Expression and Preparation of a G-Protein-Coupled Cannabinoid Receptor CB2 for NMR Structural Studies.

Current Protocols in Protein Science banner

“Cannabinoid receptor type II, or CB2 , is an integral membrane protein that belongs to a large class of G-protein-coupled receptors (GPCR)s. CB2 is a part of the endocannabinoid system, which plays an important role in the regulation of immune response, inflammation, and pain.

Information about the structure and function of CB2 is essential for the development of specific ligands targeting this receptor.

We present here a methodology for recombinant expression of CB2 and its stable isotope labeling, purification, and reconstitution into liposomes, in preparation for its characterization by nuclear magnetic resonance (NMR).

Correctly folded, functional CB2 labeled with [13 C,15 N]tryptophan or uniformly labeled with 13 C and 15 N is expressed in a medium of defined composition, under controlled aeration, pH, and temperature conditions.

The receptor is purified by affinity chromatography and reconstituted into lipid bilayers in the form of proteoliposomes suitable for analysis by NMR spectroscopy.”

https://www.ncbi.nlm.nih.gov/pubmed/30624864

https://currentprotocols.onlinelibrary.wiley.com/doi/abs/10.1002/cpps.83

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Cannabidiol in Anxiety and Sleep: A Large Case Series.

“Cannabidiol (CBD) is one of many cannabinoid compounds found in cannabis. It does not appear to alter consciousness or trigger a “high.”

A recent surge in scientific publications has found preclinical and clinical evidence documenting value for CBD in some neuropsychiatric disorders, including epilepsy, anxiety, and schizophrenia. Evidence points toward a calming effect for CBD in the central nervous system. Interest in CBD as a treatment of a wide range of disorders has exploded, yet few clinical studies of CBD exist in the psychiatric literature.

OBJECTIVE:

To determine whether CBD helps improve sleep and/or anxiety in a clinical population.

DESIGN:

A large retrospective case series at a psychiatric clinic involving clinical application of CBD for anxiety and sleep complaints as an adjunct to usual treatment. The retrospective chart review included monthly documentation of anxiety and sleep quality in 103 adult patients.

MAIN OUTCOME MEASURES:

Sleep and anxiety scores, using validated instruments, at baseline and after CBD treatment.

RESULTS:

The final sample consisted of 72 adults presenting with primary concerns of anxiety (n = 47) or poor sleep (n = 25). Anxiety scores decreased within the first month in 57 patients (79.2%) and remained decreased during the study duration. Sleep scores improved within the first month in 48 patients (66.7%) but fluctuated over time. In this chart review, CBD was well tolerated in all but 3 patients.

CONCLUSION:

Cannabidiol may hold benefit for anxiety-related disorders. Controlled clinical studies are needed.”

https://www.ncbi.nlm.nih.gov/pubmed/30624194

http://www.thepermanentejournal.org/issues/2019/winter/6960-cannabis.html

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Epidiolex (Cannabidiol): A New Hope for Patients With Dravet or Lennox-Gastaut Syndromes.

 SAGE Journals

“OBJECTIVE: To review the efficacy, safety, pharmacology and pharmacokinetics of pure, plant-derived cannabidiol (CBD; Epidiolex) in the treatment of Dravet syndrome (DS) and Lennox-Gastaut syndrome (LGS).

DATA SYNTHESIS: Pure, plant-based CBD is a pharmaceutical grade extract that exhibits clinically significant antiseizure properties, with a hypothesized multimodal mechanism of action. In the GWPCARE trial series, CBD displayed superior efficacy in reducing key seizure frequencies (convulsive seizures in DS; drop seizures in LGS) by 17% to 23% compared with placebo as adjunctive therapy to standard antiepileptic drugs in patients 2 years of age and older. Common adverse effects were somnolence, diarrhea, and elevated hepatic transaminases. Noteworthy drug-drug interactions included clobazam, valproates, and significant inducers/inhibitors of CYP2C19 and 3A4 enzymes.

Relevance to Patient Care and Clinical Practice: A discussion regarding CBD dosing, administration, adverse effects, monitoring parameters, and interactions is provided to guide clinicians. CBD offers patients with DS and LGS a new treatment option for refractory seizures.

CONCLUSION:

This is the first cannabis-derived medication with approval from the US Food and Drug Administration. This CBD formulation significantly reduces seizures as an adjunct to standard antiepileptic therapies in patients ≥2 years old with DS and LGS and is well tolerated.”

https://www.ncbi.nlm.nih.gov/pubmed/30616356

https://journals.sagepub.com/doi/abs/10.1177/1060028018822124?journalCode=aopd

“Why marijuana is headed for the mainstream. The credibility of cannabis as a source of a legitimate pharmaceutical ingredient in prescription medications took a major step forward in 2018 when the FDA approved Epidiolex (cannabidiol) for two types of severe seizures. Epidiolex was a stellar candidate for approval. It reduced convulsive seizures by about 40% and has a good safety profile.”  https://www.ncbi.nlm.nih.gov/pubmed/30620324

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The protective effects of β-caryophyllene on LPS-induced primary microglia M1/M2 imbalance: A mechanistic evaluation.

