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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.

Background: Intravenous pharmacokinetics and oral bioavailability of cannabidiol (CBD) with different formulations have not been investigated in horses and may represent a starting point for clinical studies., Objectives: To describe pharmacokinetics after intravenous and oral administrations with oil and micellar formulations and simulate different treatments., Study Design: Single intravenous experiment and two-way randomised oral experiments, Latin-square design., Methods: Eight healthy horses received intravenous CBD at 1.00 mg/kg dose, oral CBD in sesame oil and in micellar formulation, both at 10.00 mg/kg. Concentrations were measured using LC-MS/MS and fitted by nonlinear mixed effect modelling. Parameters obtained were used to simulate single and multiple treatments at steady state., Results: Intravenous and oral concentrations were simultaneously fitted using a three-compartment model. Final estimates indicate that CBD has a volume of distribution of 36 L/kg associated with a systemic clearance of 1.46 L/h/kg and half-lives ranged between 24 and 34 h. Oral bioavailability was close to 14% for both oral administrations. Simulated dose regimen of CBD every 12 and 24 h predicted similar percentages to reach effective plasma concentration with both oral formulation at 10.00 mg/kg., Main Limitations: A small horse population was used (8 horses per trial)., Conclusions and Clinical Importance: Oral bioavailability was low at the doses studied but fell within the range described for horse and other species. CBD had a high steady-state volume of distribution, a high clearance and long half-lives. No adverse reactions were detected at any dose or route. The micellar formulation showed a faster absorption and higher concentration peak, while the oil formulation presented lower levels, but more maintained over time. Simulations predicted that both could be useful in multiple oral dose treatments. These results indicated that CBD could be of interest, but further studies are needed to evaluate its clinical use in horses., (© 2023 EVJ Ltd.)

(-)-Δ 9 - trans -tetrahydrocannabinol (THC), which is the principal psychoactive constituent of Cannabis , mediates its action by binding to two members of the G-protein-coupled receptor (GPCR) family: the cannabinoid CB 1 (CB 1 R) and CB 2 (CB 2 R) receptors. Molecular dynamics simulations showed that the pentyl chain of THC could adopts an I-shape conformation, filling an intracellular cavity between Phe 3.36 and Trp 6.48 for initial agonist-induced receptor activation, in CB 1 R but not in CB 2 R. This cavity opens to the five-carbon chain of THC by the conformational change of the γ-branched, flexible, Leu 6.51 side chain of CB 1 R, which is not feasible by the β-branched, mode rigid, Val 6.51 side chain of CB 2 R. In agreement with our computational results, THC could not decrease the forskolin-induced cAMP levels in cells expressing mutant CB 1 R L6.51V receptor but could activate the mutant CB 2 R V6.51L receptor as efficiently as wild-type CB 1 R. Additionally, JWH-133, a full CB 2 R agonist, contains a branched dimethyl moiety in the ligand chain that bridges Phe 3.36 and Val 6.51 for receptor activation. In this case, the substitution of Val 6.51 to Leu in CB 2 R makes JWH-133 unable to activate CB 2 R V6.51L . In conclusion, our combined computational and experimental results have shown that the amino acid at position 6.51 is a key additional player in the initial mechanism of activation of GPCRs that recognize signaling molecules derived from lipid species.

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.)

