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Cannabidiol (CBD) is a phytocannabinoid with potential as a therapy for a variety of diseases. CBD may act via cannabinoid receptors but also via other G-protein-coupled receptors (GPCRs), including the adenosine A 2A receptor. Homogenous binding and signaling assays in Chinese hamster ovary (CHO) cells expressing the human version of the A 2A receptor were performed to address the effect of CBD on receptor functionality. CBD was not able to compete for the binding of a SCH 442416 derivative labeled with a red emitting fluorescent probe that is a selective antagonist that binds to the orthosteric site of the receptor. However, CBD reduced the effect of the selective A 2A receptor agonist, CGS 21680, on G s -coupling and on the activation of the mitogen activated kinase signaling pathway. It is suggested that CBD is a negative allosteric modulator of the A 2A receptor.

Medical cannabis ( Cannabis sativa L.) is a source of bioactive phytochemicals with promising pharmacological and therapeutic applications. Enhancing the accumulation of valuable bioactive compounds is potentially a way of increasing the economic importance of this crop. Signaling molecules like salicylic acid (SA), jasmonic acid (JA), and γ-aminobutyric acid (GABA) are involved in the regulation of plant development and responses to biotic and abiotic stresses. Moreover, several phytohormones regulate plant trichome formation and elicit the synthesis of secondary metabolites in many plant species in both in vitro and in vivo systems. Therefore, exogenously delivered plant signaling molecules have the potential to modify the chemical profiles of medical cannabis. In this study, we found that the foliar application of SA, methyl jasmonate (MeJA), and GABA produces changes in the accumulation of the two major cannabinoids, cannabidiolic acid (CBDA) and Δ 9 - tetrahydrocannabinolic acid (THCA), in leaves and inflorescences of a medical cannabis variety. MeJA at 0.1 mM increased the CBDA content in inflorescences by 15.6%, while SA and MeJA at 0.1 mM increased CBDA and THCA accumulation in leaves by up to 57.3%. Treatments did not change the expression of genes participating in the final steps of the biosynthetic pathway of cannabinoids: olivetolic acid cyclase ( CsOAC-1 and CsOAC-2 ), 2-acylphloroglucinol 4-prenyltransferase ( CsPT4 ), cannabidiolic acid synthase ( CsCBDAS ), and tetrahydrocannabinolic acid synthase ( CsTHCAS ). Trichome density was not significantly different from the control plants in any treatment. Besides, we found strong correlations between several plant growth parameters and cannabinoid yields, showing a direct link between plant fitness and the production of cannabinoids., Competing Interests: Authors JG, CC, JM-Q, and CF-V are employed by Phytoplant Research S.L.U. The remaining authors declare that the research was conducted in the absence of any commercial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Garrido, Rico, Corral, Sánchez, Vidal, Martínez-Quesada and Ferreiro-Vera.)

The substantial increase in legalization and subsequent regulation of cannabis has intensified the control and analytical monitoring of cannabis products to assure sample quality and control the cannabinoid content of the crop. In this sense, the restriction on cultivating legal cannabis plants has been limited to 0.2-0.3% of Δ 9 -THC content, depending on the host country's laws. Thereby, cannabis flowers containing more than this limit are considered illicit drug-type cultivations and require the obtention of specific permits to work with them. The official method established by the European Commission set the gas chromatography/flame ionization detector (GC-FID) as the proper instrument to analyze the delta-9 tetrahydrocannabinol (Δ 9 -THC) content. In the present work, the potential drawbacks associated with the utilization of the official method for the evaluation of the Δ9-THC content have been described. Thus, the effect of the GC injector port temperature in the degradation of cannabinoids was evaluated, observing the degradation of CBD by 20%, generating Δ 9 -THC and CBN as by-products. Likewise, 17.2% of Δ 9 -THC was degraded, producing CBN as a by-product. Therefore, despite the brief residence of cannabinoids in the GC inlet, the effect of temperature is noteworthy and must be considered. Derivatization of cannabinoids should be a mandatory step to prevent the thermal degradation of cannabinoids, assuring the accuracy of the results. Furthermore, the evaluation of cannabinoid degradation thermally treated for longer periods of time was carried out. The kinetic degradation of CBD was evaluated in this way, observing a degradation of 0.22 μg/L per second. At the same time, the kinetics of the appearance of Δ 9 -THC demonstrates the intermediate nature of this cannabinoid, being degraded at 0.03 s -1  μM -1 . The degradation of CBD also produced CBN and CBE as by-products., Competing Interests: Authors MG-V, CS-C, MD-L, VS, JH-G, XN, and CF-V were employed by the company Phytoplant Research S.L.U. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 García-Valverde, Sánchez-Carnerero Callado, Díaz-Liñán, Sánchez de Medina, Hidalgo-García, Nadal, Hanuš and Ferreiro-Vera.)

