Development and validation of an HPLC-DAD method for the quantification of cannabigerol, cannabidiol, cannabinol and cannabichromene in human plasma and mouse matrices.

Cannabigerol, cannabidiol, cannabinol and cannabichromene are non-psychoactive phytocannabinoids, highly present in Cannabis sativa, for which numerous therapeutical applications have been described. However, additional pre-clinical and clinical data, including toxicopharmacokinetic and pharmacodynamic studies, remain required to support their use in clinical practice and new therapeutic applications. To support these studies, a new high performance liquid chromatography technique (HPLC) with diode-array detection (DAD) was developed and validated to quantify these cannabinoids in human plasma and mouse matrices. Sample extraction was accomplished by protein precipitation and double liquid-liquid extraction. Simvastatin and perampanel were used as internal standards in human and mouse matrices, respectively. Chromatographic separation was achieved in 16 min on an InfinityLab Poroshell® 120 C18 column (4.6 mm × 100 mm, 2.7 µm) at 40 °C. A mobile phase composed of water/acetonitrile was pumped with a gradient elution program at 1.0 mL min-1. The technique revealed linearity in the defined concentration ranges with a determination coefficient of over 0.99. Intra and inter-day accuracy and precision values ranged from -14.83 to 13.97% and 1.08 to 13.74%, respectively. Sample stability was assessed to ensure that handling and storage conditions did not compromise analyte concentrations in different matrices. Carry-over was absent and recoveries were over 77.31%. This technique was successfully applied for the therapeutic monitoring of cannabidiol and preliminary pre-clinical studies with cannabigerol and cannabidiol. All samples were within calibration ranges, with the exception of cannabigerol after intraperitoneal administration. This is the first HPLC-DAD technique that simultaneously quantifies cannabinoids in these biological matrices, supporting future pre-clinical and clinical investigations.

Insights into the human metabolism of hexahydrocannabinol by non-targeted liquid chromatography-high-resolution tandem mass spectrometry.

Hexahydrocannabinol (HHC), 6,6,9-trimethyl-3-pentyl-6a,7,8,9,10,10a-hexahydrobenzo[c]chromen-1-ol, is a semi-synthetic cannabinoid that has presented challenges to analytical laboratories due to its emergence and spread in the drug market. The lack of information on human pharmacokinetics hinders the development and application of presumptive and confirmatory tests for reliably detecting HHC consumption. To address this knowledge gap, we report the analytical results obtained from systematic forensic toxicological analysis of body-fluid samples collected from three individuals suspected of drug-impaired driving after HHC consumption. Urine and plasma samples were analyzed using non-targeted liquid chromatography-high-resolution tandem mass spectrometry. The results provided evidence that HHC undergoes biotransformation reactions similar to other well-characterized cannabinoids, such as ∆9-tetrahydrocannabinol or cannabidiol. Notably, HHC itself was only detectable in plasma samples, not in urine samples. The observed Phase I reactions involved oxidation of C11 and the pentyl side chain, leading to corresponding hydroxylated and carboxylic acid species. Additionally, extensive glucuronidation of HHC and its Phase I metabolites was evident.

Hexahydrocannabinol (HHC) and Δ9-tetrahydrocannabinol (Δ9-THC) driven activation of cannabinoid receptor 1 results in biased intracellular signaling.

The Cannabis sativa plant has been used for centuries as a recreational drug and more recently in the treatment of patients with neurological or psychiatric disorders. In many instances, treatment goals include relief from posttraumatic disorders, anxiety, or to support treatment of chronic pain. Ligands acting on cannabinoid receptor 1 (CB1R) are also potential targets for the treatment of other health conditions. Using an evidence-based approach, pharmacological investigation of CB1R agonists is timely, with the aim to provide chronically ill patients relief using well-defined and characterized compounds from cannabis. Hexahydrocannabinol (HHC), currently available over the counter in many countries to adults and even children, is of great interests to policy makers, legal administrators, and healthcare regulators, as well as pharmacologists. Herein, we studied the pharmacodynamics of HHC epimers, which activate CB1R. We compared their key CB1R-mediated signaling pathway activities and compared them to the pathways activated by Δ9-tetrahydrocannabinol (Δ9-THC). We provide evidence that activation of CB1R by HHC ligands is only broadly comparable to those mediated by Δ9-THC, and that both HHC epimers have unique properties. Together with the greater chemical stability of HHC compared to Δ9-THC, these molecules have a potential to become a part of modern medicine.