We have here assessed, using Δ 9 -tetrahydrocannabinol (Δ 9 -THC) for comparison, the effect of Δ 9 -tetrahydrocannabinolic acid (Δ 9 -THCA) and of Δ 9 -tetrahydrocannabivarin (Δ9-THCV) that is mediated by human versions of CB 1 , CB 2 , and CB 1 -CB 2 receptor functional units, expressed in a heterologous system. Binding to the CB 1 and CB 2 receptors was addressed in living cells by means of a homogeneous assay. A biphasic competition curve for the binding to the CB 2 receptor, was obtained for Δ 9 -THCV in cells expressing the two receptors. Signaling studies included cAMP level determination, activation of the mitogen-activated protein kinase pathway and ß-arrestin recruitment were performed. The signaling triggered by Δ 9 -THCA and Δ 9 -THCV via individual receptors or receptor heteromers disclosed differential bias, i.e. the bias observed using a given phytocannabinoid depended on the receptor (CB 1 , CB 2 or CB 1 -CB 2 ) and on the compound used as reference to calculate the bias factor (Δ 9 -THC, a selective agonist or a non-selective agonist). These results are consistent with different binding modes leading to differential functional selectivity depending on the agonist structure, and the state (monomeric or heteromeric) of the cannabinoid receptor. In addition, on studying Gi-coupling we showed that Δ 9 -THCV and Δ 9 -THCA and Δ 9 -THCV were able to revert the effect of a selective CB 2 receptor agonist, but only Δ9-THCV, and not Δ9-THCA, reverted the effect of arachidonyl-2'-chloroethylamide (ACEA 100 nM) a selective agonist of the CB 1 receptor. Overall, these results indicate that cannabinoids may have a variety of binding modes that results in qualitatively different effects depending on the signaling pathway that is engaged upon cannabinoid receptor activation., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Recent approved medicines whose active principles are Δ 9 Tetrahidrocannabinol (Δ 9 -THC) and/or cannabidiol (CBD) open novel perspectives for other phytocannabinoids also present in Cannabis sativa L. varieties. Furthermore, solid data on the potential benefits of acidic and varinic phytocannabinoids in a variety of diseases are already available. Mode of action of cannabigerol (CBG), cannabidiolic acid (CBDA), cannabigerolic acid (CBGA), cannabidivarin (CBDV) and cannabigerivarin (CBGV) is, to the very least, partial., Hypothesis/purpose: Cannabinoid CB 1 or CB 2 receptors, which belong to the G-protein-coupled receptor (GPCR) family, are important mediators of the action of those cannabinoids. Pure CBG, CBDA, CBGA, CBDV and CBGV from Cannabis sativa L. are differentially acting on CB 1 or CB 2 cannabinoid receptors., Study Design: Determination of the affinity of phytocannabinoids for cannabinoid receptors and functional assessment of effects promoted by these compounds when interacting with cannabinoid receptors., Methods: A heterologous system expressing the human versions of CB 1 and/or CB 2 receptors was used. Binding to membranes was measured using radioligands and binding to living cells using a homogenous time resolved fluorescence resonance energy transfer (HTRF) assay. Four different functional outputs were assayed: determination of cAMP levels and of extracellular-signal-related-kinase phosphorylation, label-free dynamic mass redistribution (DMR) and ß-arrestin recruitment., Results: Affinity of cannabinoids depend on the ligand of reference and may be different in membranes and in living cells. All tested phytocannabinoids have agonist-like behavior but behaved as inverse-agonists in the presence of selective receptor agonists. CBGV displayed enhanced potency in many of the functional outputs. However, the most interesting result was a biased signaling that correlated with differential affinity, i.e. the overall results suggest that the binding mode of each ligand leads to specific receptor conformations underlying biased signaling outputs., Conclusion: Results here reported and the recent elucidation of the three-dimensional structure of CB 1 and CB 2 receptors help understanding the mechanism of action that might be protective and the molecular drug-receptor interactions underlying biased signaling., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

While natural Δ 9 -tetrahidrocannabinol (Δ 9 THC), cannabidiol (CBD), and their therapeutic potential have been extensively researched, some cannabinoids have been less extensively investigated. The present article compiles data from the literature that highlight the health benefits and therapeutic potential of lesser known phytocannabinoids, which we have divided into varinic, acidic, and "minor" (i.e., cannabinoids that are not present in high quantities in common varieties of Cannabis sativa L). A growing interest in these compounds, which are enriched in some cannabis varieties, has already resulted in enough preclinical information to show that they are promising therapeutic agents for a variety of diseases. Every phytocannabinoid has a "preferential" mechanism of action, and often targets the cannabinoid receptors, CB 1 and/or CB 2 . The recent resolution of the structure of cannabinoid receptors demonstrates the atypical nature of cannabinoid binding, and that different binding modes depend on the agonist or partial agonist/inverse agonist, which allows for differential signaling, even acting on the same cannabinoid receptor. In addition, other players and multiple signaling pathways may be targeted/engaged by phytocannabinoids, thereby expanding the mechanistic possibilities for therapeutic use., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Medicinal cannabis has remarkable therapeutic potential, but its clinical use is limited by the psychotropic activity of Δ9-tetrahydrocannabinol (Δ9-THC). However, the biological profile of the carboxylated, non-narcotic native precursor of Δ9-THC, the Δ9-THC acid A (Δ9-THCA-A), remains largely unexplored. Here we present evidence that Δ9-THCA-A is a partial and selective PPARγ modulator, endowed with lower adipogenic activity than the full PPARγ agonist rosiglitazone (RGZ) and enhanced osteoblastogenic effects in hMSC. Docking and in vitro functional assays indicated that Δ9-THCA-A binds to and activates PPARγ by acting at both the canonical and the alternative sites of the ligand-binding domain. Transcriptomic signatures in iWAT from mice treated with Δ9-THCA-A confirmed its mode of action through PPARγ. Administration of Δ9-THCA-A in a mouse model of HFD-induced obesity significantly reduced fat mass and body weight gain, markedly ameliorating glucose intolerance and insulin resistance, and largely preventing liver steatosis, adipogenesis and macrophage infiltration in fat tissues. Additionally, immunohistochemistry, transcriptomic, and plasma biomarker analyses showed that treatment with Δ9-THCA-A caused browning of iWAT and displayed potent anti-inflammatory actions in HFD mice. Our data validate the potential of Δ9-THCA-A as a low adipogenic PPARγ agonist, capable of substantially improving the symptoms of obesity-associated metabolic syndrome and inflammation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Neuroprotective M2-skewed microglia appear as promising to alter the course of neurodegenerative diseases and G protein-coupled receptors (GPCRs) are potential targets to achieve such microglial polarization. A common feature of adenosine A 2A (A 2A R) and cannabinoid CB 2 (CB 2 R) GPCRs in microglia is that their expression is upregulated in Alzheimer's disease (AD). On the one hand, CB 2 R seems a target for neuroprotection, delaying neurodegenerative processes like those associated to AD or Parkinson's diseases. A 2A R antagonists reduce amyloid burden and improve cognitive performance and memory in AD animal models. We here show a close interrelationship between these two receptors in microglia; they are able to physically interact and affect the signaling of each other, likely due to conformational changes within the A 2A -CB 2 receptor heteromer (A 2A -CB 2 Het). Particularly relevant is the upregulation of A 2A -CB 2 Het expression in samples from the APP Sw , Ind AD transgenic mice model. The most relevant finding, confirmed in both heterologous cells and in primary cultures of microglia, was that blockade of A 2A receptors results in increased CB 2 R-mediated signaling. This heteromer-specific feature suggests that A 2A R antagonists would potentiate, via microglia, the neuroprotective action of endocannabinoids with implications for AD therapy., (© 2019 Wiley Periodicals, Inc.)

