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- P03-10 Synthetic cannabinoids & neuronal senescence: distinctive responses of in vitro models to AMB-FUBINACAPublication . Bravo, R. Roque; Carmo, H.; Silva, J.P.; Carvalho, F.; Silva, Diana Dias da; Dias da Silva, Diana CristinaAmong the array of new psychoactive substances, synthetic cannabinoids (SC) stand out as highly popular among consumers. These substances closely resemble, in terms of their pharmacology, Δ9-tetrahydrocannabinol (THC), cannabis’ main active principle, albeit exhibiting full agonism at the cannabinoid receptors 1 and 2. In light of recent scientific findings suggesting that cannabis use can exacerbate ageing-related parameters, the present work was designed to explore whether SC share similar potential effects. For this purpose, we employed two distinct in vitro models. The first model involved primary hippocampal cultures (PHC), isolated from Wistar rat embryos at embryonic day 18–19; after seeding, cells were kept in culture and exposed to the popular SC AMB-FUBINACA (AMB-FUB) at 1 pM, 1 nM and 1 µM, starting at day-in-vitro (DIV) 3 or DIV7, and perpetuated until DIV21. DMSO at 0.02% was used as the solvent control. At the end of the exposure, ß-galactosidase activity (a common first-line cell senescence biomarker) was assessed using a commercially-available kit. Our findings under these experimental conditions, revealed that PHC exposed to all AMB-FUB concentrations had less ß-galactosidase activity than the control condition (p<0.01, 1 pM; p<0.001, 1 nM and 1 µM). The other in vitro model used herein was the human neuroblastoma cell line SH-SY5Y. Beginning at passage 24, cells were seeded and exposed to 1 nM and 1 µM AMB-FUB. At passages 24 (48h after drug exposure) and 28, samples were collected for the analysis of several senescence-related endpoints, namely ß-galactosidase activity, cell cycle analysis (via flow cytometry, following DNA staining with propidium iodide) and relative telomere length measurement (using qPCR). Surprisingly, no discernible effect of AMB-FUBINACA was observed for any of the endpoints examined. The apparent observed “anti-ageing” effect of AMB-FUB on PHC warrants further investigation. Moreover, the differential reponse observed between the two in vitro models also requires scrutiny, particularly in light of recent published findings suggesting that THC has the potential to increase ß-galactosidase activity. Further experiments will ensue to hopefully shed some light on these interesting results.
- Optimization of the derivatization procedure for the separation of the stereoisomers of 1,3-dimethylamylamine (1,3-DMAA) by gas chromatography - preliminary dataPublication . Almeida, Maria Mexia de; Silva, Diana Dias da; Oliveira, Ricardo Jorge Dinis; Ribeiro, Cláudia; Dias da Silva, Diana Cristina1,3-Dimethylamylamine (1,3-DMAA),also known as methylhexanamine, is a central nervous system stimulant with structural similarities with amphetamines and therefore presenting over-lapping biological and detrimental effects [1]. Despite being banned, the presence of 1,3-DMAA in dop-ing controls and dietary supplements continues to be of significant concern. This molecule has two stere-ogenic centres and thus four stereoisomers [2]. It is widely recognized that enantiomers may exhibit dif-ferent biological activity, including pharmacokinetics, pharmacodynamics, and toxicity. Consequently, the development of analytical methods for enantioselective separation of 1,3-DMAA is crucial for an accurate determination of the risks associated with each of these stereoisomers. To develop an indirect method by gas chromatography coupled to mass spectrometry (GC-MS) for the separation and identification of the stereoisomers of the 1,3-DMAA. 1,3-DMAA was regenerated with sodium hydroxide, extracted with 0.1% triethylamine in hexane and then derivatized using the enantiomeric pure reagent (R)-(-)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride ((R)-MTPA-Cl). Subsequently, the sample was evaporated, reconstituted in anhydrous ethyl acetate, and analyzed by GC-MS. The chroma-tographic conditions were established using a capillary column containing 5% diphenyl-95% dime-thylpolysiloxane (30 m × 0.25 mm × 0.25 μm), an injector temperature set to 280 ºC, with a temperature ramp starting at 140 ºC and increasing up to 215 ºC at a flow rate of 1 mL/min to a total run of 12.32 min. Results: As preliminary data indicate, the derivatization procedure allowed the formation of 4 diastere-omers of 1,3-DMAA. The chromatographic conditions were optimised, allowing for the separation of the four diastereomers within 12 min. Derivatization and chromatographic conditions were established for enantioselective separation of 1,3-DMAA by GC-MS. Further validation of the method will be crucial for understanding the diastereomers' differential pharmacokinetics and pharmacodynamics, and consequently, the perils associated with their presence in food supplement samples.