Fragment-based drug discovery and biological evaluation of novel cannabinol-based inhibitors of oxytosis/ferroptosis for neurological disorders.

The oxytosis/ferroptosis regulated cell death pathway is an emerging field of research owing to its pathophysiological relevance to a wide range of neurological disorders, including Alzheimer's and Parkinson's diseases and traumatic brain injury. Developing novel neurotherapeutics to inhibit oxytosis/ferroptosis offers exciting opportunities for the treatment of these and other neurological diseases. Previously, we discovered cannabinol (CBN) as a unique, potent inhibitor of oxytosis/ferroptosis by targeting mitochondria and modulating their function in neuronal cells. To further elucidate which key pharmacophores and chemical space are essential to the beneficial effects of CBN, we herein introduce a fragment-based drug discovery strategy in conjunction with cell-based phenotypic screens using oxytosis/ferroptosis to determine the structure-activity relationship of CBN. The resulting information led to the development of four new CBN analogs, CP1-CP4, that not only preserve the sub-micromolar potency of neuroprotection and mitochondria-modulating activities seen with CBN in neuronal cell models but also have better druglike properties. Moreover, compared to CBN, the analog CP1 shows improved in vivo efficacy in the Drosophila model of mild traumatic brain injury. Together these studies identify the key molecular scaffolds of cannabinoids that contribute to neuroprotection against oxytosis/ferroptosis. They also highlight the advantageous approach of combining in vitro cell-based assays and rapid in vivo studies using Drosophila models for evaluating new therapeutic compounds.

Appearance of hexahydrocannabinols as recreational drugs and implications for cannabis drug testing - focus on HHC, HHC-P, HHC-O and HHC-H.

This study investigated the effects of hexahydrocannabinol (HHC) and other unclassified cannabinoids, which were recently introduced to the recreational drug market, on cannabis drug testing in urine and oral fluid samples. After the appearance of HHC in Sweden in 2022, the number of posts about HHC on an online drug discussion forum increased significantly in the spring of 2023, indicating increased interest and use. In parallel, the frequency of false positive screening tests for tetrahydrocannabinol (THC) in oral fluid, and for its carboxy metabolite (THC-COOH) in urine, rose from <2% to >10%. This suggested that HHC cross-reacted with the antibodies in the immunoassay screening, which was confirmed in spiking experiments with HHC, HHC-COOH, HHC acetate (HHC-O), hexahydrocannabihexol (HHC-H), hexahydrocannabiphorol (HHC-P), and THC-P. When HHC and HHC-P were classified as narcotics in Sweden on 11 July 2023, they disappeared from the online and street shops market and were replaced by other unregulated variants (e.g. HHC-O and THC-P). In urine samples submitted for routine cannabis drug testing, HHC-COOH concentrations up to 205 (mean 60, median 27) µg/L were observed. To conclude, cannabis drug testing cannot rely on results from immunoassay screening, as it cannot distinguish between different tetra- and hexahydrocannabinols, some being classified but others unregulated. The current trend for increased use of unregulated cannabinols will likely increase the proportion of positive cannabis screening results that need to be confirmed with mass spectrometric methods. However, the observed cross-reactivity also means a way to pick up use of new cannabinoids that otherwise risk going undetected.

A GraphSAGE-based model with fingerprints only to predict drug-drug interactions.

