Development and validation of LC-MS/MS methods for the simultaneous quantification of sofosbuvir and its major metabolite (GS-331007) in blood plasma and cerebrospinal and seminal fluid: Application to a pilot clinical trial with a focus on Zika.

Zika still poses a threat to global health owing to its association with serious neurological conditions and the absence of a vaccine and treatment. Sofosbuvir, an anti-hepatitis C drug, has shown anti-Zika effects in animal and cell models. Thus, this study aimed to develop and validate novel LC-MS/MS methods for the quantification of sofosbuvir and its major metabolite (GS-331007) in human plasma and cerebrospinal (CSF) and seminal fluid (SF), and apply the methods to a pilot clinical trial. The samples were prepared by liquid-liquid extraction and separated using isocratic mode on Gemini C18 columns. Analytical detection was performed using a triple quadrupole mass spectrometer equipped with an electrospray ionization source. The validated ranges for sofosbuvir were 0.5-2,000 ng/mL (plasma) and 0.5-100 ng/mL (CSF and SF), while for the metabolite they were 2.0-2,000 ng/mL (plasma), 5.0-200 ng/mL (CSF) and 10-1,500 ng/mL (SF). The intra-day and inter-day accuracies (90.8-113.8%) and precisions (1.4-14.8%) were within the acceptance range. The developed methods fulfilled all validation parameters concerning selectivity, matrix effect, carryover, linearity, dilution integrity, precision, accuracy and stability, confirming the suitability of the method for the analysis of clinical samples.

Protective effect of methyl gallate on murine antigen-induced arthritis by inhibiting inflammatory process and bone erosion.

Methyl gallate (MG) is a plant-derived phenolic compound known to present remarkable anti-inflammatory effect in different experimental models, such as paw oedema, pleurisy, zymosan-induced arthritis and colitis. Herein we investigated the effect of MG in the mice model of antigen-induced arthritis (AIA), a model with complex inflammatory response, driven primally by immune process and that cause bone and cartilage erosion similarly found in rheumatoid arthritis. Arthritis was induced by intra-articular injection of albumin methylated from bovine serum (mBSA) in C57BL/6 male mice previously immunized. The dose-response analysis of MG (0.7-70 mg/kg; p.o) showed that maximum inhibition was reached with the dose of 7 mg/kg on paw oedema and cell infiltration induced by AIA at 7 h. Treatment with MG (7 mg/kg; p.o) or with the positive control, dexamethasone (Dexa, 10 mg/kg, ip) reduced AIA oedema formation, leukocyte infiltration, release of extracellular DNA and cytokine production 7 and 24 h (acute response). Mice treated daily with MG for 7 days showed no significant weight loss or liver and kidney toxicity contrary to dexamethasone that induced some degree of toxicity. Prolonged treatment with MG inhibited the late inflammatory response (28 days) reducing oedema formation, cell infiltration, synovial hyperplasia, pannus formation and cartilage degradation as observed in histopathological analyses. Ultimately, MG reduced bone resorption as evidenced by a decrease in tartrate-resistant acid phosphate (TRAP)-positive cells number in femur histology. Altogether, we demonstrate that MG ameliorates the inflammatory reaction driven primarily by the immune process, suggesting a potential therapeutic application in arthritis treatment.

Anti-Inflammatory Activity and Chemical Analysis of Different Fractions from Solidago chilensis Inflorescence.

