Investigation of Ehrlich ascites tumor cell death mechanisms induced by Synadenium umbellatum Pax.

AbstractEthnopharmacological relevanceSynadenium umbellatum Pax. is widely found in South America and empirically used in Brazil for the treatment of several diseases, mainly cancer. The aim of the study was to investigate cell death mechanisms induced by Synadenium umbellatum Pax. using Ehrlich ascites tumor (EAT) cells, as well as the myelotoxicity potential of this plant.Materials and methodsS. umbellatum cytotoxicity was evaluated in EAT cells by trypan blue exclusion and MTT reduction test and the mechanisms involved in EAT cell death were investigated by light and fluorescence microscopy, flow cytometry and immunocytochemistry. Investigation of S. umbellatum myelotoxicity was performed by clonogenic assay of colony forming unit- granulocyte macrophage (CFU-GM).Results and ConclusionOur results demonstrated that S. umbellatum decreased the viability of EAT cells using both methods. Morphological analyses revealed that S. umbellatum-treatment induced EAT cell death by apoptotic pathway. We demonstrated the occurrence of reactive oxygen species (ROS) overgeneration, increased intracellular Ca2+ concentration, alteration in mitochondrial membrane potential, phosphatydylserine externalization, and activation of caspases 3, 8, and 9. However, S. umbellatum produced myelotoxicity in bone marrow cells in a concentration-dependent manner. In comparison to EAT cells, the effects of S. umbellatum in bone marrow cells were 8-fold lower. Taken together, our results showed that S. umbellatum induced apoptosis in EAT cells at several levels and seems more toxic to tumor cells than to normal bone marrow cells

Aspectos da irrigação do nó atrioventricular e tronco do fascículo atrioventricular em bovinos mestiços Girolando

Aspects of blood supply of the atriumventricular node and trunk of the atriumventricular fascicle, components of the cardiac conducting system were studied. Thirty female hearts of Girolando crossbred bovines, with ages varying between 36 and 48 month-old were utilized. They were dissecated, intervening previous injection of the right and left coronary arteries with coloured solution of Neoprene latex 450, and posterior fixation in aqueous solution of 15.00% formaldehyde, by immersion. The atriumventicular node was irrigated, isolatedly or in association, by the first septal branch of interventricular paraconal branch (3.33%), distal branch of the left atrium (6.66%), septal branch of the right coronary artery (46.66%), proximal branch of the right atrium (76.66%) and by the right ventricular branch. (76.66%). The trunk of atriumventricular fascicle was irrigated, isolatedly or in association, by the first septal branch of interventricular paraconal branch (3.33%), right ventricular branch (60.00%), proximal branch of the right atrium (60.00%) and more often by the septal branch of the right coronary artery (70.00%). Arterial anastomoses were present in 76.66% of the sample, and commonly they formed circuits around the margins of the cardiac conducting system.Estudaram-se aspectos da irrigação do nó atrioventricular e tronco do fascículo atrioventricular, componentes do sistema de condução do coração. Para tanto, utilizaram-se trinta corações de bovinos fêmeas, mestiças Girolando, com idade entre 36 e 48 meses. Os mesmos foram dissecados, após prévia injeção das artérias coronárias direita e esquerda, com solução corada de Neoprene látex 450, seguida pela fixação dos corações em solução aquosa de formol a 15,00%, pelo método de imersão. O nó atrioventricular foi irrigado, isoladamente ou em associação, pelo primeiro ramo septal do ramo interventricular paraconal (3,33%), ramo distal do átrio esquerdo (6,66%), ramo septal da artéria coronária direita (46,66%), ramo proximal do átrio direito (76,66%) e ramo ventricular direito (76,66%). O tronco do fascículo atrioventricular foi irrigado, isoladamente ou em associação, pelo primeiro ramo septal do ramo interventricular paraconal (3,33%), ramo ventricular direito (60,00%), ramo proximal do átrio direito (60,00%), e com maior freqüência pelo ramo septal da artéria coronária direita (70,00%). As anastomoses arteriais estiveram presentes em 76,66% dos casos e geralmente formaram circuitos junto às margens do sistema de condução cardíaco