Background: Cannabidiol (CBD) is a phytocannabinoid with potential in one of the most prevalent syndromes occurring at birth, the hypoxia of the neonate. CBD targets a variety of proteins, cannabinoid CB 2 and serotonin 5HT 1A receptors included. These two receptors may interact to form heteromers (CB 2 -5HT 1A -Hets) that are also a target of CBD. Aims: We aimed to assess whether the expression and function of CB 2 -5HT 1A -Hets is affected by CBD in animal models of hypoxia of the neonate and in glucose- and oxygen-deprived neurons. Methods: We developed a quantitation of signal transduction events in a heterologous system and in glucose/oxygen-deprived neurons. The expression of receptors was assessed by immuno-cyto and -histochemistry and, also, by using the only existing technique to visualize CB 2 -5HT 1A -Hets fixed cultured cells and tissue sections (in situ proximity ligation PLA assay). Results: CBD and cannabigerol, which were used for comparative purposes, affected the structure of the heteromer, but in a qualitatively different way; CBD but not CBG increased the affinity of the CB 2 and 5HT 1A receptor-receptor interaction. Both cannabinoids regulated the effects of CB 2 and 5HT 1A receptor agonists. CBD was able to revert the upregulation of heteromers occurring when neurons were deprived of oxygen and glucose. CBD significantly reduced the increased expression of the CB 2 -5HT 1A -Het in glucose/oxygen-deprived neurons. Importantly, in brain sections of a hypoxia/ischemia animal model, administration of CBD led to a significant reduction in the expression of CB 2 -5HT 1A -Hets. Conclusions: Benefits of CBD in the hypoxia of the neonate are mediated by acting on CB 2 -5HT 1A -Hets and by reducing the aberrant expression of the receptor-receptor complex in hypoxic-ischemic conditions. These results reinforce the potential of CBD for the therapy of the hypoxia of the neonate.

The aim of this study was to propagate axillary shoots of Cannabis sativa L. using liquid medium in temporary immersion bioreactors. The effect of immersion frequency (3 or 6 immersions per day), explant type (apical or basal sections), explant number (8, 10, and 16 explants), mineral medium (Murashige and Skoog half-strength nitrates, β -A and β -H, all supplemented with 2-μM metatopoline), sucrose supplementation (2, 0.5, and 0% sucrose), culture duration (4 and 6 weeks), and bioreactor type (RITA® and Plantform™) were investigated. As a result, we propose a protocol for the proliferation of cannabis apical segments in RITA® or Plantform™ bioreactors. The explants (8 per RITA® and 24 per Plantform™) are immersed for 1 min, 3 times per day in β -A medium supplemented with 2-μM metatopoline and 0.5% of sucrose and subcultured every 4 weeks. This is the first study using temporary immersion systems in C. sativa production, and our results provide new opportunities for the mass propagation of this species., Competing Interests: JG, CF-V, and VC are employed by Phytoplant Research SLU. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Rico, Garrido, Sánchez, Ferreiro-Vera, Codesido and Vidal.)