Solidago chilensis Meyen (Compositae) is a species native to South America (Brazil) popularly known as arnica. In Brazilian popular medicine, inflorescences and rhizomes of this plant have been used since the end of the 19th century to replace the exogenous and hepatotoxic Arnica montana L. in the treatment of edema and inflammatory pathologies. Although the anti-inflammatory activity of S. chilensis is evidenced in the literature, there is a lack of studies with enriched fractions or compounds isolated from it. The objective of the current study was to characterize phytochemically and to evaluate the pharmacological action in vivo and in vitro of the crude extract and the different fractions (hexane, dichloromethane, acetal, butanolic, and aqueous) isolated from the inflorescence of S. chilensis. The inflorescence crude extract (ScIE) and fractions were administered by intraperitoneal route to mice at different doses. In an LPS-induced pleurisy model, inhibition of leukocyte influx was observed for the ScIE and all fractions tested, as compared to controls. Dichloromethane (ScDicF), butanolic (ScButF), and aqueous (ScAquF) were selected for further analysis as they showed the best inhibitory effects in leukocyte migration and inflammatory cytokine and chemokine production: TNF-α, CXCL1/KC, CXCL2/MIP-2, and CCL11/eotaxin-1. In LPS-stimulated J774A.1 cell line, ScIE and the ScDicF exhibited an inhibitory effect on nitric oxide (NO) production and downmodulated the COX-2 expression; ScAquF failed to modulate NO production and COX-2 expression. In phytochemical analysis, HPLC-UV-DAD chromatograms of ScDicF and ScAquF showed the main peaks with UV spectrum characteristics of flavonoids; chlorogenic acid and isoquercetin were the most present phytochemicals identified in the ScAquF, and a high number of n-alkanes was found in ScHexF. Our study was the first to address biological effects and correlate them to phytochemically characterized fractions from inflorescences of S. chilensis.

Pharmacological profiling of JME-173, a novel mexiletine derivative combining dual anti-inflammatory/anti-spasmodic functions and limited action in Na+ channels.

Existing evidence suggests that the local anaesthetic mexiletine can be beneficial for patients with asthma. However, caution is required since anaesthesia of the airways inhibits protective bronchodilator neuronal reflexes, limiting applications in conditions of hyperirritable airways. Here, we describe the synthesis of a new series of mexiletine analogues, which were screened for reduced activity in Na+ channels and improved smooth muscle relaxant effects, that were evaluated using the patch-clamp technique and an isolated tracheal organ bath, respectively. JME-173 (1-(4-bromo-3,5-dimethylphenoxy)propan-2-amine) was the most effective among the four mexiletine analogues investigated. JME-173 was then studied in vivo using a murine model of lung inflammation induced by cigarette smoke (CS) and in vitro using neutrophil chemotaxis and mast cell degranulation assays. Finally, the JME-173 pharmacokinetic profile was assessed using HPLC-MS/MS bioanalytical method. JME-173 directly inhibited IL-8 (CXCL8)- and FMLP-induced human neutrophil chemotaxis and allergen-induced mast cell degranulation. After oral administration 1 h before CS exposure, JME-173 (50 mg/kg) strongly reduced the increased number of macrophages and neutrophils recovered in the bronchoalveolar effluent without altering lymphocyte counts. Pharmacokinetic experiments of JME-173 (10 mg/kg, orally) showed values of maximum concentration (Cmax), maximum time (Tmax), area under the blood concentration-time curve (AUC0-t) and area under the blood concentration-time curve from 0-Inf (AUC0-inf) of 163.3 ± 38.3 ng/mL, 1.2 ± 0.3 h, 729.4 ± 118.3 ng*h/ml and 868.9 ± 117.1 ng*h/ml (means ± S.E.M.), respectively. Collectively, these findings suggest that JME-173 has the potential to be an effective oral treatment for diseases associated with bronchoconstriction and inflammation.

Extract of Solidago chilensis Meyen inflorescences: cytotoxicity and inhibitory activity on nitric oxide synthesis in activated macrophage cell line J774A. 1

Solidago chilensis Meyen (= Solidago microglossa) popularly known as "Brazilian arnica" is used to treat of inflammatory disorders. S. chilensis is constant in the Therapeutic Memento of the Rio de Janeiro city and belongs to the medicinal species of Brazilian National List of Medicinal Plants of Interest of the Unified National Health System (SUS). There are no studies in the literature showing the direct activity of this plant species on immune system cells. The present study evaluated the chemical composition as well as the cytotoxic and pharmacological activity of the ether-ethanol extract from S. chilensis inflorescences (SCIE) in murine macrophage cell line J774A.1. The results showed that higher concentrations (50 to 200 µg/mL) of SCIE had significant cytotoxicity on J774A.1 cells, however, lower concentrations (from 10 to 0.1 µg/mL) did not produce significant cytotoxic effects and exhibited an inhibitory effect on nitric oxide production in LPS-stimulated J774A.1 cell line. The chemical analysis by HPLC-UV-PDA indicated that the SCIE contains flavonoid derived from quercetin and kaempferol; and diterpenes, probably labdanes. These findings complement data in the literature regarding the activity of this plant species on an important cell from the immune system involved in the innate and acquired immune response, the macrophages.

