Carreadores lip?dicos nanoestruturados contendo benznidazol como estrat?gia para aumentar a efic?cia no tratamento da doen?a de Chagas

?rea de concentra??o: Pesquisa e Desenvolvimento de Insumos, F?rmacos e Medicamentos.A doen?a de Chagas (DC) ? uma antropozoonose causada pelo protozo?rio Trypanosoma cruzi que atinge milh?es de pessoas principalmente na Am?rica Latina e cujo tratamento permanece insatisfat?rio. O benznidazol (BZN) ? considerado a primeira escolha para o tratamento devido a sua maior tolerabilidade pelos pacientes. Entretanto, este f?rmaco apresenta baixa solubilidade aquosa, absor??o oral irregular, farmacocin?tica desfavor?vel, al?m de ocasionar v?rios efeitos adversos. A incorpora??o de f?rmacos com estas caracter?sticas em sistemas lip?dicos nanoestruturados representa uma alternativa para melhorar estes fatores limitantes. Diante disso, o objetivo deste estudo foi desenvolver, caracterizar e avaliar a efic?cia tripanocida in vitro de carreadores lip?dicos nanoestruturados (CLN) contendo BZN. O desenvolvimento da formula??o avaliou a influ?ncia do l?pide l?quido, da composi??o do sistema tensoativo, da concentra??o de l?pides e da concentra??o de BZN no tamanho, potencial zeta (PZ) e incorpora??o do f?rmaco no nanossistema. A formula??o otimizada tamb?m foi caracterizada por espectroscopia no infravermelho, difra??o de raios-X, calorimetria explorat?ria diferencial, termogravimetria e microscopia eletr?nica de varredura. Avaliou-se tamb?m a estabilidade preliminar, o potencial hemol?tico, toxicidade em c?lulas L929 e atividade tripanocida em formas epimastigotas do T. cruzi. O CLN-BZN desenvolvido apresentou morfologia esf?rica, di?metro m?dio compat?vel ? administra??o oral (110 ? 3 nm), PZ negativo e preditivo de estabilidade (-18,0 ? 2,6 mV), al?m de alto teor de encapsula??o de BZN (82,6 ? 2,1%). A atividade tripanocida do CLN-BZN foi similar ao f?rmaco livre at? a concentra??o de 31 ?g/mL. Contudo a formula??o apresentou baixo potencial hemol?tico e menor toxicidade em c?lulas L929 em compara??o ao BZN livre. Estes resultados sugerem que a formula??o desenvolvida apresenta caracter?sticas adequadas para a administra??o oral, sendo assim uma alternativa promissora para o tratamento da DC.Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES)Disserta??o (Mestrado) ? Programa de P?s-gradua??o em Ci?ncias Farmac?uticas, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2019.Chagas disease (CD) is an anthropozoonosis caused by the protozoan Trypanosoma cruzi, which affects millions of people mainly in Latin America with unsatisfactory treatment. Benznidazole (BZN) is related as first choice for treatment since presents greater tolerability by patients. However, this drug shows low aqueous solubility, irregular oral absorption, unfavorable pharmacokinetics, resulting several adverse effects. In that way, incorporation of drugs with these characteristics into nanostructured lipid systems represents an alternative to improve these limiting factors. In this context, the aim of this study is based on development, characterization and evaluation of in vitro trypanocidal efficacy of nanostructured lipid carriers (NLC) loaded with BZN. The influence of liquid lipid, surfactant system composition, lipid concentration and BZN concentration on size, zeta potential (ZP) and incorporation of the drug in the nanosystem were evaluated for each formulation. The optimized formulation was also characterized by infrared spectroscopy, X-ray diffraction, differential scanning calorimetry, thermogravimetry and scanning electron microscopy. Preliminary stability, hemolytic potential, toxicity in L929 cells and trypanocidal activity in T. cruzi epimastigote forms were also carried out. NLC-BZN showed spherical morphology, mean diameter compatible with oral administration (110 ? 3 nm), Zp negative and predictive of stability (-18,0 ? 2,6 mV), and high encapsulation efficiency of BZN (82,6 ? 2,1%), which was proved by the other characterizations. The optimized formulation showed low hemolytic potential and lower toxicity in L929 cells compared to free BZN, and trypanocidal activity was similar to the free drug up to the concentration of 31 ?g/mL. These results suggest that the optimized formulation presents highlight characteristics for oral administration, representing a promising alternative for the treatment of DC

A Three-Site Mechanism for Agonist/Antagonist Selective Binding to Vasopressin Receptors

