Design and physicochemical stability studies of paediatric oral formulations of sildenafil

Abstract Personalized medicine is a challenging research area in paediatric treatments. Elaborating new paediatric formulations when no commercial forms are available is a common practice in pharmacy laboratories; among these, oral liquid formulations are the most common. But due to the lack of specialized equipment, frequently studies to assure the efficiency and safety of the final medicine cannot be carried out. Thus the purpose of this work was the development, characterization and stability evaluation of two oral formulations of sildenafil for the treatment of neonatal persistent pulmonary hypertension. After the establishment of a standard operating procedure (SOP) and elaboration, the physicochemical stability parameters appearance, pH, particle size, rheological behaviour and drug content of formulations were evaluated at three different temperatures for 90 days. Equally, prediction of long term stability, as well as, microbiological stability was performed. Formulations resulted in a suspension and a solution slightly coloured exhibiting fruity odour. Formulation I (suspension) exhibited the best physicochemical properties including Newtonian behaviour and uniformity of API content above 90% to assure an exact dosification process.

Development, physical-chemical stability and release studies of four alcohol-free spironolactone suspensions for use in pediatrics

Dissolution studies have become of great significance because, in most cases, drug dissolution is the rate-limiting step in the absorption process. As occurs with solid oral dosage forms, heterogeneous disperse systems (suspensions) could also have some problems with their in vitro dissolution. The objective of this study was to evaluate influence of the excipients on the release of spironolactone from four alcohol free suspensions (pharmaceutical compounding) of spironolactone 5 mg/mL suitable for pediatric use. Also the comparison of the physical and chemical stability of the suspensions stored at 4, 25 and 40 ºC over a 60- day period has been studied. Rheological behavior, particle size, a prediction of long-term physical stability, pH and assay of spironolactone by HPLC were assessed at prefixed times. The dissolution profile of each suspension was determined and compared with that of the commercial tablets. A microbiological study of the best formula was also performed. Chemically, the four spironolactone suspensions were stable for 60 days stored at three temperatures; Suspension IV had optimum pH values and the highest recovery percentage. In terms of physical stability, sedimentation occurred in Suspension IV and flotation of spironolactone in Suspensions I, II and III. Suspension III had the highest viscosity and the slowest drug release. Suspension IV was also microbiologically stable for 60 days. In conclusion, Suspension IV had the best properties and the least suitable form was Suspension III, as its high viscosity made it difficult to achieve homogeneous redispersion, and it had the slowest dissolution profile.

A system to assess the stability of airborne nanoparticle agglomerates under aerodynamic shear

Stability of airborne nanoparticle agglomerates is important for occupational exposure and risk assessment in determining particle size distribution of nanomaterials. In this study, we developed an integrated method to test the stability of aerosols created using different types of nanomaterials. An aerosolization method, that resembles an industrial fluidized bed process, was used to aerosolize dry nanopowders. We produced aerosols with stable particle number concentrations and size distributions, which was important for the characterization of the aerosols' properties. Next, in order to test their potential for deagglomeration, a critical orifice was used to apply a range of shear forces to them. The mean particle size of tested aerosols became smaller, whereas the total number of particles generated grew. The fraction of particles in the lower size range increased, and the fraction in the upper size range decreased. The reproducibility and repeatability of the results were good. Transmission electron microscopy imaging showed that most of the nanoparticles were still agglomerated after passing through the orifice. However, primary particle geometry was very different. These results are encouraging for the use of our system for routine tests of the deagglomeration potential of nanomaterials. Furthermore, the particle concentrations and small quantities of raw materials used suggested that our system might also be able to serve as an alternative method to test dustiness in existing processes.