Life Sciences

“Neuroinflammation is observed as a routine characterization of neurodegenerative disorders such as dementia, multiple sclerosis (MS) and Alzheimer’s diseases (AD). Scientific evidence propounds both of the neuromodulatory and immunomodulatory effects of CB2 in the immune system. β-Caryophyllene (BCP) is a dietary selective CB2 agonist, which deserves the anti-inflammatory and antioxidant effects at both low and high doses through activation of the CB2 receptor.

METHODS:

In this study, we investigated the protective effects of a broad range concentration of BCP against LPS-induced primary microglia cells inflammation and M1/M2 imbalance and identifying the portion of the involvement of related signaling pathways on BCP effects using pharmacological antagonists of CB2, PPAR-γ, and sphingomyelinase (SMase).

KEY FINDINGS:

The protective effects of BCP on LPS-induced microglia imbalance is provided by the M2 healing phenotype of microglia, releasing the anti-inflammatory (IL-10, Arg-1, and urea) and anti-oxidant (GSH) parameters and reducing the inflammatory (IL-1β, TNF-α, PGE2, iNOS and NO) and oxidative (ROS) biomarkers. Moreover, we showed that BCP exerts its effects through CB2receptors which overproduction of ceramides by SMase at middle to higher concentrations of BCP reduce the protective activity of BCP and results in the activation of the PPAR-γ pathway.

SIGNIFICANCE:

In conclusion, the low concentration of BCP has higher selective anti-inflammatory effects rather than high levels. On this occasion, BCP by modulating the microglia is able to have potential therapeutic effects in neuro-inflammation conditions and microglia cells such as MS and AD.”

https://www.ncbi.nlm.nih.gov/pubmed/30620895

https://www.sciencedirect.com/science/article/abs/pii/S0024320518308610?via%3Dihub

“β-caryophyllene (BCP) is a common constitute of the essential oils of numerous spice, food plants and major component in Cannabis.”   http://www.ncbi.nlm.nih.gov/pubmed/23138934

“Beta-caryophyllene is a dietary cannabinoid.”  https://www.ncbi.nlm.nih.gov/pubmed/18574142

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Changes in Monoaminergic Neurotransmission in an Animal Model of Osteoarthritis: The Role of Endocannabinoid Signaling.

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“Chronic pain is a main symptom of osteoarthritis (OA). Moreover, a high percentage of OA patients suffer from mental health problems.

The endocannabinoid (EC) system has attracted attention as an emerging drug target for pain treatment together with its activity on the mesolimbic reward system.

Understanding the circuits that govern the reward of pain relief is crucial for the search for effective analgesics. Therefore, we investigated the role of the EC system on dopamine (DA) and noradrenaline (NA) in an animal model of OA-related chronic pain.

Our results demonstrated that chronic pain in OA rats was reflected by the inhibition of mesolimbic and mesocortical dopaminergic transmission, and may indicate the pro-pain role of NA in the FCx.

The data provide understanding about changes in neurotransmission in chronic pain states and may explain the clinical improvement in perceived life quality following cannabinoid treatment.

Additional mechanistic studies in preclinical models examining the intersection between chronic pain and reward circuits may offer new approaches for improving pain therapy.”

https://www.ncbi.nlm.nih.gov/pubmed/30618615

https://www.frontiersin.org/articles/10.3389/fnmol.2018.00466/full

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The effects of cannabinoids on the endocrine system.

“Cannabinoids are the derivatives of the cannabis plant, the most potent bioactive component of which is tetrahydrocannabinol (THC). The most commonly used drugs containing cannabinoids are marijuana, hashish, and hashish oil.

These compounds exert their effects via interaction with the cannabinoid receptors CB1 and CB2. Type 1 receptors (CB1) are localised mostly in the central nervous system and in the adipose tissue and many visceral organs, including most endocrine organs. Type 2 cannabinoid receptors (CB2) are positioned in the peripheral nervous system (peripheral nerve endings) and on the surface of the immune system cells.

Recently, more and more attention has been paid to the role that endogenous ligands play for these receptors, as well as to the role of the receptors themselves. So far, endogenous cannabinoids have been confirmed to participate in the regulation of food intake and energy homeostasis of the body, and have a significant impact on the endocrine system, including the activity of the pituitary gland, adrenal cortex, thyroid gland, pancreas, and gonads.

Interrelations between the endocannabinoid system and the activity of the endocrine system may be a therapeutic target for a number of drugs that have been proved effective in the treatment of infertility, obesity, diabetes, and even prevention of diseases associated with the cardiovascular system.”

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Cannabinoids-induced peripheral analgesia depends on activation of BK channels.