Drugs are an effective way to treat various diseases. Some diseases are so complicated that the effect of a single drug for such diseases is limited, which has led to the emergence of combination drug therapy. The use multiple drugs to treat these diseases can improve the drug efficacy, but it can also bring adverse effects. Thus, it is essential to determine drug-drug interactions (DDIs). Recently, deep learning algorithms have become popular to design DDI prediction models. However, most deep learning-based models need several types of drug properties, inducing the application problems for drugs without these properties. In this study, a new deep learning-based model was designed to predict DDIs. For wide applications, drugs were first represented by commonly used properties, referred to as fingerprint features. Then, these features were perfectly fused with the drug interaction network by a type of graph convolutional network method, GraphSAGE, yielding high-level drug features. The inner product was adopted to score the strength of drug pairs. The model was evaluated by 10-fold cross-validation, resulting in an AUROC of 0.9704 and AUPR of 0.9727. Such performance was better than the previous model which directly used drug fingerprint features and was competitive compared with some other previous models that used more drug properties. Furthermore, the ablation tests indicated the importance of the main parts of the model, and we analyzed the strengths and limitations of a model for drugs with different degrees in the network. This model identified some novel DDIs that may bring expected benefits, such as the combination of PEA and cannabinol that may produce better effects. DDIs that may cause unexpected side effects have also been discovered, such as the combined use of WIN 55,212-2 and cannabinol. These DDIs can provide novel insights for treating complex diseases or avoiding adverse drug events.

Oil-in-water and oleogel-in-water emulsion encapsulate with hemp seed oil containing Δ9-tetrahydrocannabinol and cannabinol: Stability, degradation and in vitro simulation characteristics.

The present study focused on investigating the stability and in vitro simulation characteristics of oil-in-water (O/W) and oleogel-in-water (Og/W) emulsions. Compared with O/W emulsion, the Og/W emulsion exhibited superior stability, with a more evenly spread droplet distribution, and the Og/W emulsion containing 3 % hemp seed protein (HSP) showed better stability against environmental factors, including heat treatment, ionic strength, and changes in pH. Additionally, the stability of Δ9-tetrahydrocannabinol (Δ9-THC) and cannabinol (CBN) and the in vitro digestion of hemp seed oil (HSO) were evaluated. The half-life of CBN in the Og/W emulsion was found to be 131.82 days, with a degradation rate of 0.00527. The in vitro simulation results indicated that the Og/W emulsion effectively delayed the intestinal digestion of HSO, and the bioaccessibility of Δ9-THC and CBN reached 56.0 % and 58.0 %, respectively. The study findings demonstrated that the Og/W emulsion constructed with oleogel and HSP, exhibited excellent stability.

Evaluation of cannabimimetic effects of selected minor cannabinoids and Terpenoids in mice.

BACKGROUND: The cannabis plant contains several cannabinoids, and many terpenoids that give cannabis its distinctive flavoring and aroma. Δ9-Tetrahydrocannabinol (Δ9-THC) is the plant's primary psychoactive constituent. Given the abuse liability of Δ9-THC, assessment of the psychoactive effects of minor cannabinoids and other plant constituents is important, especially for compounds that may be used medicinally. This study sought to evaluate select minor cannabinoids and terpenes for Δ9-THC-like psychoactivity in mouse Δ9-THC drug discrimination and determine their binding affinities at CB1 and CB2 receptors. METHODS: Δ9-THC, cannabidiol (CBD), cannabinol (CBN), cannabichromene (CBC), cannabichromenevarin (CBCV), Δ8-tetrahydrocannabinol (Δ8-THC), (6aR,9R)-Δ10-tetrahydrocannabinol [(6aR,9R)-Δ10-THC], Δ9-tetrahydrocannabinol varin (THCV), ß-caryophyllene (BC), and ß-caryophyllene oxide (BCO) were examined. RESULTS: All minor cannabinoids showed measurable cannabinoid 1 (CB1) and cannabinoid 2 (CB2) receptor binding, with CBC, CBCV, and CBD, showing the weakest CB1 receptor binding affinity. BC and BCO exhibited negligible affinity for both CB1 and CB2 receptors. In drug discrimination, only Δ8-THC fully substituted for Δ9-THC, while CBN and (6aR,9R)-Δ10-THC partially substituted for Δ9-THC. THCV and BCO did not alter the discriminative stimulus effects of Δ9-THC. CONCLUSION: In summary, only some of myriad cannabinoids and other chemicals found in the cannabis plant bind potently to the identified cannabinoid receptors. Further, only four of the compounds tested herein [Δ9-THC, Δ8-THC, (6aR,9R)-Δ10-THC, and CBN] produced Δ9-THC-like discriminative stimulus effects, suggesting they may possess cannabimimetic subjective effects. Given that the medicinal properties of phytocannabinoids and terpenoids are being investigated scientifically, delineation of their potential adverse effects, including their ability to produce Δ9-THC-like intoxication, is crucial.

