Category Archives: Uncategorized

Determination of the Negative Allosteric Binding Site of Cannabidiol at the CB1 Receptor: A Combined Computational and Site-Directed Mutagenesis Study

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“Cannabinoid receptor 1 (CB1R) has been extensively studied as a potential therapeutic target for various conditions, including pain management, obesity, emesis, and metabolic syndrome. Unlike orthosteric agonists such as Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD) has been identified as a negative allosteric modulator (NAM) of CB1R, among its other pharmacological targets. Previous computational and structural studies have proposed various binding sites for CB1R NAMs. An X-ray crystal structure revealed a binding site for the NAM, ORG27569, at an extrahelical location within the inner leaflet of the membrane. In contrast, multiple computational studies have previously proposed several potential allosteric binding sites for CBD within the CB1R structure. Given that a prior structural study suggested CBD might occupy the same site as ORG27569, we conducted a comprehensive investigation of potential CBD binding sites using molecular docking, molecular dynamics (MD) simulations, metadynamics (MTD) simulations, binding free-energy calculations, and in vitro mutagenesis experiments. Molecular docking, MD, and MTD simulations results, along with binding free-energy calculations, suggest that CBD may potentially bind to either the same extrahelical site as ORG27569 or a previously unidentified intracellular site located near TMHs 2, 6, and 7 and helix 8. This intracellular site is consistent with allosteric binding sites observed in other G protein-coupled receptors (GPCRs). To establish the most favorable allosteric site for CBD, we conducted site-directed mutagenesis of key residues at each site. Mutations at S4018.47ΔA and D4038.49ΔA augmented the binding of [3H]-SR141716A, suggesting these residues play critical roles in CBD binding. As a result, the combined computational and mutagenesis results identified a binding site for CBD between TMHs 2, 6, and 7 and helix 8, involving residues Y1532.40, I1562.43, M3376.29, L3416.33, S4018.47, and D4038.49. These findings provide valuable insights into how CBD binds to CB1R, thereby informing the rational design of new, selective, and potent NAMs. Moreover, the elucidation of this previously unexplored allosteric site might explain the polypharmacology of CBD due to structural conservation among Class A GPCRs.”

https://pubmed.ncbi.nlm.nih.gov/39812521/

https://pubs.acs.org/doi/10.1021/acschemneuro.4c00343

An Unexpected Activity of a Minor Cannabinoid: Cannabicyclol (CBL) Is a Potent Positive Allosteric Modulator of Serotonin 5-HT1A Receptor

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“Cannabicyclol ((±)-CBL), a minor phytocannabinoid, is largely unexplored, with its biological activity previously undocumented. We studied its conversion from cannabichromene (CBC) using various acidic catalysts. Montmorillonite (K30) in chloroform at room temperature had the highest yield (60%) with minimal byproducts. Key reaction conditions, such as solvent, temperature, and time, significantly impacted the yield. The structure of (±)-CBL was confirmed via X-ray crystallography. Stability studies showed that (±)-CBL and its MCT oil dilution remain stable at 25-40 °C for three months. Radioligand binding assays revealed high affinity of CBL for the 5-HT1A receptor but weak interaction with CB1 and CB2 receptors. At 10 μM and 1 μM, (±)-CBL inhibited [3H]-8-hydroxy-DPAT binding to 5-HT1A by 75% and 20%, respectively. Functional assays showed that (±)-CBL acts as a weak agonist at high concentrations but a potent positive allosteric modulator of serotonin-induced activation at low concentrations. At 4 μM, (±)-CBL increased serotonin-induced β-arrestin recruitment from 20% to 80%. This unique modulatory profile highlights the potential of (±)-CBL in drug discovery targeting serotonin receptors.”

https://pubmed.ncbi.nlm.nih.gov/39811943/

https://pubs.acs.org/doi/10.1021/acs.jnatprod.4c00977

“Positive allosteric modulators of the 5-HT1A receptor can help relieve anxiety and depression.”

