Uncovering the metabolite complexity and variability of cultivated hemp (Cannabis sativa L.): A first phytochemical diversity mapping in Greece.

The high value of fiber-type Cannabis sativa L. (hemp) due to its phytochemicals has yet to be fully recognized and leveraged. Besides cannabidiol (CBD), which is the most prevalent non-psychoactive cannabinoid, hemp contains numerous other cannabinoids with unexplored bioactivities, in addition to various compound classes. Previous works have aimed to correlate chemical profiles of C. sativa inflorescences with important parameters, mostly based on experiments under controlled conditions. However, mapping studies that explore the phytochemical diversity of hemp in a more realistic context are crucial to guide decisions at multiple levels, especially in areas where hemp cultivation was recently re-authorized, including Mediterranean countries. In this work, a powerful strategy was followed to map the phytochemical diversity of cultivated hemp in Greece, being the first study of its kind for this environment. A panel of 98 inflorescence samples, covering two harvesting years, eleven geographical regions and seven commonly used EU varieties, were studied using a combination of targeted and untargeted approaches. Quantitative results based on UPLC-PDA revealed relatively constant CBD/THC (total) ratios, while profiling by LC-HRMS effectively probed the phytochemical variability of samples, and led to the annotation of 88 metabolites, including a multitude of minor cannabinoids. Multivariate analysis substantiated a strong effect of harvesting year in sample discrimination and related biomarkers were revealed, belonging to fatty acids and flavonoids. The effect of geographical region and, especially, variety on chemical variation patterns was more intricate to interpret. The results of this work are envisioned to enhance our understanding of the real-world phytochemical complexity of C. sativa (hemp), with a view to maximized utilization of hemp for the promotion of human well-being.

Evaluation of the anti-inflammatory effects of selected cannabinoids and terpenes from Cannabis Sativa employing human primary leukocytes.

Cannabis is well established as possessing immune modulating activity. The objective of this study was to evaluate the anti-inflammatory properties of selected cannabis-derived terpenes and cannabinoids. Based on their activity in cannabis-chemovar studies, α-pinene, trans-nerolidol, D-limonene, linalool and phytol were the selected terpenes evaluated. The cannabinoid compounds evaluated included cannabidivarin, cannabidiol, cannabinol, cannabichromene, cannabigerol and delta-9-tetrahydrocannabinol. Human PBMC were pretreated with each compound, individually, at concentrations extending from 0.001 to 10 µM and then stimulated with CpG (plasmacytoid dendritic cell), LPS (monocytes), or anti-CD3/CD28 (T cells). Proliferation, activation marker expression, cytokine production and phagocytosis, were quantified. Of the 21 responses assayed for each compound, cannabinoids showed the greatest immune modulating activity compared to their vehicle control. Delta-9-tetrahydrocannabinol possessed the greatest activity affecting 11 immune parameters followed by cannabidivarin, cannabigerol, cannabichromene, cannabinol and cannabidiol. α-Pinene showed the greatest immune modulating activity from the selected group of terpenes, followed by linalool, phytol, trans-nerolidol. Limonene had no effect on any of the parameters tested. Overall, these studies suggest that selected cannabis-derived terpenes displayed minimal immunological activity, while cannabinoids exhibited a broader range of activity. Compounds possessing anti-inflammatory effects may be useful in decreasing inflammation associated with a range of disorders, including neurodegenerative disorders.

Identification of minimum essential therapeutic mixtures from cannabis plant extracts by screening in cell and animal models of Parkinson's disease.

Medicinal cannabis has shown promise for the symptomatic treatment of Parkinson's disease (PD), but patient exposure to whole plant mixtures may be undesirable due to concerns around safety, consistency, regulatory issues, and psychoactivity. Identification of a subset of components responsible for the potential therapeutic effects within cannabis represents a direct path forward for the generation of anti-PD drugs. Using an in silico database, literature reviews, and cell based assays, GB Sciences previously identified and patented a subset of five cannabinoids and five terpenes that could potentially recapitulate the anti-PD attributes of cannabis. While this work represents a critical step towards harnessing the anti-PD capabilities of cannabis, polypharmaceutical drugs of this complexity may not be feasible as therapeutics. In this paper, we utilize a reductionist approach to identify minimal essential mixtures (MEMs) of these components that are amenable to pharmacological formulation. In the first phase, cell-based models revealed that the cannabinoids had the most significant positive effects on neuroprotection and dopamine secretion. We then evaluated the ability of combinations of these cannabinoids to ameliorate a 6-hydroxydopmamine (OHDA)-induced change in locomotion in larval zebrafish, which has become a well-established PD disease model. Equimolar mixtures that each contained three cannabinoids were able to significantly reverse the OHDA mediated changes in locomotion and other advanced metrics of behavior. Additional screening of sixty-three variations of the original cannabinoid mixtures identified five highly efficacious mixtures that outperformed the original equimolar cannabinoid MEMs and represent the most attractive candidates for therapeutic development. This work highlights the strength of the reductionist approach for the development of ratio-controlled, cannabis mixture-based therapeutics for the treatment of Parkinson's disease.

