moreover, these findings may apply to type 2 diabetes where islet inflammation is also a pathophysiological factor. DATA AVAILABILITY: Transcriptomic analysis of sorted human beta cells are from Gene Expression Omnibus database, accession no. GSE121863, available at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM3448161 .

Positive allosteric modulation of the cannabinoid CB1 receptor potentiates endocannabinoid signalling and changes ERK1/2 phosphorylation kinetics.

BACKGROUND AND PURPOSE: Activation of CB1 by exogenous agonists causes adverse effects in vivo. Positive allosteric modulation may offer improved therapeutic potential and a reduced on-target adverse effect profile compared with orthosteric agonists, due to reduced desensitisation/tolerance, but this has not been directly tested. This study investigated the ability of PAMs/ago-PAMs to induce receptor regulation pathways, including desensitisation and receptor internalisation. EXPERIMENTAL APPROACH: Bioluminescence resonance energy transfer (BRET) assays in HEK293 cells were performed to investigate G protein dissociation, ERK1/2 phosphorylation and ß-arrestin 2 translocation, while immunocytochemistry was performed to measure internalisation of CB1 in response to the PAMs ZCZ011, GAT229 and ABD1236 alone and in combination with the orthosteric agonists AEA, 2-AG, and AMB-FUBINACA. KEY RESULTS: ZCZ011, GAT229 and ABD1236 were allosteric agonists in all pathways tested. The ago-PAM ZCZ011 induced a biphasic ERK1/2 phosphorylation time course compared to transient activation by orthosteric agonists. In combination with 2-AG but not AEA or AMB-FUBINACA, ZCZ011 and ABD1236 caused the transient peak of ERK1/2 phosphorylation to become sustained. All PAMs increased the potency and efficacy of AEA-induced signalling in all pathways tested; however, no notable potentiation of 2-AG or AMB-FUBINACA was observed. CONCLUSION AND IMPLICATIONS: Ago-PAMs can potentiate endocannabinoid CB1 agonism by AEA to a larger extent compared with 2-AG. However, all compounds were found to be allosteric agonists and induce activation of CB1 in the absence of endocannabinoid, including ß-arrestin 2 recruitment and internalisation. Thus, the spatiotemporal signalling of endogenous cannabinoids will not be retained in vivo.

Interactions between metabotropic glutamate and CB1 receptors: implications for mood, cognition, and synaptic signaling based on data from mGluR and CB1R-targeting drugs.

Metabotropic glutamate receptors (mGluRs) are part of the G protein-coupled receptors (GPCRs) family. They are coupled to Gαq (group I) or Gi/o (groups II and III) proteins, which result in the generation of diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3) or the inhibition of adenylyl cyclase, respectively. mGluRs have been implicated in anxiety, depression, learning, and synaptic plasticity. Similarly, CB1 cannabinoid receptors (CB1Rs), also GPCRs, play roles in cognitive function and mood regulation through Gαi/o-mediated inhibition of adenylyl cyclase. Both mGluRs and CB1Rs exhibit surface labeling and undergo endocytosis. Given the similar cellular distribution and mechanisms of action, this review complies with fundamental data on the potential interactions and mutual regulation of mGluRs and CB1Rs in the context of depression, anxiety, and cognition, providing pioneering insights into their interplay.