Molecular-dynamics simulations with metadynamics enhanced sampling reveal three distinct binding sites for arginine vasopressin (AVP) within its V2 -receptor (V2 R). Two of these, the vestibule and intermediate sites, block (antagonize) the receptor, and the third is the orthosteric activation (agonist) site. The contacts found for the orthosteric site satisfy all the requirements deduced from mutagenesis experiments. Metadynamics simulations for V2 R and its V1a R-analog give an excellent correlation with experimental binding free energies by assuming that the most stable binding site in the simulations corresponds to the experimental binding free energy in each case. The resulting three-site mechanism separates agonists from antagonists and explains subtype selectivity

Process Monitoring of Moisture Content and Mass Transfer Rate in a Fluidised Bed with a Low Cost Inline MEMS NIR Sensor

Purpose The current trend for continuous drug product manufacturing requires new, affordable process analytical techniques (PAT) to ensure control of processing. This work evaluates whether property models based on spectral data from recent Fabry–Pérot Interferometer based NIR sensors can generate a high-resolution moisture signal suitable for process control. Methods Spectral data and offline moisture content were recorded for 14 fluid bed dryer batches of pharmaceutical granules. A PLS moisture model was constructed resulting in a high resolution moisture signal, used to demonstrate (i) endpoint determination and (ii) evaluation of mass transfer performance. Results The sensors appear robust with respect to vibration and ambient temperature changes, and the accuracy of water content predictions (±13 % ) is similar to those reported for high specification NIR sensors. Fusion of temperature and moisture content signal allowed monitoring of water transport rates in the fluidised bed and highlighted the importance water transport within the solid phase at low moisture levels. The NIR data was also successfully used with PCA-based MSPC models for endpoint detection. Conclusions The spectral quality of the small form factor NIR sensor and its robustness is clearly sufficient for the construction and application of PLS models as well as PCA-based MSPC moisture models. The resulting high resolution moisture content signal was successfully used for endpoint detection and monitoring the mass transfer rate

Pro-inflammatory polarization and colorectal cancer modulate alternative and intronic polyadenylation in primary human macrophages

Macrophages are essential cells of the immune system that alter their inflammatory profile depending on their microenvironment. Alternative polyadenylation in the 3'UTR (3'UTR-APA) and intronic polyadenylation (IPA) are mechanisms that modulate gene expression, in particular in cancer and activated immune cells. Yet, how polarization and colorectal cancer (CRC) cells microenvironment affect 3'UTR-APA and IPA in primary human macrophages remains unknown. Here, primary human monocytes were isolated from healthy donors, differentiated and polarized into a pro-inflammatory state and ChrRNA-Seq and 3'RNA-Seq were performed to quantify gene expression and characterize new 3’UTR-APA and IPA mRNA isoforms. Our results show that polarization of human macrophages from naïve to a pro-inflammatory state causes a marked increase both in proximal polyA site selection in the 3'UTR and in IPA events, in genes relevant for macrophage functions. Additionally, we found a negative correlation between differential gene expression and IPA during pro-inflammatory polarization of primary human macrophages. As macrophages are abundant immune cells in the CRC microenvironment that either promote or abrogate cancer progression, we investigated how indirect exposure to CRC cells affects macrophage gene expression and 3'UTR-APA and IPA mRNA events. Co-culture with CRC cells alters the inflammatory phenotype of macrophages, increases the expression of pro tumoral genes and induce 3’UTR-APA alterations. Notably, some of these gene expression differences were also found in tumour-associated macrophages of CRC patients, indicating that they are physiological relevant. Upon macrophage pro inflammatory polarization SRSF12 is the pre-mRNA processing gene that is most upregulated. After SRSF12 knockdown in M1 macrophages there is a global downregulation of gene expression, in particular in genes involved in gene expression regulation and in immune responses. Our results reveal new 48 3’UTR-APA and IPA mRNA isoforms produced during pro-inflammatory polarization of primary human macrophages and CRC co-culture that may be used in the future as diagnostic or therapeutic tools

Allosteric communication in class A β-lactamases occurs via cooperative coupling of loop dynamics

Understanding allostery in enzymes and tools to identify it, offer promising alternative strategies to inhibitor development. Through a combination of equilibrium and nonequilibrium molecular dynamics simulations, we identify allosteric effects and communication pathways in two prototypical class A β-lactamases, TEM-1 and KPC-2, which are important determinants of antibiotic resistance. The nonequilibrium simulations reveal pathways of communication operating over distances of 30 Å or more. Propagation of the signal occurs through cooperative coupling of loop dynamics. Notably, 50% or more of clinically relevant amino acid substitutions map onto the identified signal transduction pathways. This suggests that clinically important variation may affect, or be driven by, differences in allosteric behavior, providing a mechanism by which amino acid substitutions may affect the relationship between spectrum of activity, catalytic turnover and potential allosteric behavior in this clinically important enzyme family. Simulations of the type presented here will help in identifying and analyzing such differences