Plant-Derived Compounds in Hemp Seeds (Cannabis sativa L.): Extraction, Identification and Bioactivity-A Review

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“The growing demand for plant-based protein and natural food ingredients has further fueled interest in exploring hemp seeds (Cannabis sativa L.) as a sustainable source of and nutrition.

In addition to the content of proteins and healthy fats (linoleic acid and alpha-linolenic acid), hemp seeds are rich in phytochemical compounds, especially terpenoids, polyphenols, and phytosterols, which contribute to their bioactive properties.

Scientific studies have shown that these compounds possess significant antioxidant, antimicrobial, and anti-inflammatory effects, making hemp seeds a promising ingredient for promoting health. Since THC (tetrahydrocannabinol) and CBD (cannabidiol) are found only in traces, hemp seeds can be used in food applications because the psychoactive effects associated with cannabis are avoided.

Therefore, the present article reviews the scientific literature on traditional and modern extraction methods for obtaining active substances that meet food safety standards, enabling the transformation of conventional foods into functional foods that provide additional health benefits and promote a balanced and sustainable diet.

Also, the identification methods of biologically active compounds extracted from hemp seeds and their bioactivity were evaluated. Mechanical pressing extraction, steam distillation, solvent-based methods (Soxhlet, maceration), and advanced techniques such as microwave-assisted and supercritical fluid extraction were evaluated. Identification methods such as high-performance liquid chromatography (HPLC) and mass spectrometry (MS) allowed for detailed chemical profiling of cannabinoids, terpenes, and phenolic substances.

Optimizing extraction parameters, including solvent type, temperature, and time, is crucial for maximizing yield and purity, offering the potential for developing value-added foods with health benefits.”

https://pubmed.ncbi.nlm.nih.gov/39795183/

https://www.mdpi.com/1420-3049/30/1/124

Effects of a Cannabinoid-Based Phytocomplex (Pain ReliefTM) on Chronic Pain in Osteoarthritic Dogs

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“Twenty-one adult crossbreed dogs with chronic pain due to severe osteoarthrosis were enrolled in the study (placebo vs. treatment groups). The dogs in the experimental group received the dietary supplement (Pain ReliefTM, Giantec, Isernia, Italy) for 30 days to evaluate its effects on metabolism and pain relief. During the trial, the Helsinki Chronic Pain Index significantly decreased (p < 0.01) in the experimental group, indicating reduced pain and improved quality of life. Additionally, the treated group showed improvements in oxidative stress, demonstrated by a reduction in reactive oxygen metabolites, and an increase in biological antioxidant potential. Interleukins 6 levels decreased in the treated group, while interleukins 10 levels increased, thus suggesting an anti-inflammatory effect of the supplement. Importantly, no adverse effects were observed. Results suggest that Pain ReliefTM is effective in ameliorating osteoarthritis in dogs, improving their quality of life.”

https://pubmed.ncbi.nlm.nih.gov/39795044/

“Chronic pain is one of the most disabling conditions in dogs, as it affects various aspects of a dog’s life and should be managed regardless of the severity of symptoms. This research investigates the effects of a cannabidiol-based nutritional supplement in dogs affected by severe osteoarthritis. The treated group showed a reduction in pain due to an improvement of some cytokines expression and oxidative status. This suggests that Pain ReliefTM possesses an anti-inflammatory effect and reduces pain perception in dogs, thereby enhancing their quality of life.”

https://www.mdpi.com/2076-2615/15/1/101

Chronic oral dosing of cannabidiol and cannabidiolic acid full-spectrum hemp oil extracts has no adverse effects in horses: a pharmacokinetic and safety study

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“Objective: To compare the pharmacokinetics of cannabidiol (CBD) and cannabidiolic acid (CBDA) in horses and to evaluate the safety of their chronic administration.

Methods: CBD- and CBDA-rich oil (1 mg/kg) were administered orally twice daily to 7 adult horses over 6 weeks in a randomized, crossover design with a 2-week washout period. A 12-hour pharmacokinetic analysis was conducted on day 1 of each 6-week trial, followed by the measurement of peak and trough concentrations at weeks 1, 2, 4, and 6. The cannabinoids safety was assessed via daily physical examination, periodic bloodwork, and liver biopsy at the beginning and end of the study.

