Cannabinoid Activation Research Articles

The Modulatory Effect of Exercise on the Endocannabinoid Signaling Pathway in the Epileptic Rats

Background: Regular moderate exercise and endogenous cannabinoid activity have independently been shown to alleviate seizure (SE) attacks. Objectives: This study aimed to investigate the effect of physical activity on the expression levels of cannabinoid CB1 and CB2 receptors in the brains Methods: Male Wistar rats were divided into five groups: Sham, SE, physical activity (PA), PA + SE, and PA before SE. Epileptic SEs were induced by administering pentylenetetrazol (PTZ; intraperitoneally, 35 mg/kg) every other day for four weeks in the SE, PA + SE, and PA before SE groups. Animals in the PA, PA + SE, and PA before SE groups participated in treadmill running (30 minutes per day, five days a week). The mean number of cortical and hippocampal (CA1, CA3) CB1 and CB2 receptors was assessed using immunohistochemistry. Results: The study data revealed a significant reduction of CB1 and CB2 receptors in the CA1, CA3, and cortex of the SE group compared to the sham group. A significant increase in CB1 receptors was observed in the PA and PA before SE groups compared to the SE group in both cortical and hippocampal areas. Physical activity significantly increased hippocampal and cortical CB2 receptor distribution in the PA, PA + SE, and PA before SE groups compared to the SE group. Conclusions: These findings suggest that exercise modulates the expression of hippocampal and cortical cannabinoid receptors in epileptic rats, highlighting the involvement of the endocannabinoid pathway in the anti-epileptic effects of exercise.

Exploring the Potential of Synthetic Cannabinoids: Modulation of Biological Activity of Normal and Cancerous Human Colon Epithelial Cells.

Colorectal cancer (CRC) is a global problem. Oncology currently practices conventional methods of treating this carcinoma, including surgery, chemotherapy, and radiotherapy. Unfortunately, their efficacy is low; hence, the exploration of new therapies is critical. Recently, many efforts have focused on developing safe and effective anticancer compounds. Some of them include cannabinoids. In the present study, we obtained cannabinoids, such as cannabidiol (CBD), abnormal cannabigerol (abn-CBG), cannabichromene (CBC), and cannabicitran (CBT), by chemical synthesis and performed the biological evaluation of their activity on colon cancer cells. In this study, we analyzed the effects of selected cannabinoids on the lifespan and metabolic activity of normal colonic epithelial cells and cancer colon cells. This study demonstrated that cannabinoids can induce apoptosis in cancer cells by modulating mitochondrial dehydrogenase activity and cellular membrane integrity. The tested cannabinoids also influenced cell cycle progression. We also investigated the antioxidant activity of cannabinoids and established a relationship between the type of cannabinoid and nitric oxide (NO) production in normal and cancerous colon cells. To conclude, it seems that, due to their interesting properties, the cannabinoids studied may constitute an interesting target for further research aimed at their use in alternative or combined therapies for human colon cancer.

Cannabidiol and fluorinated derivative anti-cancer properties against glioblastoma multiforme cell lines, and synergy with imidazotetrazine agents

BackgroundGlioblastoma multiforme (GBM) is an aggressive cancer with poor prognosis, partly due to resistance to the standard chemotherapy treatment, temozolomide (TMZ). Phytocannabinoid cannabidiol (CBD) has exhibited anti-cancer effects against GBM, however, CBD’s ability to overcome common resistance mechanisms to TMZ have not yet been investigated. 4’-Fluoro-cannabidiol (4’-F-CBD, or HUF-101/PECS-101) is a derivative of CBD, that exhibits increased activity compared to CBD during in vivo behavioural studies.MethodsThis anti-cancer activity of cannabinoids against GBM cells sensitive to and representing major resistance mechanisms to TMZ was investigated. Cannabinoids were also studied in combination with imidazotetrazine agents, and advanced mass spectrometry with the 3D OrbiSIMS was used to investigate the mechanism of action of CBD.ResultsCBD and 4’-F-CBD were found to overcome two major resistance mechanisms (methylguanine DNA-methyltransferase (MGMT) overexpression and DNA mismatch repair (MMR)-deficiency). Synergistic responses were observed when cells were exposed to cannabinoids and imidazotetrazine agents. Synergy increased with T25 and 4’-F-CBD. 3D OrbiSIMS analysis highlighted the presence of methylated-DNA, a previously unknown anti-cancer mechanism of action of CBD.ConclusionsThis work demonstrates the anti-cancer activity of 4’-F-CBD and the synergy of cannabinoids with imidazotetrazine agents for the first time and expands understanding of CBD mechanism of action.

