RESUMO
Inhalation as a route for administering drugs and dietary supplements has garnered significant attention over the past decade. We performed real-time analyses of aerosols using secondary electrospray ionization (SESI) technology interfaced with high-resolution mass spectrometry (HRMS), primarily developed for exhaled breath analysis with the goal to detect the main aerosol constituents. Several commercially available inhalation devices containing caffeine, melatonin, cannabidiol, and vitamin B12 were tested. Chemical characterization of the aerosols produced by these devices enabled detection of the main constituents and screening for potential contaminants, byproducts, and impurities in the aerosol. In addition, a programmable syringe pump was connected to the SESI-HRMS system to monitor aerosolized active pharmaceutical ingredients (APIs) such as chloroquine, hydroxychloroquine, and azithromycin. This setup allowed us to detect caffeine, melatonin, hydroxychloroquine, chloroquine, and cannabidiol in the produced aerosols. Azithromycin and vitamin B12 in the aerosols could not be detected; however, our instrument setup enabled the detection of vitamin B12 breakdown products that were generated during the aerosolization process. Positive control was realized by liquid chromatography-HRMS analyses. The compounds detected in the aerosol were confirmed by exact mass measurements of the protonated and/or deprotonated species, as well as their respective collision-induced dissociation tandem mass spectra. These results reveal the potential wide application of this technology for the real-time monitoring of aerosolized active pharmaceutical ingredients that can be administered through the inhalation route.
Assuntos
Canabidiol , Melatonina , Espectrometria de Massas por Ionização por Electrospray/métodos , Cafeína , Azitromicina , Hidroxicloroquina , Aerossóis/análise , Vitamina B 12RESUMO
In vitro screening for pharmacological activity of existing drugs showed chloroquine and hydroxychloroquine to be effective against severe acute respiratory syndrome coronavirus 2. Oral administration of these compounds to obtain desired pulmonary exposures resulted in dose-limiting systemic toxicity in humans. However, pulmonary drug delivery enables direct and rapid administration to obtain higher local tissue concentrations in target tissue. In this work, inhalable formulations for thermal aerosolization of chloroquine and hydroxychloroquine were developed, and their physicochemical properties were characterized. Thermal aerosolization of 40 mg/mL chloroquine and 100 mg/mL hydroxychloroquine formulations delivered respirable aerosol particle sizes with 0.15 and 0.33 mg per 55 mL puff, respectively. In vitro toxicity was evaluated by exposing primary human bronchial epithelial cells to aerosol generated from Vitrocell. An in vitro exposure to 7.24 µg of chloroquine or 7.99 µg hydroxychloroquine showed no significant changes in cilia beating, transepithelial electrical resistance, and cell viability. The pharmacokinetics of inhaled aerosols was predicted by developing a physiologically based pharmacokinetic model that included a detailed species-specific respiratory tract physiology and lysosomal trapping. Based on the model predictions, inhaling emitted doses comprising 1.5 mg of chloroquine or 3.3 mg hydroxychloroquine three times a day may yield therapeutically effective concentrations in the lung. Inhalation of higher doses further increased effective concentrations in the lung while maintaining lower systemic concentrations. Given the theoretically favorable risk/benefit ratio, the clinical significance for pulmonary delivery of aerosolized chloroquine and hydroxychloroquine to treat COVID-19 needs to be established in rigorous safety and efficacy studies. Graphical abstract.
Assuntos
Antimaláricos/administração & dosagem , Tratamento Farmacológico da COVID-19 , Cloroquina/administração & dosagem , Hidroxicloroquina/administração & dosagem , Modelos Químicos , Administração por Inalação , Animais , Antimaláricos/farmacocinética , Antimaláricos/toxicidade , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Humanos , Hidroxicloroquina/farmacocinética , Hidroxicloroquina/toxicidade , Masculino , Camundongos , Pessoa de Meia-Idade , RatosRESUMO
Alkaloids are a structurally complex group of natural products that have a diverse range of biological activities and significant therapeutic applications. In this study, we examined the acute, anxiolytic-like effects of nicotinic acetylcholine receptor (nAChR)-activating alkaloids with reported neuropharmacological effects but whose effects on anxiety are less well understood. Because α4ß2 nAChRs can regulate anxiety, we first demonstrated the functional activities of alkaloids on these receptors in vitro. Their effects on anxiety-like behavior in zebrafish were then examined using the zebrafish novel tank test (NTT). The NTT is a relatively high-throughput behavioral paradigm that takes advantage of the natural tendency of fish to dive down when stressed or anxious. We report for the first time that cotinine, anatabine, and methylanatabine may suppress this anxiety-driven zebrafish behavior after a single 20-min treatment. Effective concentrations of these alkaloids were well above the concentrations naturally found in plants and the concentrations needed to induce anxiolytic-like effect by nicotine. These alkaloids showed good receptor interactions at the α4ß2 nAChR agonist site as demonstrated by in vitro binding and in silico docking model, although somewhat weaker than that for nicotine. Minimal or no significant effect of other compounds may have been due to low bioavailability of these compounds in the brain, which is supported by the in silico prediction of blood-brain barrier permeability. Taken together, our findings indicate that nicotine, although not risk-free, is the most potent anxiolytic-like alkaloid tested in this study, and other natural alkaloids may regulate anxiety as well.