Cannabigerol (CBG) is one of the major phytocannabinoids present in Cannabis sativa L. that is attracting pharmacological interest because it is non-psychotropic and is abundant in some industrial hemp varieties. The aim of this work was to investigate in parallel the binding properties of CBG to cannabinoid CB 1 (CB 1 R) and CB 2 (CB 2 R) receptors and the effects of the compound on agonist activation of those receptors and of CB 1 -CB 2 heteroreceptor complexes. Using [ 3 H]-CP-55940, CBG competed with low micromolar K i values the binding to CB 1 R and CB 2 R. Homogeneous binding in living cells, which is only technically possible for the CB 2 R, provided a 152 nM K i value. Also interesting, CBG competed the binding of [ 3 H]-WIN-55,212-2 to CB 2 R but not to CB 1 R ( K i : 2.7 versus >30 μM). The phytocannabinoid modulated signaling mediated by receptors and receptor heteromers even at low concentrations of 0.1-1 μM. cAMP, pERK, β-arrestin recruitment and label-free assays in HEK-293T cells expressing the receptors and treated with endocannabinoids or selective agonists proved that CBG is a partial agonist of CB 2 R. The action on cells expressing heteromers was similar to that obtained in cells expressing the CB 2 R. The effect of CBG on CB 1 R was measurable but the underlying molecular mechanisms remain uncertain. The results indicate that CBG is indeed effective as regulator of endocannabinoid signaling.

Background and Purpose: Phytocannabinoids are produced in Cannabis sativa L. in acidic form and are decarboxylated upon heating, processing and storage. While the biological effects of decarboxylated cannabinoids such as Δ 9 -tetrahydrocannabinol have been extensively investigated, the bioactivity of Δ 9 -tetahydrocannabinol acid (Δ 9 -THCA) is largely unknown, despite its occurrence in different Cannabis preparations. Here we have assessed possible neuroprotective actions of Δ 9 -THCA through modulation of PPARγ pathways., Experimental Approach: The effects of six phytocannabinoids on PPARγ binding and transcriptional activity were investigated. The effect of Δ 9 -THCA on mitochondrial biogenesis and PPARγ coactivator 1-α expression was investigated in Neuro-2a (N2a) cells. The neuroprotective effect was analysed in STHdh Q111/Q111 cells expressing a mutated form of the huntingtin protein and in N2a cells infected with an adenovirus carrying human huntingtin containing 94 polyQ repeats (mHtt-q94). The in vivo neuroprotective activity of Δ 9 -THCA was investigated in mice intoxicated with the mitochondrial toxin 3-nitropropionic acid (3-NPA)., Key Results: Cannabinoid acids bind and activate PPARγ with higher potency than their decarboxylated products. Δ 9 -THCA increased mitochondrial mass in neuroblastoma N2a cells and prevented cytotoxicity induced by serum deprivation in STHdh Q111/Q111 cells and by mutHtt-q94 in N2a cells. Δ 9 -THCA, through a PPARγ-dependent pathway, was neuroprotective in mice treated with 3-NPA, improving motor deficits and preventing striatal degeneration. In addition, Δ 9 -THCA attenuated microgliosis, astrogliosis and up-regulation of proinflammatory markers induced by 3-NPA., Conclusions and Implications: Δ 9 -THCA shows potent neuroprotective activity, which is worth considering for the treatment of Huntington's disease and possibly other neurodegenerative and neuroinflammatory diseases., (© 2017 The British Pharmacological Society.)

The mechanism of action of cannabidiol (CBD), the main non-psychotropic component of Cannabis sativa L., is not completely understood. First assumed that the compound was acting via cannabinoid CB 2 receptors (CB 2 Rs) it is now suggested that it interacts with non-cannabinoid G-protein-coupled receptors (GPCRs); however, CBD does not bind with high affinity to the orthosteric site of any GPCR. To search for alternative explanations, we tested CBD as a potential allosteric ligand of CB 2 R. Radioligand and non-radioactive homogeneous binding, intracellular cAMP determination and ERK1/2 phosphorylation assays were undertaken in heterologous systems expressing the human version of CB 2 R. Using membrane preparations from CB 2 R-expressing HEK-293T (human embryonic kidney 293T) cells, we confirmed that CBD does not bind with high affinity to the orthosteric site of the human CB 2 R where the synthetic cannabinoid, [ 3 H]-WIN 55,212-2, binds. CBD was, however, able to produce minor but consistent reduction in the homogeneous binding assays in living cells using the fluorophore-conjugated CB 2 R-selective compound, CM-157. The effect on binding to CB 2 R-expressing living cells was different to that exerted by the orthosteric antagonist, SR144528, which decreased the maximum binding without changing the K D . CBD at nanomolar concentrations was also able to significantly reduce the effect of the selective CB 2 R agonist, JWH133, on forskolin-induced intracellular cAMP levels and on activation of the MAP kinase pathway. These results may help to understand CBD mode of action and may serve to revisit its therapeutic possibilities.