S2: Neuromodulation in Geriatric Psychiatry: Innovations and Insights in ECT, TMS, tDCS, and Cannabinoid Therapies

With the rapidly aging population worldwide, the psychiatric care of older adults faces increasingly complex challenges. Geriatric psychiatry must adapt to address the rising prevalence of depression, anxiety, and cognitive impairments with innovative and effective treatments. Neuromodulation in geriatrics encompasses a variety of innovative treatments designed to help manage symptoms of various neurological and psychiatric conditions common in older adults. This symposium will explore the mechanisms, clinical applications, safety, and up-to-date research in Electroconvulsive Therapy (ECT), Transcranial Magnetic Stimulation (TMS), Transcranial Direct Current Stimulation (tDCS), and cannabinoid therapies in older adults.ECT, conducted under anesthesia, induces seizures via small electric currents through the brain. It is proven to be safe and efficacious in severe depression, catatonia, agitation, aggression in dementia, and other neuropsychiatric conditions especially in the elderly. TMS is a non-invasive procedure that applies electromagnetic pulses to stimulate nerve cells in specific areas of the brain, thought to change neural activity and is FDA-approved for treatment resistant depression. tDCS, employing low direct current via scalp electrodes, is being increasingly explored for depressive disorders and cognitive disorders. Cannabinoids, activating cannabinoid receptors, emerge as a promising option for dementia-related agitation.In this symposium, Dr. Georgios Petrides will present updated research on the effectiveness of ECT to treat agitation in patients with dementia. Dr. Adriana Hermida will review theTMS literature and share clinical outcomes from Emory University. Dr. Maria Lapid will discuss tDCS effectiveness in depression among older adults. Dr. Brent Forester will examine cannabinoids’ role in dementia-related agitation, including ongoing clinical trials with dronabinol.The learning Objectives for this session is to understand the principles, applications, benefits, and potential side effects of various neuromodulation techniques and cannabinoids in geriatric care. By the end of the presentation, attendees should be able to explain the mechanisms of action of ECT, TMS, tDCS, and cannabinoids, identify the conditions they can treat, and discuss their relevance and effectiveness in geriatric psychiatry. Furthermore, attendees should be able to compare these techniques and make informed decisions about their use in clinical practice.

Bioelectronic sensing platform emulating the human endocannabinoid system for assessing and modulating of cannabinoid activity

Cannabinoids are involved in physiological and neuromodulatory processes through their interactions with the human cannabinoid receptor-based endocannabinoid system. Their association with neurodegenerative diseases and brain reward pathways underscores the importance of evaluating and modulating cannabinoid activity for both understanding physiological mechanisms and developing therapeutic drugs. The use of agonists and antagonists could be strategic approaches for modulation. In this study, we introduce a bioelectronic sensor designed to monitor cannabinoid binding to receptors and assess their agonistic and antagonistic properties. We produced human cannabinoid receptor 1 (hCB1R) via an Escherichia coli expression system and incorporated it into nanodiscs (NDs). These hCB1R-NDs were then immobilized on a single-walled carbon nanotube field-effect transistor (swCNT-FET) to construct a bioelectronic sensing platform. This novel system can sensitively detect the cannabinoid ligand anandamide (AEA) at concentrations as low as 1 fM, demonstrating high selectivity and real-time response. It also successfully identified the hCB1R agonist Δ9-tetrahydrocannabinol and observed that the hCB1R antagonist rimonabant diminished the sensor signal upon AEA binding, indicating the antagonism-based modulation of ligand interaction. Consequently, our bioelectronic sensing platform holds potential for ligand detection and analysis of agonism and antagonism.