Protective effect of Myrsine parvifolia plant extract against the inflammatory process induced by Bothrops jararaca snake venom.

Accidents involving snakes from the genus Bothrops sp. constitute the most important cause of snake envenomation in Brazil. The Myrsine genus has been reported to be used in folk medicine against snakebites. In this work, the phytochemical profiles and ability of extracts from Myrsine parvifolia leaves to reduce the inflammatory process (edema, vascular permeability increase and leukocyte migration) induced by Bothrops jararaca venom were investigated in vivo. Chemical compounds were identified by chromatographic and spectroscopy techniques. Total polyphenol, tannin, and flavonoid contents were determined by spectrophotometric methods. Swiss male mice received an oral administration of extracts (100 mg/kg) in different protocols. Paw edema, intraperitoneal vascular permeability and pleurisy models in mice were used to evaluate the antiophidic potential of the extracts. Paw edema was induced by subplantar injection of B. jararaca venom and quantified as the increase in paw volume. Changes in vascular permeability were assessed by measuring the amount of Evans blue dye extravasation. Leukocyte migration was assessed by total and differential counts in the pleural cavity washes. Myricetin, myricetin-3-O-ß-arabinopyranoside, quercetin and kaempferol were isolated from the ethyl acetate extract and identified as the primary compounds of the dichloromethane extract. Terpenes and fatty acids were identified in the hexane and dichloromethane extracts. The pretreated group with hydroethanolic and dichloromethane extract reduced total edema (40 and 52%, respectively), vascular permeability increase (32.4 and 32.2%, respectively) and leukocyte influx into the pleural cavity (42 and 39%, respectively), while the group treated with hexane extract showed only reduced edema (37%) induced by B. jararaca venom. The hydroethanolic extract showed better results in all of the tests performed and was also administered by the protocol of post-poisoning, showing maintenance of paw edema reduction and cell migration. These data indicate a potential anti-inflammatory activity of M. parvifolia in poisoning by B. jararaca, especially to reduce local poison effects.

Exposure to the UV Filter Octyl Methoxy Cinnamate in the Postnatal Period Induces Thyroid Dysregulation and Perturbs the Immune System of Mice.

Evidence demonstrates the bidirectional communication and regulation between the neuroendocrine and immune systems. Thyroid hormones play key roles in nervous system development and can exert influence on various immune cells contributing to pathophysiological conditions. Octyl methoxycinnamate (OMC) is one of the most commonly used UV filters, and in vitro and in vivo studies have found thyroid disrupting effects. The present study assessed whether OMC administration in mice dams during the lactational period can cause thyroid disruption and generate immunologic alterations in the offspring. Indirect exposure to the OMC (1,000 mg/kg) in the lactational period affected neurodevelopment parameters, such as delayed eye-opening and weight gain in mice of both sexes, and these alterations are corroborated by the decrease in the T4 levels present in the pups' blood. No significant changes were observed in the thymus of these pups, but the number of lymphocytes increased in the spleen of the animals exposed to OMC, similar to the animals treated with propyl-thiouracil (PTU), a well-known thyroid disruptor. OMC modulated the percentage of leukocyte populations in peripheral blood, and the number of circulating polymorphonuclear cells increased two-fold. In vitro, OMC exhibited an inhibitory effect on splenocyte proliferation and IL-2 production induced by anti-CD3 antibody; however, this effect was reversed with the addition of T4 in the cell culture. In summary, the results of the present study demonstrate the influence of OMC on thyroid dysregulation and its impact on the modulation of the immune system in mice pups.