Caracterização do extrato de arruda e avaliação de seu potencial para o controle da brusone

The objective of this work was to purify and standardize the rue (Ruta graveolens) extract and evaluate its effect on Magnaporthe oryzae as an alternative to the integrated management of rice blast. The drug was characterized, the liquid extract was obtained, and the methodology for quantifying the standard markers psoralen and bergapten was validated. Rue extract and the markers, solely or in combination, were assayed in vitro, as well as in greenhouse conditions, for their ability to suppress leaf blast, by the evaluation of mycelial growth, conidial germination, and appressorium formation. Rue extract inhibited M. oryzae mycelial growth (100%), conidial germination (LD50=0.237 mg), and the appressorium formation (LD50=0.121 mg); besides, the extract reduced leaf blast severity by 80.84%. Fluorescence microscopy showed that rue extract did not damage M. oryzae cell wall and plasma membrane, indicating another mode of action. Rue extract has a great potential for controlling rice leaf blast.O objetivo deste trabalho foi purificar e padronizar o extrato de arruda (Ruta graveolens) e avaliar o seu efeito sobre Magnaporthe oryzae como alternativa para a gestão integrada da brusone. A droga vegetal foi caracterizada, o extrato líquido obtido, e a metodologia para quantificar os marcadores padrão psoraleno e bergapteno validada. O extrato de arruda e os marcadores, isolados ou combinados, foram ensaiados in vitro, assim como em condições de casa de vegetação, para a avaliação de sua capacidade de suprimir a brusone, pela avaliação do crescimento micelial, da germinação de conídios e da formação de apressório. O extrato de arruda inibiu o crescimento micelial de M. oryzae (100%), a germinação de conídios (DL50=0,237 mg) e a formação de apressórios (DL50=0,121 mg); além disso, o extrato reduziu a severidade da brusone em 80,84%. A microscopia de fluorescência mostrou que o extrato de arruda não danificou a parede celular e a membrana plasmática de M. oryzae, o que indica outro modo de ação. O extrato de arruda apresenta grande potencial para o controle da brusone em folhas de arroz

Rationale, study design, and analysis plan of the Alveolar Recruitment for ARDS Trial (ART): Study protocol for a randomized controlled trial

Background: Acute respiratory distress syndrome (ARDS) is associated with high in-hospital mortality. Alveolar recruitment followed by ventilation at optimal titrated PEEP may reduce ventilator-induced lung injury and improve oxygenation in patients with ARDS, but the effects on mortality and other clinical outcomes remain unknown. This article reports the rationale, study design, and analysis plan of the Alveolar Recruitment for ARDS Trial (ART). Methods/Design: ART is a pragmatic, multicenter, randomized (concealed), controlled trial, which aims to determine if maximum stepwise alveolar recruitment associated with PEEP titration is able to increase 28-day survival in patients with ARDS compared to conventional treatment (ARDSNet strategy). We will enroll adult patients with ARDS of less than 72 h duration. The intervention group will receive an alveolar recruitment maneuver, with stepwise increases of PEEP achieving 45 cmH(2)O and peak pressure of 60 cmH2O, followed by ventilation with optimal PEEP titrated according to the static compliance of the respiratory system. In the control group, mechanical ventilation will follow a conventional protocol (ARDSNet). In both groups, we will use controlled volume mode with low tidal volumes (4 to 6 mL/kg of predicted body weight) and targeting plateau pressure <= 30 cmH2O. The primary outcome is 28-day survival, and the secondary outcomes are: length of ICU stay; length of hospital stay; pneumothorax requiring chest tube during first 7 days; barotrauma during first 7 days; mechanical ventilation-free days from days 1 to 28; ICU, in-hospital, and 6-month survival. ART is an event-guided trial planned to last until 520 events (deaths within 28 days) are observed. These events allow detection of a hazard ratio of 0.75, with 90% power and two-tailed type I error of 5%. All analysis will follow the intention-to-treat principle. Discussion: If the ART strategy with maximum recruitment and PEEP titration improves 28-day survival, this will represent a notable advance to the care of ARDS patients. Conversely, if the ART strategy is similar or inferior to the current evidence-based strategy (ARDSNet), this should also change current practice as many institutions routinely employ recruitment maneuvers and set PEEP levels according to some titration method.Hospital do Coracao (HCor) as part of the Program 'Hospitais de Excelencia a Servico do SUS (PROADI-SUS)'Brazilian Ministry of Healt