- Preliminary chemical profile and in vitro pharmacological evaluation of the hallucinogenic plant Diplopterys cabreranaPublication . Garcia, Maria Rita; Dutka, Mykhaylo; Guimarães, Sofia; Andrade, Paula B.; Seabra, Vítor; Diana Dias da Silva; Gomes, Nelson G. M.; Dias da Silva, Diana CristinaFor the last few years, Ayahuasca ceremonies have been gaining popularity in recreational settings in Europe and North America [1]. Similar to Psychotria viridis, Diplopterys cabreranais also suggested to contain the psychoactive compound N,N-dimethyltryptamine, and is therefore used in Aya-huasca rituals for its ability to induce hallucinations, euphoria and entheogenic effects [1-3]. However, while information on the toxic profile of D. cabreranaremains very limited, its acquisition is easily ac-complished by consumers. We aimed to characterize the aqueous extracts of D. cabreranaleaves, mimicking those typically consumed, to identify bioactives that underlie the psychoactive or toxic effects, and evaluate their impact on neuronal function, neurotransmission and radical stress. Chemical characterization was attained by HPLC-DAD. Impact upon neuronal viability was assessed by the MTT assay (up to 1000 μg/mL) in SH-SY5Y neuroblastoma cells. Impact on neuromodulation and neuroinflammation was evaluated through acetylcholinesterase and 5-lipoxygenase inhibition, while an-tiradical properties were assessed by evaluating nitric oxide (•NO) and xanthine oxidase (XO) activity. Inhibition of the α-glucosidase enzyme was also evaluated. Statistical comparisons among groups per-formed by one-way ANOVA followed by Dunnett post hoc test. Preliminary characterization results revealed the presence of several catechin derivates, alongside two apigenin derivates and one tryp-tamine derivate. Cytotoxicity was not verified up to the highest concentration tested. Acetylcholinesterase inhibition was recorded starting at 250 μg/mL, and a concentration-dependent inhibition of 5-lipoxygen-ase was found (IC50=79.77 μg/mL). Concentration-dependent scavenging effects upon •NO and XO inhi-bition were verified at concentrations higher than 1.953 μg/mL and 31.25 μg/mL, respectively. At last, inhibition of α-glucosidase occurred with concentration-dependency and an IC50of 4.78 μg/mL. Conclu-sions:Although antiradical, anti-inflammatory and antidiabetic properties were verified, with no in vitrocytotoxicity being detected, further research is needed to elucidate the underlying mechanisms that might be involved in our preliminary results.
- In vitro and in silico evaluation of psilocybin and psilocin’s interaction with CYP450 enzymesPublication . Brito-da-Costa, Andreia Machado; Carvalho, Mariana; Dinis-Oliveira, Ricardo Jorge; Madureira-Carvalho, Áurea; Sousa, Sérgio F.; Silva, Diana Dias da; Dias da Silva, Diana CristinaPsilocybin is a hallucinogen produced by “magic mushrooms”, being rapidly metabolized in the organism into psilocin [1, 2]. A scientific gap exists regarding the possible interactions between psilocybin/psilocin and CYP450 enzymes. Given their biological importance, and since the binding of drugs to CYP450 enzymes can interfere with the metabolism of other substrates leading to drug-drug interactions, this research topic is of utmost importance. This study aimed to evaluate in silicoand in vitrothe interaction of psilocybin and psilocin with the enzymes CYP2A6/2B6/2D6/2E1/3A4. The in vitroassessment of inhibition was performed using the Vivid®CYP450 screening kits. IC50 was calculated using GraphPad Prism 9.3.0. For in silico assessment, molecular dynamics were per-formed using the PMEMD.cuda module in AMBER16. Calculations were made on the last 100 ns of the trajectory (stable zone) to assess the interaction between enzyme and ligand, namely MMGBSA, per-residue decomposition energy, and hydrogen bonds. Psilocin showed the capacity to be an in-hibitor of CYP2A6/2B6/2D6/2E1/3A4, based on the respective IC50 values (μM) of 2.06, 6.17, 11.89, 6.37, and 2.36. Considering the MMGBSA, higher values were obtained for psilocin, corroborating the stronger binding affinity of this compound. The interaction of psilocybin/psilocin with CYP2A6 is medi-ated by a hydrogen bond established with the protein residue Asn297. Other important residues include Phe107 and Ile366. For CYP2B6, the strong binding of psilocin is mediated by interactions with Ile114, Thr302 (hydrogen bond), and Leu363. For interaction with CYP2D6, the most important residue seems to be Ser304, with which it forms a hydrogen bond; for CYP2E1, key residues include Phe207, Thr303, and Phe478. A strong hydrogen bond is formed between psilocin and CYP3A4 residue Phe304, contrib-uting to the high binding affinity. The results suggest a potential for psilocin to inhibit all enzymes, especially CYP2A6 and CYP3A4.