Potential use for chronic pain: Poly(Ethylene Glycol)-Poly(Lactic-Co-Glycolic Acid) nanoparticles enhance the effects of Cannabis-Based terpenes on calcium influx in TRPV1-Expressing cells.

The objective of these in vitro studies was to investigate the impact of the encapsulation of three cannabis-based terpenes, namely ß-myrcene (MC), ß-caryophyllene (CPh), and nerolidol (NL), on their potential efficacy in pain management. Terpene-encapsulated poly(ethylene glycol)-poly(lactic-co-glycolic acid) nanoparticles (PEG-PLGA NPs) were prepared by an emulsion-solvent evaporation method. The terpene-loaded NPs were examined in HEK293 cells that express the nociceptive transient receptor potential vanilloid-1 (TRPV1), an ion channel involved in pain perception. TRPV1 activation was assessed by monitoring calcium influx kinetics over 1 h in cells pre-treated with the fluorescent indicator Fluo-4. In addition, the fluorescence intensity changes induced by the NPs in living cells were also explored by a fluorescence microscope. Furthermore, the cytotoxicity of the terpene-loaded NPs was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-3,5-diphenyl tetrazolium bromide (MTT) proliferation assay. The terpene-loaded NPs had a diameter in the range of 250-350 nm and a zeta potential of approximately -20 mV. The encapsulation efficiency was 18.5%, 51.3%, and 60.3% for MC, NL, and CPh NPs, respectively. The nano-formulations significantly increased the fluorescence intensity in comparison with free terpenes. Furthermore, combinations of terpene-loaded NPs produced significantly higher calcium responses when compared to combinations of free terpenes. Similar findings were shown by the fluorescence images. In conclusion, the terpene-PLGA NPs can be promising therapeutics for more effective pain management.

Cannabis Chemovar Nomenclature Misrepresents Chemical and Genetic Diversity; Survey of Variations in Chemical Profiles and Genetic Markers in Nevada Medical Cannabis Samples.

Introduction: Medical cannabis patients receive clinical benefits from the secondary metabolites of the plant, which contain a variety of cannabinoids and terpenoids in combinations that can be used to classify the chemovars. State-regulated medical cannabis programs rely on breeder-reported "strain" names both within diversion control systems and to describe the medical cannabis products that are sold to patients in medical cannabis dispensaries. In state-regulated medical cannabis programs, there is no conventional nomenclature system that correlates the breeder-reported names with their profiles of active ingredients, and these "strain" names are invalid as they refer to chemical differences properly referred to as to chemovars. Materials and Methods: To determine the actual levels of chemical diversity represented in 2662 samples of Cannabis flower collected between January 2016 and June of 2017 in Nevada, chemical profile data were measured from these samples by a state-qualified third-party testing laboratory. Principal component analysis (PCA) was used to define clusters in data sets representing both cannabinoids and terpenoids, cannabinoids only, or terpenoids only. Results: The PCA of the terpenoid only data set revealed three well-defined clusters. All three terpenoids only data clusters had high tetrahydrocannabinolic acid synthase, but the terpene profiles listed in reverse-order of abundance best defined these chemovars. The three chemovars in Nevada were labeled with 396 breeder-reported sample names, which overestimate the diversity and do not inform patients regarding chemical properties. Representative DNA samples were taken from each chemovar to determine whether the genetic diversity was greater than the chemical diversity. The limited genotyping experiment was based on DNA sequence polymorphisms. The genetic analysis revealed twelve distinct genetic clades, which still does not account for the entirety of the 396 reported sample names. The finite genotypes did not correlate with the chemotypes determined for the samples. This suggests that either the DNA-markers used were too narrowly restricted for factual separation or that environmental factors contributed more significantly to the chemical profiles of cannabis than genetics. Conclusion: The three chemovars and twelve genotypes reflect low medical diversity on the market in Nevada during its "medical use only" phase. Furthermore, the 396 breeder-reported sample names within this set imply a false sense of diversity of products in Nevada dispensaries.