Ensaio do linfonodo local murino (LLNA) na diferenciação entre a dermatite de contato alérgica e de contato irritante: um estudo da expressão de marcadores de superfície de linfócitos T / Local Lymph node assay in the differentiation between an allergic contact dermatitis and irritation: study of the expression of T lymphocyte surface markers

Introdução: O ensaio do linfonodo local murino (LLNA) foi desenvolvido como uma alternativa aos testes de Buhler e Maximização. O teste é utilizado com o objetivo de identificar substâncias capazes de induzir dermatite de contato e tem como desfecho a quantificação celular nos linfonodos auriculares. Embora recomendado por agências internacionais envolvidas no desenvolvimento de metodologias alternativas, o LLNA ainda necessita de aprimoramento. Objetivo: O objetivo do trabalho foi estudar possíveis diferenças nos padrões de subpopulações linfocitárias entre camundongos tratados com substâncias irritantes e dermosensibilizantes. Método: Os animais foram tratados com os sensibilizantes dinitroclorobenzeno (DNCB) e parafenilenidiamina (PPD), os irritantes lauril sulfato de sódio (LSS) e tritonX-100 (TX-100), por três dias consecutivos no dorso de ambas as orelhas. As subpopulações foram analisadas por citometria de fluxo e possíveis alterações histopatológicas nas orelhas dos animais foram também analisadas. Resultados: Foram observadas diferenças nas células CD4+CD25+ e CD4+CD69+, assim como na proliferação dessas subpopulações. Nenhuma diferença foi vista nos estudos histopatológicos das orelhas dos animais quando tratados com dermosensibilizantes ou irritantes. Conclusões: A fenotipagem de linfócitos T pode ser considerada útil no desenvolvimento de possíveis protocolos de ensaios que visem a diferenciação entre substâncias dermosensibilizantes e irritantes. Além disso, os resultados obtidos podem vir a contribuir com o aumento do conhecimento nesta área e auxiliar na busca por um ensaio in vitro correlato.

Introduction: The Local Lymph Node Assay (LLNA) was developed as an alternative to Buhler and Maximization assays. It is applied to discriminate substances that are able to induce contact dermatitis and the endpoint is cell quantification in mice auricular lymph nodes. Although recommended by international agencies involved in the development of alternative methodologies, LLNA still needs to be improved. Objective: In this context, the goal of this study was to investigate possible differences in lymphocyte subpopulation patterns among mice treated with irritants and dermosensitizers. Method: Animals were treated with sensitizers dinitrochlorobenzene (DNCB) and paraphenylenediamine (PPD) and irritants sodium lauryl sulfate (SLS) and tritonX-100 (TX-100) for 3 days, using dorsum area of both ears. The percentage of different lymphocyte subpopulations were analyzed by flow cytometry. Ears of animals were also evaluated for possible pathological alterations. Results: Differences were observed in CD4+ CD25+ and CD4+ CD69+ cells, as well as in the proliferation of these subpopulations. The histopathological analysis of the ears showed no difference between the treatment with either dermosensitizers or irritants. Conclusions: T lymphocyte phenotyping might still be a useful tool in the development of an assay to differentiate between dermosensitizers and irritants. Moreover, these results may contribute to improving knowledge on this field and helping in the search of a correlate in vitro assay.

Development of a HPLC/MS/MS methodology for determining 3-O-methyldopa in human plasma and its application in a bioequivalence study

A simple, sensitive and specific HPLC/MS/MS methodology was developed and it was validated for determining 3-O-methyldopa, the major metabolite of dopamine, in human plasma. The separation was achieved on Atlantis T3 C18 analytical column (5 μm; 150 x 4.6 mm i.d.) using a mobile phase consisted of a solution of water and methanol (85:15, v/v) and containing formic acid 0.05 %. The extraction from the analyte and the internal standard sample was performed using a simple protein plasma precipitation with perchloric acid. The detection was conducted on a triple quadrupole tandem mass spectrometer with a positive multiple reaction monitoring mode (MRM). The monitored fragmentation transitions were m/z212.0  m/z 166.0 for 3-O-methyldopa and m/z 227.10  m/z 181.0 for carbidopa (internal standard).The calibration curves were linear in the range of 50–4000 ng/mL for 3-O-methyldopa. The methodology presented a good precision and accuracy in accordance to the criteria for biomedical analysis. And it was successfully applied to the bioequivalence study of two formulations levodopa + benserazide (200 + 50mg) in plasma samples from healthy human volunteers, following the ANVISA guidelines...

Analgesic and anti-inflammatory activity of the aqueous extract of Rheedia longifolia Planch & Triana.