Functionally-selective inhibition of threshold sodium currents and excitability in dorsal root ganglion neurons by cannabinol.

Cannabinol (CBN), an incompletely understood metabolite for ∆9-tetrahydrocannabinol, has been suggested as an analgesic. CBN interacts with endocannabinoid (CB) receptors, but is also reported to interact with non-CB targets, including various ion channels. We assessed CBN effects on voltage-dependent sodium (Nav) channels expressed heterologously and in native dorsal root ganglion (DRG) neurons. Our results indicate that CBN is a functionally-selective, but structurally-non-selective Nav current inhibitor. CBN's main effect is on slow inactivation. CBN slows recovery from slow-inactivated states, and hyperpolarizes steady-state inactivation, as channels enter deeper and slower inactivated states. Multielectrode array recordings indicate that CBN attenuates DRG neuron excitability. Voltage- and current-clamp analysis of freshly isolated DRG neurons via our automated patch-clamp platform confirmed these findings. The inhibitory effects of CBN on Nav currents and on DRG neuron excitability add a new dimension to its actions and suggest that this cannabinoid may be useful for neuropathic pain.

The structurally diverse phytocannabinoids cannabichromene, cannabigerol and cannabinol significantly inhibit amyloid ß-evoked neurotoxicity and changes in cell morphology in PC12 cells.

BACKGROUND: Phytocannabinoids (pCBs) have been shown to inhibit the aggregation and neurotoxicity of the neurotoxic Alzheimer's disease protein beta amyloid (Aß). We characterized the capacity of six pCBs-cannabichromene (CBC), cannabigerol (CBG), cannabinol (CBN), cannabidivarin (CBDV), cannabidiol (CBD) and Δ9 -tetrahydrocannabinol (Δ9 -THC)-to disrupt Aß aggregation and protect against Aß-evoked neurotoxicity in PC12 cells. METHODS: Neuroprotection against lipid peroxidation and Aß-induced cytotoxicity was assessed using the MTT assay. Transmission electron microscopy was used to visualize pCB effects on Aß aggregation and fluorescence microscopy, with morphometrics and principal component analysis to assess PC12 cell morphology. RESULTS: CBD inhibited lipid peroxidation with no significant effect on Aß toxicity, whilst CBN, CBDV and CBG provided neuroprotection. CBC, CBG and CBN inhibited Aß1-42 -induced neurotoxicity in PC12 cells, as did Δ9 -THC, CBD and CBDV. CBC, CBN and CBDV inhibited Aß aggregation, whilst Δ9 -THC reduced aggregate density. Aß1-42 induced morphological changes in PC12 cells, including a reduction in neuritic projections and rounded cell morphology. CBC and CBG inhibited this effect, whilst Δ9 -THC, CBD and CBDV did not alter Aß1-42 effects on cell morphology. CONCLUSIONS: These findings highlight the neuroprotective activity of CBC, CBG and CBN as novel pCBs associated with variable effects on Aß-evoked neurite damage and inhibition of amyloid ß aggregation.

Determination of cannabinoids in 50 µL whole blood samples by online extraction using turbulent flow chromatography and LC-HRAM-Orbitrap-MS: Application on driving under the influence of drugs cases.

The analysis of cannabinoids in whole blood is usually done by traditional mass spectrometry (MS) techniques, after offline cleanup or derivatization steps which can be lengthy, laborious, and expensive. We present a simple, fast, highly specific, and sensitive method for the determination of Δ9 -tetrahydrocannabinol (THC), cannabidiol (CBD), cannabinol (CBN), 11-hydroxy-Δ9 -tetrahydrocannabinol (11-OH-THC), and 11-nor-9-carboxy-Δ9 -tetrahydrocannabinol (THC-COOH) in 50 µL whole blood samples. After the addition of deuterated internal standards (IS) and a simple protein precipitation step, an online extraction of sample supernatants using turbulent flow chromatography (TurboFlow-Thermo Scientific) was carried out. Analytes were separated on a C18 analytical column and detected by LC-HRAM-Orbitrap-MS using a Thermo Scientific Q Exactive Focus MS system. MS detection was performed in polarity switching and selected ion monitoring (SIM) modes using five specific acquisition windows, at a resolution of 70,000 (FWHM). Total run time was about 10 min including preanalytical steps. Method validation was carried out by determining limit of detection (LOD), lower limit of quantitation (LLOQ), linearity range, analytical accuracy, intra-assay and interassay precision, carry-over, matrix effect, extraction recovery, and selectivity, for all analytes. Measurement uncertainties were also evaluated, and a decision rule was set with confidence for forensic purposes. The method may become suitable for clinical and forensic toxicology applications, taking advantage of the small matrix volume required, the simple and cost-effective sample preparation procedure, and the fast analytical run time. Performances were monitored over a long-term period and tested on 7620 driving under the influence of drugs (DUID) samples, including 641 positive samples.