"TRPV1 is a component of the atrial natriuretic signaling complex, and using orally delivered antagonists, presents a valid therapeutic target in the longitudinal reversal and treatment of cardiac hypertrophy and heart failure".

Activation of the atrial natriuretic signaling pathway is intrinsic to the pathological responses associated with a range of cardiovascular diseases that stress the heart, especially those involved in sustained cardiac pressure overload which induces hypertrophy and the pathological remodeling that frequently leads to heart failure. We identify transient receptor potential cation channel, subfamily V, member 1, as a regulated molecular component, and therapeutic target of this signaling system. Data show that TRPV1 is a physical component of the natriuretic peptide A, cGMP, PKG signaling complex, interacting with the Natriuretic Peptide Receptor 1 (NPR1), and upon binding its ligand, Natriuretic Peptide A (NPPA, ANP) TRPV1 activation is subsequently suppressed through production of cGMP and PKG mediated phosphorylation of the TRPV1 channel. Further, inhibition of TRPV1, with orally delivered drugs, suppresses chamber and myocyte hypertrophy, and can longitudinally improve in vivo heart function in mice exposed to chronic pressure overload induced by transverse aortic constriction, reversing pre-established hypertrophy induced by pressure load while restoring chamber function. TRPV1 is a physical and regulated component of the natriuretic peptide signaling system, and TRPV1 inhibition may provide a new treatment strategy for treating, and reversing the loss of function associated with cardiac hypertrophy and heart failure.

Advantages of the AMDL-ELISA DR-70 (FDP) assay over carcinoembryonic antigen (CEA) for monitoring colorectal cancer patients.

The DR-70 (FDP) test was the first cancer test cleared by USFDA for monitoring colorectal cancer (CRC) since Carcinoembryonic Antigen (CEA) in 1982. Conservatively, 50% of biopsy-positive CRC patients have negative CEA values. DR-70 and CEA values were compared for 113 CRC monitoring patients. Total concordance rates for DR-70 and CEA were 0.665 and 0.686, respectively. CRC patient pairs were grouped based on their CEA value to deduce DR-70's effectiveness at monitoring patients with low CEA values. DR-70 had 12% to 100% greater positive concordance rates than CEA in this group. DR-70 is a welcome new option for CRC patients.

Secretogranin III directs secretory vesicle biogenesis in mast cells in a manner dependent upon interaction with chromogranin A.

Mast cells are granular immunocytes that reside in the body's barrier tissues. These cells orchestrate inflammatory responses. Proinflammatory mediators are stored in granular structures within the mast cell cytosol. Control of mast cell granule exocytosis is a major therapeutic goal for allergic and inflammatory diseases. However, the proteins that control granule biogenesis and abundance in mast cells have not been elucidated. In neuroendocrine cells, whose dense core granules are strikingly similar to mast cell granules, granin proteins regulate granulogenesis. Our studies suggest that the Secretogranin III (SgIII) protein is involved in secretory granule biogenesis in mast cells. SgIII is abundant in mast cells, and is organized into vesicular structures. Our results show that over-expression of SgIII in mast cells is sufficient to cause an expansion of a granular compartment in these cells. These novel granules store inflammatory mediators that are released in response to physiological stimuli, indicating that they function as bona fide secretory vesicles. In mast cells, as in neuroendocrine cells, we show that SgIII is complexed with Chromogranin A (CgA). CgA is granulogenic when complexed with SgIII. Our data show that a novel non-granulogenic truncation mutant of SgIII (1-210) lacks the ability to interact with CgA. Thus, in mast cells, a CgA-SgIII complex may play a key role in secretory granule biogenesis. SgIII function in mast cells is unlikely to be limited to its partnership with CgA, as our interaction trap analysis suggests that SgIII has multiple binding partners, including the mast cell ion channel TRPA1.

Supramolecular complexes mediate selenocysteine incorporation in vivo.

