Estudo de síntese e estabilidades química e enzimática de fármaco dirigido dendrimérico potencialmente ativo em doença de Chagas / Synthesis, chemical and enzymatic stability studies of targeted dendrimer potentially active in Chagas disease

A doença de Chagas representa um problema de saúde pública em muitos países e regiões. O tratamento consiste em fármacos tóxicos, com eficácia discutível, principalmente, na fase crônica da doença. Assim, faz-se necessário o planejamento de novos quimioterápicos, mais seguros e eficazes. Os dendrímeros são novas arquiteturas moleculares formadas por um foco central e ramificações partindo desse foco. Apresentam diversas aplicações biológicas como, por exemplo, atuar como transportadores de fármacos. Face ao exposto, o objetivo deste trabalho foi o estudo de condições para ligar o ácido anacárdico (AA) em derivado dendrimérico com potencial ação na doença de Chagas, o qual tem como foco central o ácido succínico (AS) e ramificações compostas por arginina (Arg) e lisina (Lys). Sabe-se que a cruzaína, uma cisteíno-protease do T. cruzi, catalisa a hidrólise de ligação peptídica entre lisina e arginina. A síntese dos compostos em fase sólida forneceu os derivados brutos: (1) pró-fármaco AA-K-R-NH2 e (2) G.05 AA-K(AS)-R-NH2, que foram purificados e caracterizados por Cromatografia Líquida de Alta Eficiência e espectrometria de massas. Os compostos purificados AA-K-R-NH2 e AA-K(AS)-R-NH2 apresentaram rendimentos de 34% e 47%, com pureza de 88% e 98%, respectivamente. Os resultados dos experimentos enzimáticos utilizando o AA-K-R-NH2 não foram conclusivos. Acredita-se que a baixa solubilidade e/ou baixa concentração podem ter contribuído para tal. Já na estabilidade química em pH 7,4 (que simula pH sanguíneo), pH 1,2 (que simula pH estomacal) e pH 8,5 (que simula pH intestinal), observou-se que o AA-K(AS)-R-NH2 foi estável durante as 24 h de ensaio. Estes últimos resultados são interessantes, pois espera-se que o pró-fármaco dendrimérico alcance o T. cruzi estruturalmente integro, sofrendo hidrólise e liberação do composto ativo no interior do parasita

Chagas disease is a public health problem in many countries and regions. The treatment consists of toxic drugs, with debatable efficacy, mainly, in the chronic phase of the disease. Thus, it is necessary to plan new chemotherapeutics, safer and more effective than those drugs. Dendrimers are new molecular architectures composed by a central focus and branching from that focus. They present several biological applications, such as acting as drug carriers. Thereby, the goal of this work was the study of conditions to bind anacardic acid (AA) in a dendrimeric derivative with potential action in Chagas disease, which was composed by a central focus of succinic acid (AS) and branches of arginine (Arg) and lysine (Lys). Cruzain, a T. cruzi cysteine protease, is known to catalyze the peptide-binding hydrolysis between lysine and arginine. Synthesis of the solid phase compounds provided the crude derivatives: (1) prodrug AA-KR-NH2 and (2) G.05 AA-K(AS)-R-NH2, which were purified and characterized by High Performance Liquid Chromatography (HPLC) and mass spectrometry. The purified AA-K-R-NH2 and AA-K(AS)-R-NH2 compounds showed yields of 34% and 47%, with purity of 88% and 98% respectively. The results of the enzymatic experiments using AA-K-R-NH2 were not conclusive. It is believed that the low solubility and/or low concentration may have contributed for this. On the chemical stability at pH 7.4 (which simulates blood pH), pH 1.2 (which simulates stomach pH) and pH 8.5 (which simulates intestinal pH), it was observed that AA-K(AS)R-NH2 was stable for 24 hours. These latter results are interesting because the dendrimeric prodrug is expected to reach structurally integral T. cruzi, undergoing hydrolysis and release of the active compound within the parasite

Kinetic investigations and stability studies of two Bothrops L-amino acid oxidases