The Safety and Comparative Effectiveness of Non-Psychoactive Cannabinoid Formulations for the Improvement of Sleep: A Double-Blinded, Randomized Controlled Trial.

BACKGROUND: Clinical evidence on the use of cannabidiol (CBD) for sleep remains limited. Even fewer studies have tested the comparative effectiveness of cannabinoid formulations found within CBD products used for sleep or how they compare to other complementary therapies such as melatonin. METHODS: Participants (N = 1,793 adults experiencing symptoms of sleep disturbance) were randomly assigned to receive a 4-week supply of 1 of 6 products (all capsules) containing either 15 mg CBD or 5 mg melatonin, alone or in combination with minor cannabinoids. Sleep disturbance was assessed over a period of 5 weeks (baseline week and 4 weeks of product use) using Patient-Reported Outcomes Measurement Information System (PROMIS™) Sleep Disturbance SF 8A, administered via weekly online surveys. A linear mixed-effects regression model was used to assess the differences in the change in sleep disturbance through time between each active product arm and CBD isolate. RESULTS: All formulations exhibited a favorable safety profile (12% of participants reported a side effect and none were severe) and led to significant improvements in sleep disturbance (p < 0.001 in within-group comparisons). Most participants (56% to 75%) across all formulations experienced a clinically important improvement in their sleep quality. There were no significant differences in effect, however, between 15 mg CBD isolate and formulations containing 15 mg CBD and 15 mg cannabinol (CBN), alone or in combination with 5 mg cannabichromene (CBC). There were also no significant differences in effect between 15 mg CBD isolate and formulations containing 5 mg melatonin, alone or in combination with 15 mg CBD and 15 mg CBN. CONCLUSIONS: Our findings suggest that chronic use of a low dose of CBD is safe and could improve sleep quality, though these effects do not exceed that of 5 mg melatonin. Moreover, the addition of low doses of CBN and CBC may not improve the effect of formulations containing CBD or melatonin isolate.

Cannabigerol (CBG) signal enhancement in its analysis by gas chromatography coupled with tandem mass spectrometry.

PURPOSE: According to recent reports, cannabigerol (CBG) concentration level in blood and body fluids may have forensic utility as a highly specific albeit insensitive biomarker of recent cannabis smoking. While the analytical sensitivity of cannabidiol (CBD), Δ9-tetrahydrocannabinol (Δ9-THC), cannabichromene (CBC) or cannabinol (CBN) estimation by gas chromatography-mass spectrometry (GC-MS) is similar and sufficiently high, it is exceptionally low in the case of CBG (ca. 25 times lower than for the other mentioned cannabinoids). The purpose of this study is to explain the reasons for the extremely low analytical sensitivity of GC-MS in estimating CBG and to present possible ways of its improvement. METHODS: Nuclear magnetic resonance (NMR) data and GC-MS responses to CBG and its various derivatization and transformation products were studied. RESULTS: The validation data of individual derivatives of CBG and its transformation products were established. CBG silylation/acylation or hydration allows to decrease LOD about 3 times, whereas the formation of pyranic CBG derivative leads to 10-times decrease of LOD. The paper enriches the literature of the subject by providing MS and NMR spectra, not published so far, for derivatives of CBG and its transformation products. The most likely cause of low GC-MS response to CBG is also presented. CONCLUSIONS: The presented results shows that although the signal increase of CBG can be obtained through its derivatization by silylation and/or acylation, the greatest increase is observed in the case of its cyclization to the pyranic CBG form during the sample preparation process. The CBG cyclization procedure is very simple and workable in estimating this cannabinoid in blood/plasma samples.