A lactate-dependent shift of glycolysis mediates synaptic and cognitive processes in male mice

Astrocytes control brain activity via both metabolic processes and gliotransmission, but the physiological links between these functions are scantly known. Here we show that endogenous activation of astrocyte type-1 cannabinoid (CB1) receptors determines a shift of glycolysis towards the lactate-dependent production of d-serine, thereby gating synaptic and cognitive functions in male mice. Mutant mice lacking the CB1 receptor gene in astrocytes (GFAP-CB1-KO) are impaired in novel object recognition (NOR) memory. This phenotype is rescued by the gliotransmitter d-serine, by its precursor l-serine, and also by lactate and 3,5-DHBA, an agonist of the lactate receptor HCAR1. Such lactate-dependent effect is abolished when the astrocyte-specific phosphorylated-pathway (PP), which diverts glycolysis towards l-serine synthesis, is blocked. Consistently, lactate and 3,5-DHBA promoted the co-agonist binding site occupancy of CA1 post-synaptic NMDA receptors in hippocampal slices in a PP-dependent manner. Thus, a tight cross-talk between astrocytic energy metabolism and gliotransmission determines synaptic and cognitive processes.

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.

Synthesis and Antitumour Evaluation of Tricyclic Indole-2-Carboxamides against Paediatric Brain Cancer Cells.

Antitumour properties of some cannabinoids (CB) have been reported in the literature as early as 1970s, however there is no clear consensus to date on the exact mechanisms leading to cancer cell death. The indole-based WIN 55,212-2 and SDB-001 are both known as potent agonists at both CB1 and CB2 receptors, yet we demonstrate herein that only the former can exert in vitro antitumour effects when tested against a paediatric brain cancer cell line KNS42. In this report, we describe the synthesis of novel 3,4-fused tricyclic indoles and evaluate their functional potencies at both cannabinoid receptors, as well as their abilities to inhibit the growth or proliferation of KNS42 cells. Compared to our previously reported indole-2-carboxamides, these 3,4-fused tricyclic indoles had either completely lost activities, or, showed moderate-to-weak antagonism at both CB1 and CB2 receptors. Compound 23 displayed the most potent antitumour properties among the series. Our results further support the involvement of non-CB pathways for the observed antitumour activities of amidoalkylindole-based cannabinoids, in line with our previous findings. Transcriptomic analysis comparing cells treated or non-treated with compound 23 suggested the observed antitumour effects of 23 are likely to result mainly from disruption of the FOXM1-regulated cell cycle pathways.

Regulation of Cannabinoid and Opioid Receptor Levels by Endogenous and Pharmacological Chaperones.

Cannabinoid and opioid receptor activities can be modulated by a variety of post-translational mechanisms including the formation of interacting complexes. This study examines the involvement of endogenous and exogenous chaperones in modulating the abundance and activity of cannabinoid CB1 receptor (CB1R), δ opioid receptor (DOR), and CB1R-DOR interacting complexes. Focusing on endogenous protein chaperones, namely receptor transporter proteins (RTPs), we examined relative mRNA expression in the mouse spinal cord and found RTP4 to be expressed at higher levels compared with other RTPs. Next, we assessed the effect of RTP4 on receptor abundance by manipulating RTP4 expression in cell lines. Overexpression of RTP4 causes an increase and knock-down causes a decrease in the levels of CB1R, DOR, and CB1R-DOR interacting complexes; this is accompanied by parallel changes in signaling. The ability of small molecule lipophilic ligands to function as exogenous chaperones was examined using receptor-selective antagonists. Long-term treatment leads to increases in receptor abundance and activity with no changes in mRNA supporting a role as pharmacological chaperones. Finally, the effect of cannabidiol (CBD), a small molecule ligand and a major active component of cannabis, on receptor abundance and activity in mice was examined. We find that CBD administration leads to increases in receptor abundance and activity in mouse spinal cord. Together, these results highlight a role for chaperones (proteins and small molecules) in modulating levels and activity of CB1R, DOR, and their interacting complexes potentially through mechanisms including receptor maturation and trafficking. SIGNIFICANCE STATEMENT: This study highlights a role for chaperones (endogenous and small membrane-permeable molecules) in modulating levels of cannabinoid CB1 receptor, delta opioid receptor, and their interacting complexes. These chaperones could be developed as therapeutics for pathologies involving these receptors.