Systemat ic study of melanogenic act ion of Brosimum gaudichaudi i Trécul

O objetivo do presente trabalho foi avaliar a ação melanogênica das furanocumarinas, psoraleno e bergapteno assim como do extrato seco das raízes de Brosimum gaudichaudii Trécul, contendo 1,2 % m/m de psoraleno, e 5,2 % m/m de bergapteno. A toxidade celular in vitro foi avaliada pelo teste de incorporação do vermelho neutro em quatro linhagens de células diferentes (fibroblastos-L929, queratinócitos-HaCat, Melanoma-B16F10, e melanócitos-Melam-A) e apresentou resposta dose dependente tanto para os fármacos puros quanto para o extrato de B. gaudichaudii. Quando expostas à radiação ultravioleta do tipo A e B houve aumento da toxicidade, proporcional a dose de radiação. A mutagenicidade e genotoxicidade realizas pelos ensaios de micronúcleo e cometa, mostrou que os compostos são genotóxicos e mutagênicos em doses >= 150 ?g.mL-1.A síntese de melanina in vitro realizada em cultura de melanoma B16F10 foi dependente da dose e do tempo de exposição aos fármacos e UV. Nas concentrações máximas usadas (48 ?g.mL-1 de psoraleno, 104 ?g.mL-1 de bergapteno e 0,5 mg.mL-1 de extrato) o psoraleno aumentou a produção de melanina em 26 %, o bergapteno em 69 %, e o extrato de B.gaudichaudii em 163 %. Quando utilizado mistura equivalente 6 ?g.mL-1 de psoraleno e 26 ?g.mL-1 de bergapteno a presente em 0,5 mg.mL-1 de extrato a produção de melanina foi de 61%. A atividade da tirosinase em culturas de B16F10, tratadas com 20 ?g.mL-1 de psoraleno ou bergapteno, quando comparada ao grupo não tratado aumentou em 13% a atividade enzimatica , quando associados foi de 37%, e 0,5 mg.mL-1 de extrato em 54,1%. Com o ensaio de microdiálise in vivo observou-se que os fármacos são rapidamente absorvidos pela pele e distribuídos no plasma. Ambos os composto apresentaram um cinética linear . O estudo de produção de melanina in vivo confimou que a radiação estimula a produçao de melanina assim como o extrato de B.Gaudichaudii, e quando associados (PUVA) a sintese de melanina foi 143% maior em comparação ao controle negativo. Os resultados destre trabalho mostrou a capacidade de pgmentaçao dos fármacos assim como do B.Gaudichaudii, revelando ainda o sinergismo entre psoraleno e bergapteno.The objective of this study was to evaluate the melanogenic action of furanocoumarins, namely, psoralen and bergapten, as extract of Brosimum gaudichaudii Trécul containing 1.2% m / m (psoralen), 5.2% m / m (bergapten) . The cellular toxicity (in vitro) was evaluated by neutral red incorporation test in four different cell lines (fibroblasts, L929, keratinocytes, HaCaT, melanoma, B16F10, and melanocyte- Melam-A) and showed dose dependent response to both extracted compounds, as well as the whole extract of B. gaudichaudii. The ultraviolet radiation type A and B increased the toxicity associated with the compounds and severity of toxicity was proportional to the radiation dose. The mutagenicity and genotoxicity evaluated by the micronucleus test and comet showed that the compounds are genotoxic and mutagenic compounds at concentrations >= 150 ?g/mL. The in vitro melanin synthesis, performed in melanoma B16F10, was dose, duration and UV dependent. In the maximum concentration used (48 ?g/mL psoralen, 104 ?g/mL bergapten and 0.5 mg.mL-1 extract) psoralen increased melanin synthesis by 26%, bergapten by 69% and the B.gaudichaudii extract by 163%. When administered as an equivalent mixture of 6 ?g/mL psoralen and 26 ?g/mL of bergapten to 0.5 mg/mL of extract, melanin synthesis was increased by 61%. The enzymatic activity of tyrosinase in B16F10 cultures treated with 20 ?g/mL of psoralen or bergapten, when compared to non-treated group, increased by 13%; the increases were 37% and 54.1% in the two-compound mixture and 0.5 mg/mL of whole extract. The in vivo microdialese test in rats showed that the drugs are quickly absorbed through the skin and distributed in plasma. Both compound exhibited linear kinetics. The in vivo evaluation also showed that melanin production was stimulated by UV radiation as well as B.Gaudichaudii extract. When combined with PUVA, melanin synthesis was 143% higher compared to the negative contro