 Brain Research“The endogenous cannabinoid system is involved in the physiological inhibitory control of pain and is of particular interest for the development of therapeutic approaches for pain management.

Selective activation of the peripheral CB1 cannabinoid receptor has been shown to suppress the heightened firing of primary afferents, which is the peripheral mechanism underlying neuropathic pain after nerve injury. However, the mechanism underlying this effect of CB1 receptor remains unclear.

The large-conductance calcium-activated potassium (BK) channels have been reported to participate in anticonvulsant and vasorelaxant effects of cannabinoids. We asked whether BK channels participate in cannabinoids-induced analgesia and firing-suppressing effects in primary afferents after nerve injury.

Here, using mice with chronic constriction injury(CCI)-induced neuropathic pain, antinociception action and firing-suppressing effect of HU210 were measured before and after BK channel blocker application. We found that local peripheral application of HU210 alleviated CCI-induced pain behavior and suppressed the heightened firing of injured fibers. Co-administration of IBTX with HU210 significantly reversed the analgesia and the firing-suppressing effect of HU210.

This result indicated that the peripheral analgesic effects of cannabinoids depends on activation of BK channels.”

https://www.ncbi.nlm.nih.gov/pubmed/30615887

https://www.sciencedirect.com/science/article/pii/S0006899319300071?via%3Dihub

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Practical Perspectives in the Treatment of Nausea and Vomiting.

Image result for J Clin Gastroenterol.

“Nausea and vomiting result from complex interactions between afferent and efferent pathways of the gastrointestinal tract, central nervous system, and autonomic nervous system. Afferent pathways from the vagus nerve, vestibular system, and chemoreceptor trigger zone project to nucleus tractus solitarius, which in turn relays signals to the central pattern generator to initiate multiple downstream pathways resulting in symptoms of nausea and vomiting. There is increasing evidence that the central pathway of chronic nausea is different from that of acute nausea and vomiting-and closely resembles that of neuropathic pain. This improved understanding of chronic nausea has resulted in a paradigm shift with regard to management strategy. Although conventional therapies such as antiemetics and prokinetics are commonly used to manage acute nausea and vomiting, they are historically not as effective in treating chronic nausea. Recently, neuromodulator agents, such as tricyclic antidepressants, gabapentin, olanzapine, mirtazapine, and benzodiazepines, and cannabinoids have been shown to be efficacious in the treatment of nausea and vomiting, and may be useful in the treatment of chronic symptoms. There is a need to study these agents, especially in the management of chronic functional nausea. Improved understanding of the central and peripheral circuitry of nausea and vomiting symptoms will allow for enhanced utilization of the currently available medications, and the development of novel therapeutic options.”

https://www.ncbi.nlm.nih.gov/pubmed/30614944

https://insights.ovid.com/crossref?an=00004836-900000000-97784

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Progress in Brain Cannabinoid CB2 Receptor Research: From Genes to Behavior.

Neuroscience & Biobehavioral Reviews

“The type 2 cannabinoid receptor (CB2R) was initially regarded as a peripheral cannabinoid receptor. However, recent technological advances in gene detection, alongside the availability of transgenic mouse lines, indicate that CB2Rs are expressed in both neurons and glial cells in the brain under physiological and pathological conditions, and are involved in multiple functions at cellular and behavioral levels. Brain CB2Rs are inducible and neuroprotective via up-regulation in response to various insults, but display species differences in gene and receptor structures, CB2R expression, and receptor responses to various CB2R ligands. CB2R transcripts also differ between the brain and spleen. In the brain, CB2A is the major transcript isoform, while CB2A and CB2B transcripts are present at higher levels in the spleen. These new findings regarding brain versus spleen CB2R isoforms may in part explain why early studies failed to detect brain CB2R gene expression. Here, we review evidence supporting the expression and function of brain CB2R from gene and receptor levels to cellular functioning, neural circuitry, and animal behavior.”

https://www.ncbi.nlm.nih.gov/pubmed/30611802

https://www.sciencedirect.com/science/article/pii/S0149763418308297?via%3Dihub

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Adenosine A2A-Cannabinoid CB1 Receptor Heteromers in the Hippocampus: Cannabidiol Blunts Δ9-Tetrahydrocannabinol-Induced Cognitive Impairment

“At present, clinical interest in the plant-derived cannabinoid compound cannabidiol (CBD) is rising exponentially, since it displays multiple therapeutic properties. In addition, CBD can counteract the undesirable effects of the psychoactive cannabinoid Δ9-tetrahydrocannabinol (Δ9-THC) that hinder clinical development of cannabis-based therapies. Here, by combining in vivo and complementary molecular techniques, we demonstrate for the first time that CBD blunts the Δ9-THC-induced cognitive impairment in an adenosine A2A receptor (A2AR)-dependent manner. Overall, these data provide new evidence regarding the mechanisms of action of CBD and the nature of A2AR-CB1R interactions in the brain.”

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