The Intoxication Equivalency of 11-Hydroxy-Δ9-Tetrahydrocannabinol Relative to Δ9-Tetrahydrocannabinol.

Δ9-Tetrahydrocannabinol (THC) is a psychoactive phytocannabinoid found in the Cannabis sativa plant. THC is primarily metabolized into 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (COOH-THC), which may themselves be psychoactive. There is very little research-based evidence concerning the pharmacokinetics and pharmacodynamics of 11-OH-THC as an individual compound. Male C57BL/6 mice were treated with THC or 11-OH-THC via intraperitoneal injection, tail vein intravenous injection, or oral gavage, and whole-blood compound levels were measured to determine pharmacokinetic parameters [Cmax, time to Cmax (Tmax), elimination half-life, area under the curve, apparent volume of distribution, systemic clearance, terminal rate constant, and absolute bioavailability] while also monitoring changes in catalepsy, body temperature, and nociception. 11-OH-THC achieved a Tmax at 30 minutes for all routes of administration. The maximum concentration at 30 minutes was not different between intravenous and intraperitoneal routes, but the oral gavage Cmax was significantly lower. THC had a 10-minute time to the maximum concentration, which was the first blood collection time point, for intravenous and intraperitoneal and 60 minutes for oral gavage, with a lower Cmax for intraperitoneal and oral gavage compared with intravenous. When accounting for circulating compound levels and ED50 responses, these data suggest that 11-OH-THC was 153% as active as THC in the tail-flick test of nociception and 78% as active as THC for catalepsy. Therefore, 11-OH-THC displayed equal or greater activity than the parent compound THC, even when accounting for pharmacokinetic differences. Thus, the THC metabolite 11-OH-THC likely plays a critical role in the bioactivity of cannabis; understanding its activity when administered directly will aid in the interpretation of future animal and human studies. SIGNIFICANCE STATEMENT: This study establishes that the primary metabolite of THC, 11-OH-THC, displays equal or greater activity than THC in a mouse model of cannabinoid activity when directly administered and even when accounting for route of administration, sex, pharmacokinetic, and pharmacodynamic differences. These data provide critical insight into the bioactivity of THC metabolites that will inform the interpretation of future in vivo cannabinoid research and represent a model for how THC consumption and metabolism may affect cannabis use in humans.

High-performance supercritical fluid chromatography hyphenated to high-resolution quadrupole-time of flight hybrid mass spectrometry (SFC-HR-QTOF-MS/MS) for the determination of cannabinoids in edible products