Currently, biased agonism is at the center stage of drug development approaches. We analyzed effects of a battery of cannabinoids plus/minus cannabidiol (CBD) in four functional parameters (cAMP levels, phosphorylation of extracellular signal-regulated kinases (ERK1/2), β-arrestin recruitment and label-free/DMR) in HEK-293T cells expressing cannabinoid receptors, CB 1 or CB 2 , or CB 1 -CB 2 heteroreceptor complexes. In all cases two natural agonists plus two selective synthetic agonists were used. Furthermore, the effect of cannabidiol, at a dose (100 nM) that does not allow significant binding to the orthosteric center of either receptor, was measured. From the huge amount of generated data, we would like to highlight that the two psychotropic molecules (Δ 9 -tetrahydrocannabinol/THC and CP-55940) showed similar bias in CB 1 R and that the bias of THC was particularly relevant toward MAPK pathway. Furthermore, THC did not activate the G i protein coupled to CB 2 R. Interestingly, the biased agonism was reduced when assays were performed in cells expressing the two receptors, thus suggesting that the heteromer allows less functional selectivity. In terms of cannabidiol action, the phytocannabinoid altered the functional responses, likely by allosteric means, and modified potency, agonist IC 50 /EC 50 values and biased agonism in qualitative and/or quantitative different ways depending on the agonist. The effect of cannabidiol on anandamide actions on both cannabinoid receptors was particularly noteworthy as was significantly different from that of other compounds. Results are a compendium of data on biased agonism on cannabinoid receptors in the absence and presence of cannabidiol. In addition, for the first time, GPCR biased agonism is characterized in an heteromeric context., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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 metabolic profile of human serum after intake of breakfasts prepared with different heated vegetable oils has been studied. Four oils (olive and sunflower oils, pure and enriched with natural and artificial oxidation inhibitors) were subjected to a simulated heated process prior to breakfast preparation. A metabolomics global profiling approach performed on post-basal serum samples revealed statistical differences among individuals based on breakfast intake, and identified compounds responsible for such differences. Serum samples obtained in basal state (control samples) and 2 and 4h after programmed intakes were analyzed by LC-TOF/MS. The resulting fingerprints were compared and differences between basal and post-basal states evaluated, observing that the intake of different breakfasts altered the metabolic signature of serum. Analysis models based on PLS algorithms were developed to discriminate individuals in post-basal state for each intervention breakfast. Then, Volcano tests enabled to detect significant molecular entities explaining the variability associated to each breakfast. It is worth emphasizing the importance of fatty acids, their derivatives and phospholipids for tentative identification., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

An approach for quantitative analysis of two vitamins with different polarities (vitamins D and B9) and their metabolites is presented here. The approach is based on an experimental setup based on hyphenation of an automated workstation for preparation of liquid samples and an LC-MS/MS system with a triple quadrupole mass spectrometer. This configuration enabled development of an orthogonal protocol for sequential SPE retention of analytes with different polarities for subsequent elution and chromatographic separation prior to detection. The resulting method was validated by application to three human biofluids. Estimation of recovery factors in the SPE step led to values from 85.2 to 100% for vitamin D and metabolites and from 93.1 to 100% for vitamin B9 and metabolites (folic acid and folates). The influence of sample matrix variability by analysis of human serum, urine and breast milk was minimized with a complete optimization of the SPE step. The utility of the proposed configuration is shown by the sensitivity and precision of the method, expressed as limits of detection (between 0.2 and 0.30 ng mL(-1) or 4 and 60 pg on-column) and within-laboratory reproducibility (lower than 6.7%, as relative standard deviation). The present application represents an example of determination methods involving targeted analysis of compounds with different polarities using a single aliquot of the sample.