Advancing Virtual Bioequivalence for Orally Administered Drug Products: Methodology, Real-World Applications and Future Outlook

Bioequivalence studies are pivotal in generic drug development wherein therapeutic equivalence is provided with an innovator product. However, bioequivalence studies represent significant complexities due to the interplay of multiple factors related to drug, formulation, physiology, and pharmacokinetics. Approaches such as physiologically based biopharmaceutics modeling (PBBM) can enable virtual bioequivalence (VBE) assessment through appropriately developed and validated models. Such models are now being extensively used for bioequivalence risk assessment, internal decision-making, and the evaluation of drug and formulation factors related to bioequivalence. Depiction of the above-mentioned factors through the incorporation of variability and development of a virtual population for bioequivalence assessment is of paramount importance in utilizing such models. In this manuscript, we have portrayed our current understanding of VBE. A detailed explanation was provided with respect to study designs, in vivo variability, and the impact of physiological, drug, and formulation factors on the development of the population for VBE. Furthermore, strategies are suggested to incorporate variability in GastroPlus with an emphasis on intra-subject and inter-occasion variability. Two industrial case studies pertaining to immediate and modified release formulation were portrayed wherein VBE was utilized for decision-making and regulatory justification. Finally, regulatory understanding in the area of VBE, along with future perspectives, was detailed

Isolation and quantitative HPLC-PDA analysis of lupeol in phytopharmaceutical intermediate products from Vernonanthura ferruginea (Less.) H. Rob.

Prior to obtain a standardized dried extract from V. ferruginea, lupeol was first time isolated from leaves and used as chemical maker. An analytical method using HPLC-PDA for lupeol determination in V. ferruginea intermediate products was developed using a C8 reverse-phase column, acetonitrile-acetic acid (99.99:0.01, v/v) as mobile phase at 0.8 mL min-1, oven temperature at 23-25 ºC, sample injection volume at 30 µL and detection at 210 nm. The method presented linearity from 10 to 160 µg mL-1, accuracy, precision, robustness and suitable sensitivity proving to be a useful tool to the obtainment process of lupeol standardized dried extracts of V. ferruginea

El efecto de la división sobre la calidad y la liberación in vitro del clorhidrato de metformina de las tabletas de liberación prolongada (XR)