- In vitro and in silico evaluation of 5-MeO-DMT, LSD, and mescaline’s interaction with CYP450 enzymesPublication . Brito-da-Costa, Andreia Machado; Carvalho, Mariana; Dinis-Oliveira, Ricardo Jorge; Madureira-Carvalho, Áurea; Sousa, Sérgio F.; Silva, Diana Dias da; Dias da Silva, Diana Cristina5-Methoxy-N,N-dimethyltryptamine(5-MeO-DMT), lysergic acid diethylamide (LSD), and mescaline are classic hallucinogens known for their recreational use, which increased in the last dec-ades. Despite some available data on the metabolism of these drugs [1-3], a scientific gap exists regarding their possible interactions with CYP450 enzymes. Nevertheless, this information is of crucial relevance to predict drug-drug interactions and understand toxicological phenomena, in particular interindividual variability. This study aimed to evaluate in vitroand in silicothe interaction of 5-MeO-DMT, LSD, and mescaline with the enzymes CYP2A6/2B6/2D6/2E1/3A4. The in vitroassessment of CYP450 inhibition was performed using the Vivid®CYP450 screening kits. IC50 was calculated using GraphPad Prism 9.3.0. For in silicoassessment, molecular dynamics were performed using the PMEMD.cuda module in AMBER16. Calculations were made on the last 100 ns of the trajectory (stable zone) to assess the interaction mode/strength between enzyme and ligand, namely MMGBSA, per-residue decomposition energy, and hydrogen bonds. Based on the IC50(μM), LSD (0.35) and 5-MeO-DMT (3.47) present the capacity to be inhibitors of CYP2D6. Based on the MMGBSA (kcal/mol), LSD showed the highest binding affinities for all enzymes, while mescaline showed the lowest. The strong interaction of LSD with CYP2A6 is mediated by a hydrogen bond established with the protein residue Asn297. For interaction with CYP2B6, the residues Thr302 and Lys479 were important in mediating the interaction with 5-MeO-DMT and LSD. Key residues mediating the interaction of 5-MeO-DMT and LSD with CYP2D6 included Phe120, Leu213, and Phe483. For interaction with CYP2E1, residues Phe207, Phe298, and Thr303 are important; and for CYP3A4, an important hydrogen bond between LSD and Ala370 was identified.Conclusions: Both LSD and 5-MeO-DMT are predicted to have strong potential to be CYP2D6 inhibitors. A strong interaction was also identified in silicobetween LSD and CYP2A6.