A growing number of papers suggest the positive action of cannabinoids in the treatment of several diseases, which is ascribed to the ability of phytocannabinoids to affect the human endocannabinoid system. In particular, this activity is characteristic of CBD, a non-psychoactive variation of 9-THC. It initiated a trend in the manufacturing of edible and cosmetic products containing CBD that can be easily acquired in drugstores. However, the composition of such products has rarely been tested. Hence, there is a need for data regarding their safety. Considering the possibility of cannabinoid interconversions as well as the complexity and diversity of edible products’ matrix, their analysis requires a suitable preparation and cleanup procedure. In this paper, the perspective of the application of Supercritical Fluid Chromatography (SFC) coupled to High-Resolution (HR) Quadrupole-Time of Flight (Q-TOF) tandem mass spectrometry (MS/MS) with electrospray ionization (ESI) in quality control of cannabis-containing food products was examined. The focus is on six neutral (CBD, 9-THC, CBC, CBG, CBN, and THCV) and three acidic (CBDA, THCA, and CBGA) cannabinoids. The study covers the thorough optimization of conditions for chromatographic separation with the use of the supercritical CO2 (scCO2)-based mobile phase and the working parameters of MS/MS detection with the elucidation of fragmentation patterns. Thanks to the utilization of scCO2, a short analysis time of 17 min., high intensity and small width of peaks, as well as low consumption of toxic organic solvent have been achieved at the 2-EP column with a 5 μm grain size. To reach such performance in LC, the utilization of more expensive columns packed with sub-2 μm particles is needed. In the case of the solvent matrix, the LOD was equal to 8 ng mL−1 for CBDA, THCA, and CBGA, 15 ng mL−1 for CBG and THCV, and 21 ng mL−1 for THC, CBD, CBC, and CBN. In the case of tea, an LOD of 25 ng mL−1 for acidic and 75 ng mL−1 for neutral cannabinoids has been obtained. The accuracy and precision were within the ± 15 % tolerance limit. Finally, the devised protocol has been applied for cannabinoid determination in hemp tea infusions.

A Humanized CB1R Yeast Biosensor Enables Facile Screening of Cannabinoid Compounds.

Yeast expression of human G-protein-coupled receptors (GPCRs) can be used as a biosensor platform for the detection of pharmaceuticals. Cannabinoid receptor type 1 (CB1R) is of particular interest, given the cornucopia of natural and synthetic cannabinoids being explored as therapeutics. We show for the first time that engineering the N-terminus of CB1R allows for efficient signal transduction in yeast, and that engineering the sterol composition of the yeast membrane modulates its performance. Using an engineered cannabinoid biosensor, we demonstrate that large libraries of synthetic cannabinoids and terpenes can be quickly screened to elucidate known and novel structure-activity relationships. The biosensor strains offer a ready platform for evaluating the activity of new synthetic cannabinoids, monitoring drugs of abuse, and developing therapeutic molecules.

The Potential of Cannabichromene (CBC) as a Therapeutic Agent.

There is a growing interest in the use of medicinal plants to treat a variety of diseases, and one of the most commonly used medicinal plants globally is Cannabis sativa The two most abundant cannabinoids (Δ9-tetrahydrocannabinol and cannabidiol) have been governmentally approved to treat selected medical conditions; however, the plant produces over 100 cannabinoids, including cannabichromene (CBC). Although the cannabinoids share a common precursor molecule, cannabigerol, they are structurally and pharmacologically unique. These differences may engender differing therapeutic potentials. In this review, we will examine what is currently known about CBC with regards to pharmacodynamics, pharmacokinetics, and receptor profile. We will also discuss the therapeutic areas that have been examined for this cannabinoid, notably antinociceptive, antibacterial, and anti-seizure activities. Finally, we will discuss areas where new research is needed and potential novel medicinal applications for CBC. SIGNIFICANCE STATEMENT: Cannabichromene (CBC) has been suggested to have disparate therapeutic benefits such as anti-inflammatory, anticonvulsant, antibacterial, and antinociceptive effects. Most of the focus on the medical benefits of cannabinoids has been focused on Δ9-tetrahydrocannabinol and cannabidiol. The preliminary studies on CBC indicate that this phytocannabinoid may have unique therapeutic potential that warrants further investigation. Following easier access to hemp, CBC products are commercially available over-the-counter and are being widely utilized with little or no evidence of their safety or efficacy.

Investigation of the intrinsic cannabinoid activity of hemp-derived and semisynthetic cannabinoids with β-arrestin2 recruitment assays-and how this matters for the harm potential of seized drugs.