Selenocysteine incorporation in eukaryotes occurs cotranslationally at UGA codons via the interactions of RNA-protein complexes, one comprised of selenocysteyl (Sec)-tRNA([Ser]Sec) and its specific elongation factor, EFsec, and another consisting of the SECIS element and SECIS binding protein, SBP2. Other factors implicated in this pathway include two selenophosphate synthetases, SPS1 and SPS2, ribosomal protein L30, and two factors identified as binding tRNA([Ser]Sec), termed soluble liver antigen/liver protein (SLA/LP) and SECp43. We report that SLA/LP and SPS1 interact in vitro and in vivo and that SECp43 cotransfection increases this interaction and redistributes all three proteins to a predominantly nuclear localization. We further show that SECp43 interacts with the selenocysteyl-tRNA([Ser]Sec)-EFsec complex in vitro, and SECp43 coexpression promotes interaction between EFsec and SBP2 in vivo. Additionally, SECp43 increases selenocysteine incorporation and selenoprotein mRNA levels, the latter presumably due to circumvention of nonsense-mediated decay. Thus, SECp43 emerges as a key player in orchestrating the interactions and localization of the other factors involved in selenoprotein biosynthesis. Finally, our studies delineating the multiple, coordinated protein-nucleic acid interactions between SECp43 and the previously described selenoprotein cotranslational factors resulted in a model of selenocysteine biosynthesis and incorporation dependent upon both cytoplasmic and nuclear supramolecular complexes.

Nuclear assembly of UGA decoding complexes on selenoprotein mRNAs: a mechanism for eluding nonsense-mediated decay?

Recoding of UGA from a stop codon to selenocysteine poses a dilemma for the protein translation machinery. In eukaryotes, two factors that are crucial to this recoding process are the mRNA binding protein of the Sec insertion sequence, SBP2, and the specialized elongation factor, EFsec. We sought to determine the subcellular localization of these selenoprotein synthesis factors in mammalian cells and thus gain insight into how selenoprotein mRNAs might circumvent nonsense-mediated decay. Intriguingly, both EFsec and SBP2 localization differed depending on the cell line but significant colocalization of the two proteins was observed in cells where SBP2 levels were detectable. We identify functional nuclear localization and export signals in both proteins, demonstrate that SBP2 undergoes nucleocytoplasmic shuttling, and provide evidence that SBP2 levels and localization may influence EFsec localization. Our results suggest a mechanism for the nuclear assembly of the selenocysteine incorporation machinery that could allow selenoprotein mRNAs to circumvent nonsense-mediated decay, thus providing new insights into the mechanism of selenoprotein translation.

Exposure to tobacco-derived materials induces overproduction of secreted proteinases in mast cells.

Mast cells reside at interfaces with the environment, including the mucosa of the respiratory and gastrointestinal tracts. This localization exposes mast cells to inhaled, or ingested, environmental challenges. In the airways of smokers, resident immune cells will be in contact with the condensed components of cigarette smoke. Mast cells are of particular interest due to their ability to promote airway remodeling and mucus hypersecretion. Clinical data show increased levels of mast cell-secreted tryptase and increased numbers of degranulated mast cells in the lavage and bronchial tissue of smokers. Since mast cell-secreted proteinases (MCPTs), including tryptases, contribute to pathological airway remodeling, we investigated the relationship between mast cell proteinases and smoke exposure. We exposed a mast cell line to cigarette smoke condensate (CSC). We show that CSC exposure increases MCPT levels in mast cells using an assay for tryptase-type MCPT activity. We hypothesized that this increase in MCPT activity reflects a CSC-induced increase in the cytosolic pool of proteinase molecules, via stimulation of MCPT transcription. Transcript array data suggested that mRNA changes in response to CSC were limited in number and peaked after 3 h of CSC exposure. However, we noted marked transcriptional regulation of several MCPT genes. CSC-induced changes in the mRNA levels for MCPTs were confirmed using quantitative RT-PCR. Taken together, our data suggest that chronic exposure to cigarette smoke up-regulates MCPT levels in mast cells at both the protein and the mRNA level. We suggest that the pathological airway remodeling that has been described in clinical studies of smoke inhalation may be attributable to MCPT overproduction in vivo.

The responses of Ht22 cells to oxidative stress induced by buthionine sulfoximine (BSO).