- Simultaneous determination of dapsone and clofazimine in nanoformulations by HPLCPublication . Fernandes, Sara R.; Fernandes, Sara; Chaves, Luíse L.; Lima, Sofia A. C.; Silva, Eduarda M. P.; Barreiros, Luísa; Reis, Salette; Segundo, Marcela A.The multidrug therapy with dapsone (DAP) and clofazimine (CLZ) is known as an effective treatment against Mycobacterium leprae. However, the low bioavailability and non-specific distribution can reduce therapy efficacy and produce side effects. The use of nanotechnological approaches was explored as a promising carrier for delivery enhancement of these drugs. Therefore, a simple and precise highperformance liquid chromatography (HPLC) method with UV/Vis detection has been developed and validated for the simultaneous determination of DAP and CLZ loaded in solid dispersion and poly(D,L-lactide-co-glycolic acid) nanoparticles, respectively, targeting therapy improvement. A reversed phase Kinetex core-shell C18 column at room temperature followed by UV/Vis detection at 280 nm was used for chromatographic separation. The elution was performed in gradient mode using aqueous acetate buffer (50 mol L-1, pH 4.8) and an increasing acetonitrile content from 27 to 63% (v/v), at a flow rate of 1.0 mL min-1. The injection volume was fixed at 20 µL and total run time was 23.0 min, with a retention time of 6.0 min for DAP and 14.0 min for CLZ. The method was validated according to EMA guideline and showed specificity, accuracy (between 99.6 and 114.0% of nominal values) and precision for intra-day (RSD ≤1.8%) and inter-day assays (RSD ≤12.5%). Calibration curves were linear (r2 >0.9979) and LOD ≤0.03 and LOQ ≤0.06 mg L-1 were obtained. Stability was studied after 24 h at room temperature and over three freeze-thaw cycles, and recovery values ≥86.2% were obtained. Precipitation of CLZ was observed at low temperatures (4 °C). Entrapment efficiency in nanoformulations was evaluated as 54.8 ± 0.1% for DAP and 24.9 ± 0.2% for CLZ. The developed method was successfully validated for the simultaneous determination of DAP and CLZ in nanoparticles.
- Quantification of tranexamic acid in human plasma: development and validation of UHPLC-MS/MS methodPublication . Barreiros, Luisa; Amoreira, Júlia L.; Machado, Sandia; Fernandes, Sara R.; Silva, Eduarda M. P.; Sá, Paula; Kietaibl, Sibylle; Segundo, Marcela A.; Fernandes, SaraTranexamic acid (TXA), an antifibrinolytic drug with the ability to inhibit lysine binding at plasminogen receptors, can be used in different settings such as trauma, cardiac surgery, major orthopedic surgery, obstetric when perioperative bleeding is concerned [1]. Effective methods for determination of TXA in biological samples are still required to understand the pharmacokinetics and pharmacodynamics of this drug in variable age groups undergoing surgeries with high blood loss [2]. The development and validation of a method based on ultra-high performance liquid chromatography coupled to triple quadrupole-tandem mass spectrometry (UHPLCMS/MS) to quantify TXA in human plasma is described herein. A simple, inexpensive and efficient sample treatment involving protein precipitation with acetonitrile containing 0.5% (v/v) formic acid was implemented using volumes within the microliter range. Separation was achieved using a hydrophilic interaction based stationary phase and ammonium bicarbonate in the mobile phase that permitted a more efficient separation of the analyte from the matrix interferences, thus reducing matrix effects and increasing method sensitivity. The method was validated according to the European Medicines Agency guideline [3]. Excellent linearity was achieved (r2 > 0.997) for TXA concentrations ranging from 30 to 600 ng mL-1 with LOD and LOQ of 3 and 6 ng mL-1 in plasma extracts, respectively. The developed method proved to be selective, sensitive, accurate (96.4-105.7% of nominal concentration values) and precise (RSD ≤ 4.5%).
- Bioacessibility of zinc in pet food determined by a dynamic leaching methodPublication . Fernandes, Sara R.; Pereira, Ana Margarida; Matos, Elisabete; Castanheira, Francisco; Baptista, Cláudia S.; Cabrita, Ana Rita J.; Segundo, Marcela A.; Fernandes, SaraIn dynamic leaching methods, portions of extractant reagents are continuously provided to the solid sample contained in flow-through microcolumns or chambers, enabling the renewal of extracting fluid and avoiding saturation effects from fluid stagnation. These methods are also suitable for fast measurements in real time with small extract manipulation, especially when coupled online with suitable detectors [1]. In this work, the bioaccessible fraction and kinetic leaching profile of zinc in pet food was determined using a robust flow-through device, composed by two filters placed in polypropylene holders to entrap the solid sample, designed for dynamic leaching experiments [2]. Continuous extraction flow was ensured by a peristaltic pump connecting the extraction reservoir and the extraction chamber, at a flow rate of 0.5 mL min-1. Synthetic fluids simulating digestive compartments were applied as extractants. The kinetic extraction profile of fast leachable Zn was evaluated by flame atomic absorption. Operational conditions, including filters’ composition and pore size, were tested. Preliminary results have shown that different extracting fluids (with and without digestive enzymes) had an influence on the total amount and on the leaching kinetic profile of Zn. In fact, higher values were obtained when enzymes were present in the extracting fluids. The proposed dynamic leaching method was suitable for evaluation of bioaccessible Zn in pet food. This information will be applied for the improvement of Zn supplementation in dog foods and for designing new products with enhanced mineral delivery.