The transcriptomic response to cannabidiol of Treponema denticola, a phytocannabinoid-resistant periodontal pathogen.

AIM: The use of cannabis, which contains multiple antimicrobials, may be a risk factor for periodontitis. We hypothesized that multiple oral spirochetes would be phytocannabinoid-resistant and that cannabidiol (CBD) would act as an environmental stressor to which Treponema denticola would respond transcriptionally, thereby providing first insights into spirochetal survival strategies. MATERIALS AND METHODS: Oral spirochete growth was monitored spectrophotometrically in the presence and absence of physiologically relevant phytocannabinoid doses, the transcriptional response to phytocannabinoid exposure determined by RNAseq, specific gene activity fluxes verified using qRT-PCR and orthologues among fully sequenced oral spirochetes identified. RESULTS: Multiple strains of oral treponemes were resistant to CBD (0.1-10 µg/mL), while T. denticola ATCC 35405 was resistant to all phytocannabinoids tested (CBD, cannabinol [CBN], tetrahydrocannabinol [THC]). A total of 392 T. denticola ATCC 35405 genes were found to be CBD-responsive by RNAseq. A selected subset of these genes was independently verified by qRT-PCR. Genes found to be differentially activated by both methods included several involved in transcriptional regulation and toxin control. Suppressed genes included several involved in chemotaxis and proteolysis. CONCLUSIONS: Oral spirochetes, unlike some other periodontal bacteria, are resistant to physiological doses of phytocannabinoids. Investigation of CBD-induced transcriptomic changes provided insight into the resistance mechanisms of this important periodontal pathogen. These findings should be considered in the context of the reported enhanced susceptibility to periodontitis in cannabis users.

Past-Year Use Prevalence of Cannabidiol, Cannabigerol, Cannabinol, and Δ8-Tetrahydrocannabinol Among US Adults.

This survey study characterizes past-year use prevalence and factors associated with use of cannabidiol, cannabigerol, cannabinol, and Δ8-tetrahydrocannabinol among US adults.

The dual role of cannabidiol on monocyte-derived dendritic cell differentiation and maturation.

Introduction: Extracts and compounds isolated from hemp (Cannabis sativa) are increasingly gaining popularity in the treatment of a number of diseases, with topical formulations for dermatological conditions leading the way. Phytocannabinoids such as ( )-cannabidiol, ( )-cannabinol and ( )-Δ9-tetrahydrocannabivarin (CBD, CBN, and THCV, respectively), are present in variable amounts in the plant, and have been shown to have mostly anti-inflammatory effects both in vitro and in vivo, albeit dominantly in murine models. The role of phytocannabinoids in regulating responses of dendritic cells (DCs) remains unclear. Methods: Our research aimed to investigate the effects of CBD, CBN, and THCV on human DCs differentiated from monocytes (moDCs). moDCs were treated with up to 10 µM of each phytocannabinoid, and their effects on viability, differentiation, and maturation were assessed both alone, and in conjunction with TLR agonists. The effects of CBD on cytokine production, T cell activation and polarization as well as the transcriptome of moDCs was also determined. Results: Phytocannabinoids did not influence the viability of moDCs up to 10 µM, and only CBD had effects on maturational markers of moDCs, and neither compound influenced LPS-induced activation at 10 µM. Since only CBD had measurable effects on moDCs, in our subsequent experiments we tested the effect only of that pCB. On moDCs differentiated in the presence of CBD subsequent activation by LPS induced a markedly different, much more tolerogenic response. CBD-treated moDCs also produced significantly more interleukin (IL)-6, TNFα and, importantly, IL-10 in response to LPS, which shows a shift toward anti-inflammatory signaling, as well as a more robust secretory response in general. To rule out the possibility that these effects of CBD are specific to TLR4 signaling, we determined the effect of CBD on TLR7/8-induced maturation as well, and saw similar, although less marked responses. CBD-treated moDCs were also less efficient at activating naïve T cells after LPS stimulation, further supporting the tolerogenic effect of this phytocannabinoid on moDCs. Reactome pathway analysis showed an inflammatory response to LPS in moDCs, and to a lesser extent to CBD as well. In contrast CBD-treated moDCs responded to LPS with a shift towards a more tolerogenic phenotype, as IL-10 signaling was the most prominently induced pathway in this group. Discussion: Our results show that CBD achieves an anti-inflammatory effect on adaptive immune responses only in the presence of an activating stimuli on moDCs by reprogramming cells during long-term treatment, and not through acute, short-term effects.