Results: 12-hour pharmacokinetics revealed a higher maximum serum concentration (103 vs 12 ng/mL) and greater area under the curve (259 vs 62 ng·h/mL) for CBDA when compared to CBD. Cannabidiolic acid nadir and peak serum levels over time ranged from 46 to 122 ng/mL, which was higher than CBD (12 to 38 ng/mL). Complete blood count and serum chemistry revealed no clinically relevant changes with either CBD or CBDA. No significant abnormalities were detected on liver ultrasonographic and histopathologic evaluation on day 0 and after both phases of the study.

Conclusions: A dose of either 1 mg/kg of CBD or CBDA administered long term appears safe; however, CBDA serum concentrations suggest superior absorption/retention.

Clinical relevance: Chronic cannabinoid supplementation in horses is safe. Considering the higher absorption of CBDA, its use is recommended to evaluate the therapeutic efficacy of this common hemp derived cannabinoid.”

https://pubmed.ncbi.nlm.nih.gov/39787699/

https://avmajournals.avma.org/view/journals/ajvr/aop/ajvr.24.08.0235/ajvr.24.08.0235.xml

CBD and the 5-HT1A receptor: A medicinal and pharmacological review

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“Cannabidiol (CBD), a phytocannabinoid, has emerged as a promising candidate for addressing a wide array of symptoms.

It has the ability to bind multiple proteins and receptors, including 5-HT1AR, transient receptor potential vanilloid 1 (TRPV1), and cannabinoid receptors. However, CBD’s pharmacodynamic interaction with 5-HT1AR and its medicinal outcomes are still debated.

This review explores recent literature to elucidate these questions, highlighting the neurotherapeutic outcomes of this pharmacodynamic interaction and proposing a signaling pathway underlying the mechanism by which CBD desensitizes 5-HT1AR signaling.

A comprehensive survey of the literature underscores CBD’s multifaceted neurotherapeutic effects, encompassing antidepressant, anxiolytic, neuroprotective, antipsychotic, antiemetic, anti-allodynic, anti-epileptic, anti-degenerative, and addiction-treating properties, attributable in part to its interactions with 5-HT1AR.

Furthermore, evidence suggests that the pharmacodynamic interaction between CBD and 5-HT1AR is contingent upon dosage. Moreover, we propose that CBD can induce desensitization of 5-HT1AR via both homologous and heterologous mechanisms. Homologous desensitization involves the recruitment of G protein-coupled receptor kinase 2 (GRK2) and β-arrestin, leading to receptor endocytosis. In contrast, heterologous desensitization is mediated by an elevated intracellular calcium level or activation of protein kinases, such as c-Jun N-terminal kinase (JNK), through the activity of other receptors.”

https://pubmed.ncbi.nlm.nih.gov/39778776/

“Cannabis was one of the first inhaled drugs utilized by humans, with evidence of use for gout, rheumatism, and malaria dating to 2737 BCE”

“The concurrent literature revealed that CBD produces several therapeutic effects through its complex pharmacodynamic interactions with 5-HT1AR. Therapeutic applications of CBD, including its anxiolytic, antidepressant, antipsychotic, anti-degenerative, neuroprotective, anti-epileptic, and anti-addictive properties were mediated, at least in part, by its binding to 5-HT1AR.”

https://www.sciencedirect.com/science/article/abs/pii/S0006295225000048?via%3Dihub

Medicinal Cannabis and the Intestinal Microbiome

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“Historically, the multiple uses of cannabis as a medicine, food, and for recreational purposes as a psychoactive drug span several centuries.

The various components of the plant (i.e., seeds, roots, leaves and flowers) have been utilized to alleviate symptoms of inflammation and pain (e.g., osteoarthritis, rheumatoid arthritis), mood disorders such as anxiety, and intestinal problems such as nausea, vomiting, abdominal pain and diarrhea.