BACKGROUND: glutathione (GSH) is the most abundant thiol antioxidant in mammalian cells. It directly reacts with reactive oxygen species (ROS), functions as a cofactor of antioxidant enzymes, and maintains thiol redox potential in cells. GSH depletion has been implicated in the pathogenesis of neurological diseases, particularly to Parkinson's disease (PD). The purpose of this study was to investigate the change of cellular antioxidant status and basic cell functions in the relatively early stages of GSH depletion. RESULTS: in this study, GSH was depleted by inhibition of glutamylcysteine synthetase using buthionine sulfoximine (BSO) treatment in Ht22, a neuronal cell line derived from mouse hippocampus. Treatment with BSO produced dose-dependent decreases in total GSH level, Fe3+-reducing ability (FRAP assay), Cu2+-reducing ability (Antioxidant Potential, AOP assay), and ABTS free radical scavenging ability (ABTS assay) of the cells, but the sensitivity of these indicators to dosage varied considerably. Most of the changes were completed during the first 8 hours of treatment. Cell viability was tested by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromid) assay, and cells at lower density in culture were found to be more sensitive to GSH depletion. The activity of antioxidant enzymes, such as glutathione peroxidase (GPx), glutathione reductase (GR), and copper/zinc superoxide dismutase (Cu/Zn-SOD) were affected by GSH depletion. A cDNA expression array assay of the effects of BSO treatment showed significantly decreased mRNA level for 3 genes, and significantly increased mRNA level for 10 genes, including the antioxidant enzymes Cu/Zn-SOD and thioredoxin peroxidase 2 (TPxII). CONCLUSIONS: the study suggests that there are BSO-sensitive and BSO-resistant pools of GSH in Ht22 cells, and that different categories of antioxidant react differently to GSH depletion. Further, the effect of GSH status on cell viability is cell density dependent. Finally, the alterations in expression or activity of several antioxidant enzymes provide insight into the various cellular responses to GSH depletion.

Anti-inflammatory potential of CB1-mediated cAMP elevation in mast cells.

Cannabinoids are broadly immunosuppressive, and anti-inflammatory properties have been reported for certain marijuana constituents and endogenously produced cannabinoids. The CB2 cannabinoid receptor is an established constituent of immune system cells, and we have recently established that the CB1 cannabinoid receptor is expressed in mast cells. In the present study, we sought to define a role for CB1 in mast cells and to identify the signalling pathways that may mediate the suppressive effects of CB1 ligation on mast cell activation. Our results show that CB1 and CB2 mediate diametrically opposed effects on cAMP levels in mast cells. The observed long-term stimulation of cAMP levels by the Galpha(i/o)-coupled CB1 is paradoxical, and our results indicate that it may be attributed to CB1-mediated transcriptional regulation of specific adenylate cyclase isoenzymes that exhibit superactivatable kinetics. Taken together, these results reveal the complexity in signalling of natively co-expressed cannabinoid receptors and suggest that some anti-inflammatory effects of CB1 ligands may be attributable to sustained cAMP elevation that, in turn, causes suppression of mast cell degranulation.

Differential roles of CB1 and CB2 cannabinoid receptors in mast cells.

Cannabinoid modulation of immune responses is a pathological consequence of marijuana abuse and a potential outcome of therapeutic application of the drug. Moreover, endogenous cannabinoids are physiological immune regulators. In the present report, we describe alterations in gene transcription that occur after cannabinoid exposure in a mast cell line, RBL2H3. Cannabinoid exposure causes marked changes in the transcript levels for numerous genes, acting both independently of and in concert with immunoreceptor stimulation via Fc epsilon RI. In two mast cell lines, we observed mRNA and protein expression corresponding to both CB1 and CB2 cannabinoid receptor isoforms, contrary to the prevailing view that CB1 is restricted to the CNS. We show that coexpression of the two isoforms is not functionally redundant in mast cells. Analysis of signaling pathways downstream of cannabinoid application reveals that activation of extracellular signal-regulated kinase, AKT, and a selected subset of AKT targets is accomplished by CB2 ligands and nonselective CB1/CB2 agonists in mast cells. CB1 inhibition does not affect AKT or extracellular signal-regulated kinase activation by cannabinoids, indicating that CB2 is the predominant regulatory receptor for these kinases in this cell context. CB1 receptors are, however, functional in these mast cells, since they can contribute to suppression of secretory responses.