Anti-Inflammatory Effects of Minor Cannabinoids CBC, THCV, and CBN in Human Macrophages.

Inflammation is a natural response of the body to signals of tissue damage or infection caused by pathogens. However, when it becomes imbalanced, it can lead to various disorders such as cancer, obesity, cardiovascular problems, neurological conditions, and diabetes. The endocannabinoid system, which is present throughout the body, plays a regulatory role in different organs and influences functions such as food intake, pain perception, stress response, glucose tolerance, inflammation, cell growth and specialization, and metabolism. Phytocannabinoids derived from Cannabis sativa can interact with this system and affect its functioning. In this study, we investigate the mechanisms underlying the anti-inflammatory effects of three minor phytocannabinoids including tetrahydrocannabivarin (THCV), cannabichromene (CBC), and cannabinol (CBN) using an in vitro system. We pre-treated THP-1 macrophages with different doses of phytocannabinoids or vehicle for one hour, followed by treating the cells with 500 ng/mL of LPS or leaving them untreated for three hours. To induce the second phase of NLRP3 inflammasome activation, LPS-treated cells were further treated with 5 mM ATP for 30 min. Our findings suggest that the mitigation of the PANX1/P2X7 axis plays a significant role in the anti-inflammatory effects of THCV and CBC on NLRP3 inflammasome activation. Additionally, we observed that CBC and THCV could also downregulate the IL-6/TYK-2/STAT-3 pathway. Furthermore, we discovered that CBN may exert its inhibitory impact on the assembly of the NLRP3 inflammasome by reducing PANX1 cleavage. Interestingly, we also found that the elevated ADAR1 transcript responded negatively to THCV and CBC in LPS-macrophages, indicating a potential involvement of ADAR1 in the anti-inflammatory effects of these two phytocannabinoids. THCV and CBN inhibit P-NF-κB, downregulating proinflammatory gene transcription. In summary, THCV, CBC, and CBN exert anti-inflammatory effects by influencing different stages of gene expression: transcription, post-transcriptional regulation, translation, and post-translational regulation.

Phytocannabinoids Reduce Seizures in Larval Zebrafish and Affect Endocannabinoid Gene Expression.

Cannabis has demonstrated anticonvulsant properties, and about thirty percent of epileptic patients do not have satisfactory seizure management with standard treatment and could potentially benefit from cannabis-based intervention. Here, we report the use of cannabinoids to treat pentylenetetrazol (PTZ)-induced convulsions in a zebrafish model, their effect on gene expression, and a simple assay for assessing their uptake in zebrafish tissues. Using an optimized behavioral assay, we show that cannabidiol (CBD) and cannabichromene (CBC) and cannabinol (CBN) are effective at reducing seizures at low doses, with little evidence of sedation, and our novel HPLC assay indicates that CBC is effective with the lowest accumulation in larval tissues. All cannabinoids tested were effective at higher concentrations. Pharmacological manipulation of potential receptors demonstrates that Gpr55 partially mediates the anticonvulsant effects of CBD. Treatment of zebrafish larvae with endocannabinoids, such as 2-arachidonoylglycerol (2-AG) and anandamide (AEA), altered larvae movement, and the expression of genes that regulate their metabolism was affected by phytocannabinoid treatment, highlighting the possibility that changes to endocannabinoid levels may represent one facet of the anticonvulsant effect of phytocannabinoids.

Pharmacokinetics of Oral Minor Cannabinoids in Blood and Brain.