El clorhidrato de metformina (MetCl) se utiliza en todo el mundo para el tratamiento de la diabetes mellitus tipo 2. Aunque se comercializan tabletas de liberación prolongada (XR) que contienen 500 mg, 850 mg y 1000 mg de MetCl, no es raro dividir la tableta cuando la forma de dosificación de la concentración requerida no está disponible comercialmente. El objetivo del trabajo fue evaluar los efectos de la división en el rendimiento in vitro de tabletas XR que contienen MetCl. Realizamos ensayos de control de calidad y estudios comparativos de perfil de disolución entre varios medicamentos que contienen 500 mg y 1000 mg de MetCl, comercializados en Brasil y Argentina. Las tabletas que contenían 1000 mg de MetCl se dividieron a la mitad (muestras de prueba) y se compararon con tabletas enteras de productos que contenían 500 mg de MetCl (muestras de referencia). Las tabletas partidas a la mitad eran más frágiles y presentaban menor uniformidad de masa y unidad de dosificación en comparación con las enteras. Sin embargo, el parámetro de arranque F2 demostró que la división de las tabletas de MetCl XR no tuvo un impacto significativo en los perfiles de liberación de fármacos in vitro en comparación con las tabletas completas. Además, ni el mecanismo de liberación del fármaco ni la cinética de liberación se vieron afectados significativamente. Las tabletas XR que contienen 1000 mg de clorhidrato de metformina podrían ser elegibles para la división, sin embargo, los fabricantes deber proporcionar surcos apropiados en las superficies de las tabletas para que la división sea adecuada y más segura para los pacientes.O cloridrato de metformina (MetCl) é usado mundialmente no tratamento do diabetes mellitus tipo 2. Embora os comprimidos de liberação prolongada (XR) contendo 500 mg, 850 mg e 1000 mg de MetCl sejam comercializados, não é incomum dividir o comprimido quando a dosagem necessária não está disponível comercialmente. O objetivo do trabalho foi avaliar os efeitos da divisão no desempenho in vitro de comprimidos XR contendo MetCl. Realizamos ensaios de controle de qualidade e estudos de perfil de dissolução comparativos entre diversos medicamentos contendo 500 mg e 1000 mg de MetCl, comercializados no Brasil e na Argentina. Comprimidos contendo 1000 mg de MetCl foram divididos pela metade (amostras de teste) e comparados com comprimidos inteiros de produtos contendo 500 mg de MetCl (amostras de referência). Os comprimidos reduzidos ao meio são mais frágeis e apresentam menor uniformidade de massa e unidade de dosagem em relação aos inteiros. No entanto, foi evidenciado pelo parâmetro de F2 bootstrapping que a divisão dos comprimidos de MetCl XR não afetou significativamente os perfis de liberação do fármaco in vitro em comparação com os comprimidos inteiros. Além disso, nem o mecanismo de liberação do fármaco nem a cinética de liberação foram significativamente afetados. Os comprimidos XR contendo 1000 mg de cloridrato de metformina podem ser elegíveis para divisão, no entanto, os fabricantes devem fornecer sulcos adequadas nas superfícies dos comprimidos para tornar a divisão adequada e mais segura para os pacientes.Metformin hydrochloride (MetCl) is used worldwide in the treatment of type-2 diabetes mellitus. Although extendedrelease (XR) tablets containing 500 mg, 850 mg, and 1000 mg of MetCl are marketed, it is not uncommon to split the tablet when the dosage form of the required strength is not available commercially. The aim of work was to evaluate the effects of splitting on the in vitro performance of XR tablets containing MetCl. We performed quality control assays and comparative dissolution profile studies among several medicines containing 500 mg and 1000 mg of MetCl, marketed in Brazil and Argentina. Tablets containing 1000 mg of MetCl were halved (test samples) and compared with whole tablets of products containing 500 mg of MetCl (reference samples). The halved tablets were more fragile and presented lower uniformity of mass and dosage unit as compared to the whole ones. However, it was evidenced by the F2 bootstrapping parameter that splitting MetCl XR tablets did not significantly impact the in vitro drug release profiles as compared to the whole tablets. In addition, nor the drug release mechanism neither the release kinetics were significantly affected. The XR tablets containing 1000 mg of metformin hydrochloride might be eligible for division, however, manufacturers could provide appropriate scores in the tablets surfaces to make the splitting proper and safer for the patients.Fil: Santos, Luiz Gustavo Fonseca. Universidade Federal de São João del-Rei; BrasilFil: Segall, Adriana Ines. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Tecnología Farmacéutica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Han, Yong Ki. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Tecnología Farmacéutica; ArgentinaFil: Kizelman, Maria Patricia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Tecnología Farmacéutica; ArgentinaFil: Ferreira, Maira Peres. Universidade de Sao Paulo; BrasilFil: Silva, Amanda Cristina Funari. Universidade de Sao Paulo; BrasilFil: Martins, Frederico Severino. Universidade de Sao Paulo; BrasilFil: Castro, Whocely Victor de. Universidade Federal de São João del-Rei; BrasilFil: Freitas, Osvaldo de. Universidade de Sao Paulo; BrasilFil: Couto, Rene Oliveira do. Universidade Federal de São João del-Rei; Brasi