It has been established that the intestinal microbiota progresses neurological, endocrine, and immunological network effects through the gut-microbiota-brain axis, serving as a bilateral communication pathway between the central and enteric nervous systems.

An expanding body of clinical evidence emphasizes that the endocannabinoid system has a fundamental connection in regulating immune responses. This is exemplified by its pivotal role in intestinal metabolic and immunity equilibrium and intestinal barrier integrity.

This neuromodulator system responds to internal and external environmental signals while also serving as a homeostatic effector system, participating in a reciprocal association with the intestinal microbiota.

We advance an exogenous cannabinoid-intestinal microbiota-endocannabinoid system axis potentiated by the intestinal microbiome and medicinal cannabinoids supporting the mechanism of action of the endocannabinoid system. An integrative medicine model of patient care is advanced that may provide patients with beneficial health outcomes when prescribed medicinal cannabis.”

https://pubmed.ncbi.nlm.nih.gov/39770543/

“Furthermore, other modes of delivery of medicinal cannabis, such as oro-buccal, sublingual and inhaled/smoked alternatives, provide cannabinoids that have rapid access to the systemic circulation, bypassing the intestinal tract.”

https://www.mdpi.com/1424-8247/17/12/1702

Protective Action of Cannabidiol on Tiamulin Toxicity in Humans-In Vitro Study

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“The growing awareness and need to protect public health, including food safety, require a thorough study of the mechanism of action of veterinary drugs in consumers to reduce their negative impact on humans. Inappropriate use of veterinary drugs in animal husbandry, such as tiamulin, leads to the appearance of residues in edible animal tissues.

The use of natural substances of plant origin, extracted from hemp (Cannabis sativa L.), such as cannabidiol (CBD), is one of the solutions to minimize the negative effects of tiamulin.

This study aimed to determine the effect of CBD on the cytotoxicity of tiamulin in humans.

The cytotoxic activity of tiamulin and the effect of its mixtures with CBD were tested after 72 h exposure to three human cell lines: SH-SY5Y, HepG2 and HEK-293. Cytotoxic concentrations (IC50) of the tested drug and in combination with CBD were assessed using five biochemical endpoints: mitochondrial and lysosomal activity, proliferation, cell membrane integrity and effects on DNA synthesis. Oxidative stress, cell death and cellular morphology were also assessed. The nature of the interaction between the veterinary drug and CBD was assessed using the combination index. The long-term effect of tiamulin inhibited lysosomal (SH-SY5SY) and mitochondrial (HepG2) activity and DNA synthesis (HEK-293). IC50 values for tiamulin ranged from 2.1 to >200 µg/mL (SH-SY5SY), 13.9 to 39.5 µg/mL (HepG2) and 8.5 to 76.9 µg/mL (HEK-293). IC50 values for the drug/CBD mixtures were higher.

Reduced levels of oxidative stress, apoptosis and changes in cell morphology were demonstrated after exposure to the mixtures. Interactions between the veterinary drug and CBD showed a concentration-dependent nature of tiamulin in cell culture, ranging from antagonistic (low concentrations) to synergistic effects at high drug concentrations.

The increased risk to human health associated with the presence of the veterinary drug in food products and the protective nature of CBD use underline the importance of these studies in food toxicology and require further investigation.”

https://pubmed.ncbi.nlm.nih.gov/39769305/

https://www.mdpi.com/1422-0067/25/24/13542

Cannabichromene as a Novel Inhibitor of Th2 Cytokine and JAK/STAT Pathway Activation in Atopic Dermatitis Models

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“Cannabichromene (CBC) is one of the main cannabinoids found in the cannabis plant, and although less well known than tetrahydrocannabinol (THC) and cannabidiol (CBD), it is gaining attention for its potential therapeutic benefits.