Cultivation of industrial low-Δ9-tetrahydrocannabinol (Δ9-THC) hemp has created an oversupply of cannabidiol (CBD)-rich products. The fact that phytocannabinoids, including CBD, can be used as precursors to synthetically produce a range of THC variants-potentially located in a legal loophole-has led to a diversification of cannabis recreational drug markets. 'Hemp-compliant', 'hemp-derived' and 'semisynthetic' cannabinoid products are emerging and being advertised as (legal) alternatives for Δ9-THC. This study included a large panel (n = 30) of THC isomers, homologs, and analogs that might be derived via semisynthetic procedures. As a proxy for the abuse potential of these compounds, we assessed their potential to activate the CB1 cannabinoid receptor with a β-arrestin2 recruitment bioassay (picomolar-micromolar concentrations). Multiple THC homologs (tetrahydrocannabihexol, THCH; tetrahydrocannabiphorol, THCP; tetrahydrocannabinol-C8, THC-C8) and THC analogs (hexahydrocannabinol, HHC; hexahydrocannabiphorol, HHCP) were identified that showed higher potential for CB1 activation than Δ9-THC, based on either higher efficacy (Emax) or higher potency (EC50). Structure-activity relationships were assessed for Δ9-THC and Δ8-THC homologs encompassing elongated alkyl chains. Additionally, stereoisomer-specific differences in CB1 activity were established for various THC isomers (Δ7-THC, Δ10-THC) and analogs (HHC, HHCP). Evaluation of the relative abundance of 9(S)-HHC and 9(R)-HHC epimers in seized drug material revealed varying epimeric compositions between batches. Increased abundance of the less active 9(S)-HHC epimer empirically resulted in decreased potency, but sustained efficacy for the resulting diastereomeric mixture. In conclusion, monitoring of semisynthetic cannabinoids is encouraged as the dosing and the relative composition of stereoisomers can impact the harm potential of these drugs, relative to Δ9-THC products.

Pharmacological Evaluation of Cannabinoid Receptor Modulators Using GRABeCB2.0 Sensor.

Cannabinoid receptors CB1R and CB2R are G-protein coupled receptors acted upon by endocannabinoids (eCBs), namely 2-arachidonoylglycerol (2-AG) and N-arachidonoyl ethanolamine (AEA), with unique pharmacology and modulate disparate physiological processes. A genetically encoded GPCR activation-based sensor that was developed recently-GRABeCB2.0-has been shown to be capable of monitoring real-time changes in eCB levels in cultured cells and preclinical models. However, its responsiveness to exogenous synthetic cannabinoid agents, particularly antagonists and allosteric modulators, has not been extensively characterized. This current study expands upon the pharmacological characteristics of GRABeCB2.0 to enhance the understanding of fluorescent signal alterations in response to various functionally indiscriminate cannabinoid ligands. The results from this study could enhance the utility of the GRABeCB2.0 sensor for in vitro as well as in vivo studies of cannabinoid action and may aid in the development of novel ligands.

Cannabinoid type 2 receptor inhibition enhances the antidepressant and proneurogenic effects of physical exercise after chronic stress

Chronic stress is a major risk factor for neuropsychiatric conditions such as depression. Adult hippocampal neurogenesis (AHN) has emerged as a promising target to counteract stress-related disorders given the ability of newborn neurons to facilitate endogenous plasticity. Recent data sheds light on the interaction between cannabinoids and neurotrophic factors underlying the regulation of AHN, with important effects on cognitive plasticity and emotional flexibility. Since physical exercise (PE) is known to enhance neurotrophic factor levels, we hypothesised that PE could engage with cannabinoids to influence AHN and that this would result in beneficial effects under stressful conditions. We therefore investigated the actions of modulating cannabinoid type 2 receptors (CB2R), which are devoid of psychotropic effects, in combination with PE in chronically stressed animals. We found that CB2R inhibition, but not CB2R activation, in combination with PE significantly ameliorated stress-evoked emotional changes and cognitive deficits. Importantly, this combined strategy critically shaped stress-induced changes in AHN dynamics, leading to a significant increase in the rates of cell proliferation and differentiation of newborn neurons, overall reduction in neuroinflammation, and increased hippocampal levels of BDNF. Together, these results show that CB2Rs are crucial regulators of the beneficial effects of PE in countering the effects of chronic stress. Our work emphasises the importance of understanding the mechanisms behind the actions of cannabinoids and PE and provides a framework for future therapeutic strategies to treat stress-related disorders that capitalise on lifestyle interventions complemented with endocannabinoid pharmacomodulation.