Introduction: Minor cannabinoids are increasingly being consumed in oral formulations (i.e., edibles, tinctures) for medical and nonmedical purposes. This study examined the pharmacokinetics (PKs) of cannabinoids tetrahydrocannabivarin (THCV), cannabichromene (CBC), cannabinol (CBN), and delta-8-tetrahydrocannabinol (D8-THC) after the first and last oral dose during a 14-day administration period. Materials and Methods: Sprague-Dawley rats (N=6 animals/dose, 50% female) were given an assigned dose of one of four cannabinoids (THCV=3.2-100 mg/kg, CBC=3.2-100 mg/kg, CBN=1-100 mg/kg, or D8-THC=0.32-10 mg/kg) or vehicle (medium-chain triglyceride oil) through oral gavage once daily for 14 days. Blood was collected 45 min and 1.5, 3, and 24 h following the first dose (day 1) and the last dose (day 14) of repeated oral cannabinoid treatment for PK analysis. Outcomes of interest included time to maximum concentration (Tmax), maximum concentration (Cmax), and area under the concentration versus time curve (AUClast). Dose-normalized (DN) Cmax and DN AUClast were also calculated. Brain tissue was collected 24 h post-administration of the first (day 1) and the last (day 14) dose of each cannabinoid to determine concentrations in brain. Results: All cannabinoids tested were detectable in plasma after single and 14-day repeated dosing. DN Cmax and DN AUClast were highest for D8-THC, followed by CBC, CBN, and THCV. There was no sex difference observed in cannabinoid kinetics. Accumulation of D8-THC in plasma was observed after 14 days of administration. THCV levels in plasma were lower on day 14 compared to day 1, indicating potential adaptation of metabolic pathways and increased drug elimination. Cannabinoids were detected in brain tissue 24 h post-administration of the first and the last dose of 17-100 mg/kg THCV, 3.2-100 mg/kg CBC, 10-100 mg/kg CBN, and 10 mg/kg D8-THC. Conclusions: THCV, CBC, CBN, and D8-THC produced detectable levels in plasma and translocated to brain tissue after the first dose (day 1) and the last dose (day 14) of repeated oral dosing. Examination of PKs of these minor cannabinoids in blood and brain provides a critical step for informing target dose ranges and dosing schedules in future studies that evaluate the potential effects of these compounds.

Toxicological Evaluation and Pain Assessment of Four Minor Cannabinoids Following 14-Day Oral Administration in Rats.

Introduction: Despite growing consumer interest and market availability, the safety of minor cannabinoids, generally present in low concentrations in Cannabis sativa L., is not well understood. Materials and Methods: Cannabichromene (CBC; 3.2, 10, 17, 22, 32, or 100 mg/kg-bw/day), cannabinol (CBN; 1, 3.2, 10, 17, 32, or 100 mg/kg-bw/day), delta-8-tetrahydrocannabinol (D8-THC; 0.32, 1, 3.2, or 10 mg/kg-bw/day), tetrahydrocannabivarin (THCV; 3.2, 10, 17, 22, 32, or 100 mg/kg-bw/day), and vehicle (medium-chain triglyceride oil) preparations were administered via oral gavage once daily for 14 days to Sprague Dawley rats. Changes in behavior, body weight, food consumption, clinical pathology, organ weights, body temperature, and thermal pain sensitivity (tail flick assay) were assessed. Select organ tissues were collected at terminal necropsy and fixed for histopathological examination. Results: No treatment-related deaths were observed throughout the study, and cannabinoids were generally well tolerated. While some significant trends in body weight differences from controls (increases and decreases) were observed, these occurred independently of food consumption. Overall, differences in serum chemistry and hematology parameters between cannabinoid groups and their respective control groups were considered to occur due to biological variation among rats. No treatment-related gross abnormalities were observed in examined organs. Significant changes in absolute and relative organ weights occurred primarily in males and were generally of negligible magnitude. There were no biologically significant histopathological observations. While pain tolerance was significantly improved in animals treated with D8-THC (3.2 and 10 mg/kg-bw/day, day 14), results across minor cannabinoids were inconsistent and warrant further study. Conclusion: Minor cannabinoids were well tolerated across 14 days of daily oral administration at the doses assessed. Modest, dose-dependent trends in relative organ weights and serum chemistry parameters warrant exploration at higher oral doses. These data will assist in dose selection for future studies investigating the long-term safety and effects of CBC, CBN, D8-THC, and THCV.