Assuntos
Alcaloides , Receptores Nicotínicos , Alcaloides/farmacologia , Animais , Ansiedade/tratamento farmacológico , Nicotina , Peixe-ZebraRESUMO
Mitochondria are among the first responders to various stress factors that challenge cell and tissue homeostasis. Various plant alkaloids have been investigated for their capacity to modulate mitochondrial activities. In this study, we used isolated mitochondria from mouse brain and liver tissues to assess nicotine, anatabine and anabasine, three alkaloids found in tobacco plant, for potential modulatory activity on mitochondrial bioenergetics parameters. All alkaloids decreased basal oxygen consumption of mouse brain mitochondria in a dose-dependent manner without any effect on the ADP-stimulated respiration. None of the alkaloids, at 1 nM or 1.25 µM concentrations, influenced the maximal rate of swelling of brain mitochondria. In contrast to brain mitochondria, 1.25 µM anatabine, anabasine and nicotine increased maximal rate of swelling of liver mitochondria suggesting a toxic effect. Only at 1 mM concentration, anatabine slowed down the maximal rate of Ca2+-induced swelling and increased the time needed to reach the maximal rate of swelling. The observed mitochondrial bioenergetic effects are probably mediated through a pathway independent of nicotinic acetylcholine receptors, as quantitative proteomic analysis could not confirm their expression in pure mitochondrial fractions isolated from mouse brain tissue.
Assuntos
Alcaloides/toxicidade , Mitocôndrias/efeitos dos fármacos , Plantas/química , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Metabolismo Energético/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitocôndrias/metabolismo , Proteômica , Receptores Nicotínicos/metabolismoRESUMO
Within the traditional pharmacopeia, tobacco (Nicotiana spp.) is often cited as an efficient pesticide. This activity is generally attributed to nicotine, but tobacco plants contain other alkaloids that could potentially contribute to this effect. In this study, we tested methanolic extracts of N. glutinosa, N. glauca, N. debneyi, and N. tabacum (putrescine N-methyltransferase line, burley TN90 and Stella, Virginia ITB 683 and K326), selected according to alkaloid content. Their antiparasitic activity was evaluated in bioassays against adult fleas (Ctenocephalides felis), blowfly (Lucilia cuprina) larvae, nematodes (Caenorhabditis elegans), and ticks (Rhipicephalus sanguineus larvae and adults, Ixodes ricinus nymphs). None of the extracts killed fleas and blowfly larvae effectively at the concentrations tested. Only N. tabacum K326 and N. glutinosa exhibited moderate anthelmintic activity. All extracts significantly repelled R. sanguineus ticks, but not I. ricinus, and the nicotine-rich extracts rapidly knocked down all tick species and stages at high concentrations. The link between nicotine and tick knockdown was confirmed by successfully testing the pure alkaloid at concentrations found in the tobacco extracts. In contrast, repellent activity could not be correlated to the individually tested alkaloids (nicotine, nornicotine, anabasine, anatabine), although anatabine and nornicotine were active in the tick bioassay at high concentrations.
Assuntos
Antiparasitários/farmacologia , Nicotiana/química , Extratos Vegetais/farmacologia , Folhas de Planta/química , Animais , Bioensaio , Feminino , Insetos , Nematoides , Carrapatos , Nicotiana/classificaçãoRESUMO
Swedish snus is a smokeless tobacco product that contains reduced levels of harmful compounds compared with cigarette smoke. In Sweden, where snus use exceeds smoking among men, relatively low rates of major smoking-related diseases have been recorded. To better understand how snus use could align with current tobacco harm reduction strategies, its potential mechanisms of toxicity must be investigated. This study aimed to determine, via a systems toxicology approach, the biological impact of repeated 72-hour exposure of human gingival epithelial organotypic cultures to extracts from both a commercial and a reference snus and the total particulate matter (TPM) from cigarette smoke. At concentrations relevant for human use, cultures treated with snus extracts induced mild, generally reversible biological changes, while TPM treatment induced substantial morphological and inflammatory alterations. Network enrichment analysis and integrative analysis of the global mRNA and miRNA expression profiles indicated a limited and mostly transient impact of the snus extracts, in particular on xenobiotic metabolism, while the effects of TPM were marked and sustained over time. High-confidence miRNAs that might be related to pathological conditions in vivo were identified. This study highlights the limited biological impact of Swedish snus extract on human organotypic gingival cultures.