Differential regulation of horizontally acquired and core genome genes by the bacterial modulator H-NS

Horizontal acquisition of DNA by bacteria dramatically increases genetic diversity and hence successful bacterial colonization of several niches, including the human host. A relevant issue is how this newly acquired DNA interacts and integrates in the regulatory networks of the bacterial cell. The global modulator H-NS targets both core genome and HGT genes and silences gene expression in response to external stimuli such as osmolarity and temperature. Here we provide evidence that H-NS discriminates and differentially modulates core and HGT DNA. As an example of this, plasmid R27-encoded H-NS protein has evolved to selectively silence HGT genes and does not interfere with core genome regulation. In turn, differential regulation of both gene lineages by resident chromosomal H-NS requires a helper protein: the Hha protein. Tight silencing of HGT DNA is accomplished by H-NS-Hha complexes. In contrast, core genes are modulated by H-NS homoligomers. Remarkably, the presence of Hha-like proteins is restricted to the Enterobacteriaceae. In addition, conjugative plasmids encoding H-NS variants have hitherto been isolated only from members of the family. Thus, the H-NS system in enteric bacteria presents unique evolutionary features. The capacity to selectively discriminate between core and HGT DNA may help to maintain horizontally transmitted DNA in silent form and may give these bacteria a competitive advantage in adapting to new environments, including host colonization.

Differential regulation of horizontally acquired and core genome genes by the bacterial modulator H-NS

Horizontal acquisition of DNA by bacteria dramatically increases genetic diversity and hence successful bacterial colonization of several niches, including the human host. A relevant issue is how this newly acquired DNA interacts and integrates in the regulatory networks of the bacterial cell. The global modulator H-NS targets both core genome and HGT genes and silences gene expression in response to external stimuli such as osmolarity and temperature. Here we provide evidence that H-NS discriminates and differentially modulates core and HGT DNA. As an example of this, plasmid R27-encoded H-NS protein has evolved to selectively silence HGT genes and does not interfere with core genome regulation. In turn, differential regulation of both gene lineages by resident chromosomal H-NS requires a helper protein: the Hha protein. Tight silencing of HGT DNA is accomplished by H-NS-Hha complexes. In contrast, core genes are modulated by H-NS homoligomers. Remarkably, the presence of Hha-like proteins is restricted to the Enterobacteriaceae. In addition, conjugative plasmids encoding H-NS variants have hitherto been isolated only from members of the family. Thus, the H-NS system in enteric bacteria presents unique evolutionary features. The capacity to selectively discriminate between core and HGT DNA may help to maintain horizontally transmitted DNA in silent form and may give these bacteria a competitive advantage in adapting to new environments, including host colonization.

A measure of stability as a criterion for the verification and analysis of simulation models

The aim of this study is to define a new statistic, PVL, based on the relative distance between the likelihood associated with the simulation replications and the likelihood of the conceptual model. Our results coming from several simulation experiments of a clinical trial show that the PVL statistic range can be a good measure of stability to establish when a computational model verifies the underlying conceptual model. PVL improves also the analysis of simulation replications because only one statistic is associated with all the simulation replications. As well it presents several verification scenarios, obtained by altering the simulation model, that show the usefulness of PVL. Further simulation experiments suggest that a 0 to 20 % range may define adequate limits for the verification problem, if considered from the viewpoint of an equivalence test.

An affected core drives network integration deficits of the structural connectome in 22q11.2 deletion syndrome.

Chromosome 22q11.2 deletion syndrome (22q11DS) is a genetic disease known to lead to cerebral structural alterations, which we study using the framework of the macroscopic white-matter connectome. We create weighted connectomes of 44 patients with 22q11DS and 44 healthy controls using diffusion tensor magnetic resonance imaging, and perform a weighted graph theoretical analysis. After confirming global network integration deficits in 22q11DS (previously identified using binary connectomes), we identify the spatial distribution of regions responsible for global deficits. Next, we further characterize the dysconnectivity of the deficient regions in terms of sub-network properties, and investigate their relevance with respect to clinical profiles. We define the subset of regions with decreased nodal integration (evaluated using the closeness centrality measure) as the affected core (A-core) of the 22q11DS structural connectome. A-core regions are broadly bilaterally symmetric and consist of numerous network hubs - chiefly parietal and frontal cortical, as well as subcortical regions. Using a simulated lesion approach, we demonstrate that these core regions and their connections are particularly important to efficient network communication. Moreover, these regions are generally densely connected, but less so in 22q11DS. These specific disturbances are associated to a rerouting of shortest network paths that circumvent the A-core in 22q11DS, "de-centralizing" the network. Finally, the efficiency and mean connectivity strength of an orbito-frontal/cingulate circuit, included in the affected regions, correlate negatively with the extent of negative symptoms in 22q11DS patients, revealing the clinical relevance of present findings. The identified A-core overlaps numerous regions previously identified as affected in 22q11DS as well as in schizophrenia, which approximately 30-40% of 22q11DS patients develop.