The effect of breakfast intake of fried oils containing natural antioxidants or a synthetic autooxidation inhibitor on the metabolism of essential fatty acids focused on obese individuals. Serum levels of eicosanoids were compared in individuals before and after intake of different breakfasts. Univariate descriptive analysis was used to characterise the cohort selected for this study and multivariate analysis to reveal statistical differences of normalised eicosanoids concentrations (determined by solid-phase extraction coupled to LC-MS/MS) depending on the edible oil used for breakfast preparation. The results showed that the intake of breakfast prepared with pure sunflower oil subjected to deep frying causes an effect over the eicosanoids profile that enables discrimination versus the rest of individuals. The effect was a significant increase in the concentration of hydroxyoctadecadienoic acid (HODE) metabolites, indicative markers of the intake of fried oils. The concentration of HODE metabolites was lower when the oil contained either natural antioxidants from olive-oil pomace or a synthetic autooxidation inhibitor as dimethylsiloxane. The comparison of the effect of fried sunflower oils with fried extra virgin olive oil shows the benefits associated to the consumption of the latter., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Most research on the extraction of high-priced compounds from vineyard/wine byproducts has traditionally been focused on grape seeds and skins as raw materials. Vine-shoots can represent an additional source to those materials, the characteristics of which could depend on the cultivar. A comparative study of hydroalcoholic extracts from 18 different vineyard cultivars obtained by superheated liquid extraction (SHLE), microwave-assisted extraction (MAE), and ultrasound-assisted extraction (USAE) is here presented. The optimal working conditions for each type of extraction have been investigated by using multivariate experimental designs to maximize the yield of total phenolic compounds, measured by the Folin-Ciocalteu method, and control hydroxymethylfurfural because of the organoleptic properties of furanic derivatives and toxicity at given levels. The best values found for the influential variables on each extraction method were 80% (v/v) aqueous ethanol at pH 3, 180 °C, and 60 min for SHLE; 140 W and 5 min microwave irradiation for MAE; and 280 W, 50% duty cycle, and 7.5 min extraction for USAE. SHLE reported better extraction efficiencies as compared to the other two approaches, supporting the utility of SHLE for scaling-up the process. The extracts were dried in a rotary evaporator, reconstituted in 5 mL of methanol, and finally subjected to liquid-liquid extraction with n-hexane to remove nonpolar compounds that could complicate chromatographic separation. The methanolic fractions were analyzed by both LC-DAD and LC-TOF/MS, and the differences in composition according to the extraction conditions were studied. Compounds usually present in commercial wood extracts (mainly benzoic and hydroxycinnamic acids and aldehydes) were detected in vine-shoot extracts.

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.

The in vivo metabolism of the xenobiotic agent 2-ethylhexyl 4-(N,N-dimethylamino)benzoate (EDP), a UV filter commonly used in sunscreen cosmetic products, was studied by targeting metabolomics analysis in human urine. The metabolomic study involved the use of urine from male and female volunteers before and after application of an EDP-containing sunscreen cosmetic. The metabolism of EDP in urine was studied by using the triple quadrupole detector in a combination of Precursor Ion Scanning and Neutral Loss Scanning modes, with and without enzymatic hydrolysis. Detected metabolites were subsequently confirmed as glucuronide conjugates of 4-(N,N-dimethylamino)benzoic acid and 4-(N-methylamino)benzoic acid by liquid chromatography-time-of-flight/mass spectrometry (LC-TOF/MS) in the accurate mass mode. In this way, the existence of phase II metabolism in the detoxification of EDP by effects of the lipophilic character of this sunscreen agent was confirmed. Hence, to study the in vivo metabolism of EDP, a fully automated method using a solid-phase extraction (SPE) workstation connected on-line to a liquid chromatograph and a triple quadrupole mass analyzer (LC-MS/MS) was developed. The ensuing hyphenated method is very simple and requires minimal human intervention. Following thorough optimization of the SPE and LC-MS/MS conditions, the analytical procedure was validated and standard addition calibration used for the quantitative correction of matrix effects. The proposed method was applied to determine the phase I metabolites of EDP in urine samples and afforded limits of detection from 0.1 to 1.1 ng and accuracy of 91-107% with relative standard deviations in the range 1.5-8.7% (sample volume: 100 μL). Based on the results of in vivo percutaneous absorption of a single application of the sunscreen, about 0.5% of the amount of the applied EDP is excreted in urine., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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.