Assuntos
Gengiva/efeitos dos fármacos , Material Particulado/análise , Extratos Vegetais/efeitos adversos , Tabaco sem Fumaça/efeitos adversos , Células Cultivadas , Gengiva/patologia , Humanos , Inflamação/genética , Inflamação/metabolismo , Mediadores da Inflamação/metabolismo , Masculino , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Nicotina/análise , Extratos Vegetais/análise , Extratos Vegetais/química , Suécia , Fatores de Tempo , Tabaco sem Fumaça/análise , Transcriptoma/efeitos dos fármacosRESUMO
Cigarette smoking causes cardiovascular diseases. Heating tobacco instead of burning it reduces the amount of toxic compounds in the aerosol and may exert a reduced impact on health compared with cigarette smoke. Aqueous extract from the aerosol of a potential modified risk tobacco product, the Carbon Heated Tobacco Product (CHTP) 1.2, was compared in vitro with aqueous extract from the smoke of a 3R4F reference cigarette for its impact on the adhesion of monocytic cells to artery endothelial cells. Human coronary artery endothelial cells (HCAEC) were treated for 4â¯h with conditioned media from human monocytic Mono Mac 6 (MM6) cells exposed to CHTP1.2 or 3R4F extracts for 2â¯h or directly with those extracts freshly generated. In vitro monocyte-endothelial cell adhesion was measured concomitantly with inflammatory, oxidative stress, cytotoxicity, and death markers. Furthermore, transcriptomics analyses enabled to quantify the level of perturbation in HCAECs, and provide biological interpretation for the underlying molecular changes following exposure to 3R4F or CHTP1.2 extract. Our systems toxicology study demonstrated that approximately 10-15-fold higher concentrations of the CHTP 1.2 aerosol extract were needed to elicit similar effects as the 3R4F smoke extract on cardiovascular disease-relevant inflammation and cytotoxicity-related mechanisms and markers investigated in vitro.
Assuntos
Adesão Celular/efeitos dos fármacos , Vasos Coronários/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Nicotiana/química , Extratos Vegetais/toxicidade , Vasculite/induzido quimicamente , Células Cultivadas , Vasos Coronários/citologia , Endotélio Vascular/citologia , Humanos , Monócitos/citologia , Fumaça/efeitos adversos , Testes de ToxicidadeRESUMO
The allotetraploid plant Nicotiana tabacum (common tobacco) is a major crop species and a model organism, for which only very fragmented genomic sequences are currently available. Here we report high-quality draft genomes for three main tobacco varieties. These genomes show both the low divergence of tobacco from its ancestors and microsynteny with other Solanaceae species. We identify over 90,000 gene models and determine the ancestral origin of tobacco mosaic virus and potyvirus disease resistance in tobacco. We anticipate that the draft genomes will strengthen the use of N. tabacum as a versatile model organism for functional genomics and biotechnology applications.
Assuntos
Resistência à Doença/genética , Nicotiana/genética , Doenças das Plantas/imunologia , Solanum lycopersicum/genética , Solanum tuberosum/genética , Sequência de Bases , DNA de Plantas/genética , Resistência à Doença/imunologia , Perfilação da Expressão Gênica , Ligação Genética , Genoma de Planta , Doenças das Plantas/virologia , Folhas de Planta/genética , Raízes de Plantas/genética , Potyvirus/patogenicidade , Alinhamento de Sequência , Análise de Sequência de DNA , Nicotiana/classificação , Vírus do Mosaico do Tabaco/patogenicidadeRESUMO
Recent advances in the 'omics' technologies, scientific computing and mathematical modeling of biological processes have started to fundamentally impact the way we approach drug discovery. Recent years have witnessed the development of genome-scale functional screens, large collections of reagents, protein microarrays, databases and algorithms for data and text mining. Taken together, they enable the unprecedented descriptions of complex biological systems, which are testable by mathematical modeling and simulation. While the methods and tools are advancing, it is their iterative and combinatorial application that defines the systems biology approach.