Stability of nanoparticle agglomerates under mechanical stress and its effects on their release into the air

L'exposition professionnelle aux nanomatériaux manufacturés dans l'air présente des risques potentiels pour la santé des travailleurs dans les secteurs de la nanotechnologie. Il est important de comprendre les scénarios de libération des aérosols de nanoparticules dans les processus et les activités associées à l'exposition humaine. Les mécanismes de libération, y compris les taux de libération et les propriétés physico-chimiques des nanoparticules, déterminent leurs comportements de transport ainsi que les effets biologiques néfastes. La distribution de taille des particules d'aérosols est l'un des paramètres les plus importants dans ces processus. La stabilité mécanique d'agglomérats de nanoparticules affecte leurs distributions de tailles. Les potentiels de désagglomération de ces agglomérats déterminent les possibilités de leur déformation sous énergies externes. Cela rend les changements possibles dans leur distribution de taille et de la concentration en nombre qui vont finalement modifier leurs risques d'exposition. Les conditions environnementales, telles que l'humidité relative, peuvent influencer les processus de désagglomération par l'adhérence de condensation capillaire de l'humidité. L'objectif général de cette thèse était d'évaluer les scénarios de libération des nanomatériaux manufacturés des processus et activités sur le lieu de travail. Les sous-objectifs étaient les suivants: 1. Etudier les potentiels de désagglomération des nanoparticules dans des conditions environnementales variées. 2. Etudier la libération des nano-objets à partir de nanocomposites polymères; 3. Evaluer la libération de nanoparticules sur le lieu de travail dans des situations concrètes. Nous avons comparé différents systèmes de laboratoire qui présentaient différents niveau d'énergie dans l'aérosolisation des poudres. Des nanopoudres de TiO2 avec des hydrophilicités de surface distinctes ont été testées. Un spectromètre à mobilité électrique (SMPS), un spectromètre à mobilité aérodynamique (APS) et un spectromètre optique (OPC) ont été utilisés pour mesurer la concentration de particules et la distribution de taille des particules. La microscopie électronique à transmission (TEM) a été utilisée pour l'analyse morphologique d'échantillons de particules dans l'air. Les propriétés des aérosols (distribution de taille et concentration en nombre) étaient différentes suivant la méthode employée. Les vitesses des flux d'air d'aérosolisation ont été utilisées pour estimer le niveau d'énergie dans ces systèmes, et il a été montré que les tailles modales des particules étaient inversement proportionnelles à la vitesse appliquée. En général, les particules hydrophiles ont des diamètres plus grands et des nombres inférieurs à ceux des particules hydrophobes. Toutefois, cela dépend aussi des méthodes utilisées. La vitesse de l'air peut donc être un paramètre efficace pour le classement de l'énergie des procédés pour des systèmes d'aérosolisation similaires. Nous avons développé un système laboratoire pour tester les potentiels de désagglomération des nanoparticules dans l'air en utilisant des orifices critiques et un humidificateur. Sa performance a été comparée à un système similaire dans un institut partenaire. Une variété de nanopoudres différentes a été testée. Le niveau d'énergie appliquée et l'humidité ont été modifiés. Le SMPS et l'OPC ont été utilisés pour mesurer la concentration de particules et la distribution de la taille. Un TEM a été utilisé pour l'analyse morphologique d'échantillons de particules dans l'air. Le diamètre moyen des particules a diminué et la concentration en nombre s'est accrue lorsque des énergies externes ont été appliquées. Le nombre de particules inférieures à 100 nm a été augmenté, et celui au-dessus de 350 nm réduits. Les conditions humides ont faits exactement le contraire, en particulier pour les petites particules. En outre, ils ont réduits les effets de la différence de pression due à l'orifice. Les résultats suggèrent que la désagglomération d'agglomérats de nanoparticules dans l'air est possible dans la gamme d'énergie appliquée. Cependant, l'atmosphère humide peut favoriser leur agglomération et améliorer leurs stabilités en réduisant la libération de nanoparticules dans l'environnement. Nous proposons d'utiliser notre système pour le test de routine des potentiels de désagglomération des nanomatériaux manufacturés et de les classer. Un tel classement faciliterait la priorisation de l'exposition et du risque encouru en fonction du niveau d'ENM. Un système de perçage automatique et un système de sciage manuel ont été développés pour étudier la libération de nanoparticules à partir de différents types de nanocomposites. La vitesse de perçage et taille de la mèche ont été modifiées dans les expériences. La distribution de taille des particules et leur concentration en nombre ont été mesurées par un SMPS et un miniature diffusion size classifier (DISCmini). Les distributions de nanoparticules dans les composites et les particules libérées ont été analysés par un TEM et un microscope électronique à balayage (SEM). Les tests de perçage ont libérés un plus grand nombre de particules que le sciage. Des vitesses de perçage plus rapide et les mèches plus grandes ont augmentés la génération de particules. Les charges de nanoparticules manufacturées dans les composites ne modifient pas leurs comportements de libération dans les expériences de perçage. Toutefois, le sciage différencie les niveaux de libération entre les composites et les échantillons blancs. De plus, les vapeurs de polymères ont été générées par la chaleur de sciage. La plupart des particules libérées sont des polymères contenant des nanoparticules ou sur leurs surface. Les résultats ont souligné l'importance du type de processus et paramètres pour déterminer la libération de nanoparticules de composites. Les émissions secondaires telles que les fumées polymères appellent à la nécessité d'évaluations de l'exposition et de risque pour de tels scénarios. Une revue systématique de la littérature sur le sujet de libérations de nanoparticules dans l'air dans les secteurs industriels et laboratoires de recherche a été effectuée. Des stratégies de recherche des informations pertinentes et de stockage ont été développées. Les mécanismes de libération, tels que la taille de particules d'aérosol et de leur concentration en nombre, ont été comparés pour différentes activités. La disponibilité de l'information contextuelle qui est pertinente pour l'estimation de l'exposition humaine a été évaluée. Il a été constaté que les données relatives à l'exposition ne sont pas toujours disponibles dans la littérature actuelle. Les propriétés des aérosols libérés semblent dépendre de la nature des activités. Des procédés à haute énergie ont tendance à générer des plus hauts niveaux de concentrations de particules dans les gammes de plus petite taille. Les résultats peuvent être utiles pour déterminer la priorité des procédés industriels pour l'évaluation les risques associés dans une approche à plusieurs niveaux. Pour l'évaluation de l'exposition, la disponibilité de l'information peut être améliorée par le développement d'une meilleure méthode de communication des données.