To date, CBC’s known mechanisms of action include anti-inflammatory, analgesic, antidepressant, antimicrobial, neuroprotective, and anti-acne effects through TRP channel activation and the inhibition of inflammatory pathways, suggesting that it may have therapeutic potential in the treatment of inflammatory skin diseases, such as atopic dermatitis (AD), but its exact mechanism of action remains unclear. Therefore, in this study, we investigated the effects of CBC on Th2 cytokines along with the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathways involved in AD pathogenesis. We used a 2,4-Dinitrochlorobenzene (DNCB)-induced BALB/c mouse model to topically administer CBC (0.1 mg/kg or 1 mg/kg).

The results showed that skin lesion severity, ear thickness, epithelial thickness of dorsal and ear skin, and mast cell infiltration were significantly reduced in the 0.1 mg/kg CBC-treated group compared with the DNCB-treated group (p < 0.001). In addition, real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis showed a significant decrease in the mRNA expression of Th2 cytokines (TSLP, IL-4, IL-13) and inflammatory mediators (IFN-γ, IL-1β, IL-6, IL-17, IL-18, and IL-33) (p < 0.05). Western blot analysis also revealed a significant decrease in JAK1, JAK2, STAT1, STAT2, STAT3, and STAT6 protein expression (p < 0.05).

These results suggest that CBC is a promising candidate for the treatment of AD and demonstrates the potential to alleviate AD symptoms by suppressing the Th2 immune response.”

https://pubmed.ncbi.nlm.nih.gov/39769302/

https://www.mdpi.com/1422-0067/25/24/13539

An In Vitro Evaluation of Industrial Hemp Extracts Against the Phytopathogenic Bacteria Erwinia carotovora, Pseudomonas syringae pv. tomato, and Pseudomonas syringae pv. tabaci

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“Pests and diseases have caused significant problems since the domestication of crops, resulting in economic loss and hunger. To overcome these problems, synthetic pesticides were developed to control pests; however, there are significant detrimental side effects of synthetic pesticides on the environment and human health. There is an urgent need to develop safer and more sustainable pesticides.

Industrial hemp is a reservoir of compounds that could potentially replace some synthetic bactericides, fungicides, and insecticides.

We determined the efficacy of industrial hemp extracts against Pseudomonas syringae pv. tabaci (PSTA), Pseudomonas syringae pv. tomato (PSTO), and Erwinia carotovora (EC).

The study revealed a minimum inhibitory concentration (MIC) of 2.05 mg/mL and a non-inhibitory concentration (NIC) of 1.2 mg/mL for PSTA, an MIC of 5.7 mg/mL and NIC of 0.66 mg/mL for PSTO, and an MIC of 12.04 mg/mL and NIC of 5.4 mg/mL for EC. Time-kill assays indicated the regrowth of E. carotovora at 4 × MIC after 15 h and P. syringae pv. tomato at 2 × MIC after 20 h; however, P. syringae pv. tabaci had no regrowth. The susceptibility of test bacteria to hemp extract can be ordered from the most susceptible to the least susceptible, as follows: P. syringae pv. tabaci > P. syringae pv. tomato > E. carotovora.

Overall, the data indicate hemp extract is a potential source of sustainable and safe biopesticides against these major plant pathogens.”

https://pubmed.ncbi.nlm.nih.gov/39769990/

“The data show the susceptibility of the test bacteria to hemp extract, ordered from the most susceptible to the least susceptible, as follows: P. syringae pv. tabaci > P. syringae pv. tomato > E. carotovora.

This study indicates that hemp extract is effective in controlling E. carotovora, P. syringae pv. tabaci, and P. syringae pv. tomato. However, the hemp extract is more effective against both Pseudomonas spp. than E. carotovora. The difference could be due to their difference in cell wall structure, resistance mechanisms, and metabolic pathways.

More studies are needed to determine how hemp extract causes stress to bacteria such as interference with quorum sensing, biofilm formation, and oxidative stress. Moreover, to ensure sustainable agricultural practices that are safe and affordable for low-income farmers, synergistic effect studies are needed between hemp compounds in the extract and, more importantly, between hemp extracts and other plant extracts.”

https://www.mdpi.com/1420-3049/29/24/5902