Rheedia longifolia Planch et Triana belongs to the Clusiaceae family. This plant is widely distributed in Brazil, but its chemical and pharmacological properties have not yet been studied. We report here that leaves aqueous extract of R. longifolia (LAE) shows analgesic and anti-inflammatory effects. Oral or intraperitoneal administration of this extract dose-dependently inhibited the abdominal constrictions induced by acetic acid in mice. The analgesic effect and the duration of action were similar to those observed with sodium diclofenac, a classical non-steroidal analgesic. In addition to the effect seen in the abdominal constriction model, LAE was also able to inhibit the hyperalgesia induced by lipopolysaccharide from gram-negative bacteria (LPS) in rats. We also found that R. longifolia LAE inhibited an inflammatory reaction induced by LPS in the pleural cavity of mice. Acute toxicity was evaluated in mice treated with the extract for seven days with 50 mg/kg/day. Neither death, nor alterations in weight, blood leukocyte counts or hematocrit were noted. Our results suggest that aqueous extract from R. longifolia leaves has analgesic and anti-inflammatory activity with minimal toxicity and are therefore endowed with a potential for pharmacological control of pain and inflammation.

Analgesic and anti-inflammatory activity of the aqueous extract of Rheedia longifolia Planch & Triana

Rheedia longifolia Planch et Triana belongs to the Clusiaceae family. This plant is widely distributed in Brazil, but its chemical and pharmacological properties have not yet been studied. We report here that leaves aqueous extract of R. longifolia (LAE) shows analgesic and anti-inflammatory effects. Oral or intraperitoneal administration of this extract dose-dependently inhibited the abdominal constrictions induced by acetic acid in mice. The analgesic effect and the duration of action were similar to those observed with sodium diclofenac, a classical non-steroidal analgesic. In addition to the effect seen in the abdominal constriction model, LAE was also able to inhibit the hyperalgesia induced by lipopolysaccharide from gram-negative bacteria (LPS) in rats. We also found that R. longifolia LAE inhibited an inflammatory reaction induced by LPS in the pleural cavity of mice. Acute toxicity was evaluated in mice treated with the extract for seven days with 50 mg/kg/day. Neither death, nor alterations in weight, blood leukocyte counts or hematocrit were noted. Our results suggest that aqueous extract from R. longifolia leaves has analgesic and anti-inflammatory activity with minimal toxicity and are therefore endowed with a potential for pharmacological control of pain and inflammation.

Anti-inflammatory activity in the aqueous crude extract of the leaves of Nidularium procerum: A bromeliaceae from the Brazilian coastal rain forest.

Nidularium procerum LINDMAN, a common bromeliaceae from the Brazilian flora, remains poorly studied regarding its chemical and pharmacological properties. We have recently published that N. procerum has potent analgesic and anti-inflammatory activities. In the present work, we have investigated potential mechanisms involved in the anti-inflammatory effects of N. procerum aqueous extract on lipopolysaccharide (LPS)-, platelet activating factor (PAF)- or formyl-methionyl-leucyl-phenylalanine (fMLP)-induced pleurisy models of inflammation. We found that the aqueous extract of N. procerum leaves (leaf aqueous extract; LAE) inhibits the neutrophil migration, production of inflammatory cytokines interleukin-1 and -6 (IL-1 and IL-6) and the generation of prostaglandin E2 (PGE2) in LPS-induced pleural inflammation in mice. Such inhibitory effect of N. procerum on PGE2 generation was tightly correlated to the inhibition of formation of new cytoplasmic lipid bodies within recruited leukocytes. N. procerum also blocked the in vivo neutrophil influx induced by injection of PAF or fMLP into the mouse pleural cavity and directly inhibited PAF-induced neutrophil chemotaxis in vitro. The data obtained in this study indicate that N. procerum LAE exerts its anti-inflammatory effects by interfering with the capacity of the host to respond to injury at different levels. Among the different functions affected by N. procerum LAE, lipid body formation, PGE2 and cytokine production and neutrophil chemotaxis are readily evidenced in relevant surrogate models. The N. procerum bioactive profile makes it an attractive candidate for future development as a drug or phytomedicine.