Abstract 4566: Synergistic anticancer activity of cannabinoids and beta caryophyllene in xenograft mouse model of triple negative breast cancer

Abstract Combination therapy, a treatment modality that studies the effect of two or more drugs is the most common strategy used to overcome drug resistance in cancer. Cannabinoids have been proven to be potential anticancer agents. Terpenes have also been demonstrated to exhibit anticancer activity. The purpose of this study was to evaluate the effect of combinations of various cannabinoids and terpenes against drug resistant lung and breast cancer. Several cannabinoids and terpenes such as cannabidiol (CBD), cannabichromene (CBC), cannabidiolic acid (CBDA), cannabidivarin (CBDV), cannabinol (CBN), cannabigerol (CBG) and β-caryophyllene (BC), bisabolol, myrcene, linalool, limonene, geraniol, neralidol, α-pinene, α-terpineol, γ-terpinene respectively were screened for their anticancer activity against MDA MB 231 DOX RT, H1975 OSM RT, PDX Lung cancer cell lines using 2D cytotoxicity and 3D spheroids assay. It was found that the cytotoxic potential of combinations of cannabinoids with BC increased by 10 folds in comparison to their individual anticancer activity. The combinations that exhibit synergistic action were determined from combination index (CI) values using compusyn software. Other assays such as colony formation, wound healing, apoptotic assay, cell cycle analysis by flow cytometry elucidated the possible mechanism underlying the synergism of the combinations investigated. CBC+BC was proven to be the most potent combination from all the in-vitro studies and hence was further evaluated in in-vivo tumor xenografts. Briefly, 5 million MDA MB 231 DOX RT cells were injected by S.C route in the right flank region of BALB/c nude mice. The animals were treated after the tumor volume reached 180-200mm3. The animals were administered CBC (15mg/kg, i.p.), BC (100mg/kg, i.p.) and CBC+BC thrice a week for two weeks. The tumor volume in the combination group was reduced by 2 folds relative to individual treatments and 4 folds relative to control. Western blot analysis of tumor xenografts showed downregulation of apoptotic markers such as PARP, mTOR, pAMPK, Survivin, autophagy markers such as LC 3, Glypican 5 and migration markers such as vimentin and Integrin. Immune check point proteins such as PD-L1 and PD-1 were also downregulated by combination treatment. This was further validated by qPCR. In conclusion, CBC induces programmed cell death in tumors and induces autophagy whereas BC inhibits migration and thus this combination was able to overcome resistance than individual therapies in a synergistic fashion. Citation Format: Mounika Aare, Islauddin Khan, Aakash Nathani, Breana Boirie, Arvind Bagde, Mandip Singh. Synergistic anticancer activity of cannabinoids and beta caryophyllene in xenograft mouse model of triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4566.

Linking the Autotaxin-LPA Axis to Medicinal Cannabis and the Endocannabinoid System.

Over the past few decades, many current uses for cannabinoids have been described, ranging from controlling epilepsy to neuropathic pain and anxiety treatment. Medicines containing cannabinoids have been approved by both the FDA and the EMA for the control of specific diseases for which there are few alternatives. However, the molecular-level mechanism of action of cannabinoids is still poorly understood. Recently, cannabinoids have been shown to interact with autotaxin (ATX), a secreted lysophospholipase D enzyme responsible for catalyzing lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA), a pleiotropic growth factor that interacts with LPA receptors. In addition, a high-resolution structure of ATX in complex with THC has recently been published, accompanied by biochemical studies investigating this interaction. Due to their LPA-like structure, endocannabinoids have been shown to interact with ATX in a less potent manner. This finding opens new areas of research regarding cannabinoids and endocannabinoids, as it could establish the effect of these compounds at the molecular level, particularly in relation to inflammation, which cannot be explained by the interaction with CB1 and CB2 receptors alone. Further research is needed to elucidate the mechanism behind the interaction between cannabinoids and endocannabinoids in humans and to fully explore the therapeutic potential of such approaches.