- Evaluation of enzymatic digestion conditions for determination of immunoglobulins by tandem mass spectrometryPublication . Guerra, Gabriela S.; Ramos, Inês I.; Barreiros, Luísa; Silva, Eduarda M. P.; Segundo, Marcela A.Immunoassays, namely ELISA, have been the standard method for detecting clinically significant immunoglobulins (Igs). They are based on Ig-antigen interaction, often suffering interference from matrix components. New analytical approaches using detection by tandem mass spectrometry (MS/MS) search for fundamental structure information of target Igs based on protein features. In fact, there are few examples of quantitative assays achieved by liquid chromatography coupled with triple quadrupole (QqQ) mass analyzers. Due to the limited mass range of QqQ, the use of this mass analyzer requires previous tryptic digestion of IgG for analysis of highly specific surrogate peptides. In this work, initial studies on a LC-MS/MS method for the quantitative analysis of IgG are reported. The method relies upon the detection of the generic peptide DTLMISR (Fig. 1), originated from the fraction crystallizable (Fc) region of IgG after enzymatic cleavage. The multiple reaction monitoring transitions used for quantification and identification purposes were, respectively, m/z 418.20 506.10 and 418.20 619.30, corresponding to the fragmentation of double-charged molecular ions. In order to investigate the influence of trypsin concentration on digestion kinetics and efficiency, the trypsin-to-protein ratios 1:20, 1:50 and 1:100 were evaluated. Moreover, the performance of the digestion process was monitored for IgG standards and plasma samples over 18 h at 37 °C. Using a 1:50 ratio, two distinct kinetic profiles were observed for standards and plasma samples with a maximum signal intensity after 6 and 18 h, respectively.
- Determination of Vitamin C content in spinach plants grown under the use of cyanobacteria biomass as fertilizerPublication . Martins, Diogo; Massa, Anabella; Reimão, Mariana; Vasconcelos, Vítor; Campos, Alexandre; Pinto, Edgar; Freitas, Marisa; REIMÃO BORGES LOPES DA SILVA, MARIANA; Pinto, Edgar; Freitas, MarisaThe agricultural sector is continuously exploring sustainable and innovative approaches to enhance crop productivity, driving the quest for greener alternatives over synthetic fertilizers. Cyanobacteria have been reported as a promising agricultural fertilizer, with positive effects on crop yield. However, little is known about its potential effects on the produced vegetables' nutritional quality. Studies have documented that exposure of plants to cyanobacterial crude extracts stimulates the production of enzymatic and non-enzymatic antioxidants. Vitamin C, a non-enzymatic antioxidant, plays a crucial role in protecting cells from oxidative stress and spinach is known to be relatively high in vitamin C compared to other vegetables. The purpose of this study was to determine the content of vitamin C in spinach plants grown under the use of cyanobacterial biomass as fertilizer. Spinach plants were tested in soil, under six different conditions: (i) control, (ii) commercial chemical fertilizer, cyanobacterial biomass (iii) non-cyanotoxin producer, and cyanotoxin producer of (iv) Anatoxina (ANA), (v) Microcystin (MC), and (vi) Cylindrospermopsin (CYN). The analysis of vitamin C in spinach plants was performed according to EN 14130:2003. The results showed an increased content of vitamin C in all exposed groups when compared to the control (37 mg/Kg), with statistical differences in the conditions exposed to MC (56 mg/kg), ANA (76 mg/kg), and CYN (114 mg/kg). The results suggest that the rise in vitamin C may be related to a plant defence mechanism against oxidative stress potentially generated by exposure to cyanotoxins. Although the antioxidant content in spinach plants can be enhanced as a mechanism to cope with oxidative stress, prolonged exposure may increase plants' susceptibility to accumulate cyanotoxins, which may pose a risk to human health. Additional studies are needed to understand all the positive and negative effects of using cyanobacteria biomass as a biofertilizer.