L-amino acid oxidases isolated from snake venoms (SV-LAAOs) are enzymes that have great therapeutic potential and are currently being investigated as tools for developing new strategies to treat various diseases, including cancer and bacterial infections. The main objective of this study was to make a brief evaluation of the enzymatic stability of two Bothrops LAAOs, one isolated from Bothrops jararacussu (BjussuLAAO-II) and the other from Bothrops moojeni (BmooLAAO-I) venoms. Methods and results: The enzymatic activity and stability of both LAAOs were evaluated by microplate colorimetric assays, for which BjussuLAAO-II and BmooLAAO-I were incubated with different L-amino acid substrates, in the presence of different ions, and at different pH ranges and temperatures. BjussuLAAO-II and BmooLAAO-I demonstrated higher affinity for hydrophobic amino acids, such as Phe and Leu. The two enzymes showed high enzymatic activity in a wide temperature range, from 25 to 75 °C, and presented optimum pH around 7.0. Additionally, Zn2+, Al3+, Cu2+ and Ni2+ ions negatively modulated the enzymatic activity of both LAAOs. As to stability, BjussuLAAO-II and BmooLAAO-I showed high enzymatic activity for 42 days stored at 4°C in neutral pH solution. Moreover, the glycan portions of both LAAOs were analyzed by capillary electrophoresis, which revealed that BjussuLAAO-II presented two main glycan portions with relative masses of 7.78 and 8.13 CGU, while BmooLAAO-I showed three portions of 7.58, 7.94 and 8.37 CGU. Conclusions: Our results showed that, when stored properly, BjussuLAAO-II and BmooLAAO-I present enzymatic stability over a long time period, which is very important to allow the use of these enzymes in pharmacological studies of great impact in the medical field.(AU)

Estabilidad de un suero control multienzimático utilizado como material de referencia en los laboratorios clínicos / Stability of a multienzyme control serum used as reference material in clinical laboratories

Una de las vías fundamentales para garantizar la calidad de los ensayos realizados en los laboratorios clínicos es mediante el uso de materiales de referencia. Una problemática a la que nos enfrentamos es la escasez de estos productos en el mercado nacional dado su alto costo. Objetivo: evaluar la estabilidad de un suero bovino adulto enriquecido con las enzimas alanina aminotransferasa (ALAT/TGP), aspartato aminotransferasa (ASAT/TGP), fosfatasa alcalina (FA) y amilasa. Métodos: se evaluó la estabilidad a tiempo real de la matriz enriquecida con las diferentes enzimas durante 12 meses a 2 temperaturas (refrigeración y congelación). Se evaluó el efecto del glicerol sobre la actividad enzimática de los extractos, así como el efecto de los preservantes propilenglicol y etilenglicol en la estabilidad de las enzimas. Resultados: los extractos enzimáticos obtenidos comenzaron a perder la actividad biológica a partir de los 15 días, independientemente de la temperatura de almacenamiento y de la presencia o no de glicerol. Los resultados del ensayo a tiempo real realizados a la matriz enriquecida, mostraron que la estabilidad varió con el tiempo y con el tipo de enzima, independientemente del preservante ensayado, disminuyendo por debajo de los límites aceptables de actividad enzimática luego de 3 meses de almacenamiento del producto a 4 ºC. Conclusiones: se logró un material de referencia multienzimático estable por un período de 3 meses

A fundamental method to assure the quality of clinical laboratory tests is the use of reference materials. A problem we are faced with is the scarcity of these products in the domestic market, due their high cost. Objective: Evaluate the stability of an adult bovine serum enriched with the enzymes alanine aminotransferase (ALT, GPT), aspartate aminotransferase (AST, GPT), alkaline phosphatase (AP) and amylase. Methods: This enzyme-enriched matrix underwent real-time stability assessment during 12 months at two temperatures (refrigerated and frozen). An evaluation was made of the effect of glycerol on the enzymatic activity of extracts, as well as the effect of the preservatives propylene glycol and ethylene glycol on enzymatic stability. Results: The enzyme extracts obtained began to lose their biological activity at 15 days, irrespective of the storage temperature and the presence or absence of glycerol. The real time assessment of the enriched matrix showed that stability varied with time and enzyme type, irrespective of the preservative tested, and fell below acceptable limits of enzymatic activity after three months of storage at 4 ºC. Conclusions: A multienzyme reference material was obtained which was stable for a period of 3 months