Cannabinoids in the treatment of glioblastoma.

Glioblastoma (GBM) is the most prevalent primary malignant tumor of the nervous system. While the treatment of other neoplasms is increasingly more efficacious the median survival rate of GBM patients remains low and equals about 14 months. Due to this fact, there are intensive efforts to find drugs that would help combat GBM. Nowadays cannabinoids are becoming more and more important in the field of cancer and not only because of their properties of antiemetic drugs during chemotherapy. These compounds may have a direct cytotoxic effect on cancer cells. Studies indicate GBM has disturbances in the endocannabinoid system-changes in cannabinoid metabolism as well as in the cannabinoid receptor expression. The GBM cells show expression of cannabinoid receptors 1 and 2 (CB1R and CB2R), which mediate various actions of cannabinoids. Through these receptors, cannabinoids inhibit the proliferation and invasion of GBM cells, along with changing their morphology. Cannabinoids also induce an intrinsic pathway of apoptosis in the tumor. Hence the use of cannabinoids in the treatment of GBM may be beneficial to the patients. So far, studies focusing on using cannabinoids in GBM therapy are mainly preclinical and involve cell lines and mice. The results are promising and show cannabinoids inhibit GBM growth. Several clinical studies are also being carried out. The preliminary results show good tolerance of cannabinoids and prolonged survival after administration of these drugs. In this review, we describe the impact of cannabinoids on GBM and glioma cells in vitro and in animal studies. We also provide overview of clinical trials on using cannabinoids in the treatment of GBM.

Additive anxiolytic-like effect of citicoline and ACPA in the non-acute restraint stress (NARS) and acute restraint stress (ARS) mice

The cannabinoid system plays a key role in stress-related emotional symptoms such as anxiety. Citicoline is a supplemental substance with neuroprotective properties that alleviates anxiety-related behaviors. There is a relation between the actions of cannabinoids and cholinergic systems. So, we decided to evaluate the effects of intracerebroventricular (i.c.v.) infusion of cannabinoid CB1 receptor agents on citicoline-produced response to anxiety-like behaviors in the non-acute restraint stress (NARS) and acute restraint stress (ARS) mice. For i.c.v. microinjection of drugs, a guide cannula was inserted in the left lateral ventricle. ARS was induced by movement restraint for 4 h. Anxiety-related behaviors were assessed using an elevated plus maze (EPM). The results showed that induction of ARS for 4 h decreased the percentage of time spent in the open arms (%OAT) and the percentage of entries to the open arms (%OAE) without affecting locomotor activity, showing anxiogenic-like behaviors. i.c.v. infusion of ACPA (1 µg/mouse) induced an anxiolytic-like effect due to the enhancement of %OAT in the NARS and ARS mice. Nonetheless, i.c.v. microinjection of AM251 (1 µg/mouse) decreased %OAT in the NARS and ARS mice which suggested an anxiogenic-like response. Intraperitoneal (i.p.) administration of citicoline (80 mg/kg) induced an anxiolytic-like effect by the augmentation of %OAT in the ARS mice. Furthermore, when ACPA and citicoline were co-administrated, ACPA potentiated the anxiolytic-like effect induced by citicoline in the NARS and ARS mice. On the other hand, when AM251 and the citicoline were co-injected, AM251 reversed the anxiolytic-like response induced by the citicoline in the NARS and ARS mice. The results of this research exhibited an additive effect between citicoline and ACPA on the induction of anxiolytic-like response in the NARS and ARS mice. Our results indicated an interaction between citicoline and cannabinoid CB1 receptor drugs on the control of anxiety-like behaviors in the NARS and ARS mice.