Metabolites derived from the polyunsaturated fatty acids (PUFA) may modulate the mesenchymal stromal cell (MSC) differentiation. Such cells can differentiate into different cellular types, including adipocytes and osteoblasts. Aging favors the bone marrow MSC differentiation toward the former, causing a loss of bone density associated with pathologies like osteoporosis. The omega-6 arachidonic acid (AA) favors MSC adipogenesis to a greater extent than omega-3 eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). In this work, we study the joint action of both PUFA. Thus, not induced and induced to adipocyte or osteoblast MSC were treated with 20 μM of each PUFA (either AA, AA + DHA or AA + EPA). The expression of osteogenic and adipogenic molecular markers, the alox15b lipoxygenase gene expression and the 5-, 8-, 11-, 12- and 15-hydroxyeicosatetraenoic acids (HETE) derived from the AA metabolism in the culture media were determined. The results show that the adipogenesis induction of AA is not suppressed by the joint presence of EPA and DHA. In fact, both increased the adipogenic effect of AA on MSC differentiated into osteoblasts. The different HETE concentrations increased in cultures supplemented with AA, albeit such concentrations were lower in the cultures induced to differentiate, mainly at day 21 after the induction. Furthermore, the reduction in the HETE concentration was correlated with a higher expression of the alox15b gene. These results highlight the PUFA metabolism differences between uninduced and induced MSC to differentiate into adipocytes and osteoblasts, besides the relevant role of the lipoxygenase gene expression in adipogenesis induction.

The proven endocrine disruption nature of the sunscreen ingredient 2-ethylhexyl 4-(N,N-dimethylamino)benzoate (EDP) calls for research to understand its distribution and bioaccumulation in the human body. A sensitive analytical method to determine EDP and its metabolites in human semen based on online SPE-LC-MS/MS is described. The method has been fully validated and a standard addition calibration has been used for quantification to correct the observed matrix effects. The on-column detection limits of the analytes are between 0.2 and 0.6 ng, depending on the analyte and the sample. The repeatability of the method, expressed as relative standard deviation, was in the range 4.6-9.4%. The method was satisfactorily applied to semen samples from male volunteers who were subjected to single and repeated whole-body applications of an EDP-containing sunscreen product. EDP metabolites were found at different concentrations in semen samples from the repeated application study, thus showing evidences of bioaccumulation in humans.

Inflammation is a complex cascade process involved in the pathogenesis of a number of diseases or generated as response to external or internal stimuli. Current research is focused on the development of assays for fast identification and quantitation of inflammation biomarkers. Eicosanoids are the oxidation metabolites of polyunsaturated fatty acids (mainly 20-carbon fatty acids) that play a regulation role in inflammation and, therefore, they have proved to be involved in different pathological states such as cancer, atherosclerosis, arthritis and cardiovascular or immunological diseases. Eicosanoids can be metabolized by different oxygenase enzymes to prostanoids such as prostaglandins and thromboxanes or hydroxyl fatty acids such as hydroxyeicosatetraenoic acids and hydroxyoctadecadienoic acids. A high-throughput automated approach is here presented for direct eicosanoid analysis in biofluids such as human serum and cells culture media. The approach is based on a hyphenated system composed by a solid-phase extraction workstation (Prospekt 2 unit) on-line coupled to a liquid chromatograph-triple quadrupole-tandem mass spectrometer. The detection limits for the target analytes ranged from 0.009 to 204 pg on-column, with precision between 2.65% and 7.33%, expressed as relative standard deviation. Accuracy studies with a dual-cartridge configuration resulted in recoveries between 78.6% and 100%, which validated internally the proposed approach ensuring highly efficient cleanup of proteins and salts. The method is reliable, robust and endowed with a great potential for implementation in clinical and routine laboratories. Analysis of culture media of stem cells stimulated with arachidonic acid was carried out to evaluate its incidence on the eicosanoid profile of the exometabolome.