The fusion protein SS18-SSX1 employs core Wnt pathway transcription factors to induce a partial Wnt signature in synovial sarcoma.

Expression of the SS18/SYT-SSX fusion protein is believed to underlie the pathogenesis of synovial sarcoma (SS). Recent evidence suggests that deregulation of the Wnt pathway may play an important role in SS but the mechanisms whereby SS18-SSX might affect Wnt signaling remain to be elucidated. Here, we show that SS18/SSX tightly regulates the elevated expression of the key Wnt target AXIN2 in primary SS. SS18-SSX is shown to interact with TCF/LEF, TLE and HDAC but not β-catenin in vivo and to induce Wnt target gene expression by forming a complex containing promoter-bound TCF/LEF and HDAC but lacking β-catenin. Our observations provide a tumor-specific mechanistic basis for Wnt target gene induction in SS that can occur in the absence of Wnt ligand stimulation.

Step-by-step of ultrasound-guided core-needle biopsy of the breast: review and technique

Ultrasound-guided core-needle biopsy has high sensitivity in the diagnosis of breast cancer. The present study is aimed at detailing the main steps of such procedure, including indications, advantages, limitations, follow-up and description of the technique, besides presenting a checklist including the critical steps required for an appropriate practice of the technique. In the recent years, an increasing number of patients have required breast biopsy, indicating the necessity of a proportional increase in the number of skilled professionals to carry out the procedures and histological diagnoses. A multidisciplinary approach involving the tripod clinical practice-radiology-pathology is responsible for the highest rate of accuracy of the technique and must always be adopted.

Diagnostic underestimation of atypical ductal hyperplasia and ductal carcinoma in situ at percutaneous core needle and vacuum-assisted biopsies of the breast in a Brazilian reference institution

Abstract Objective: To determine the rates of diagnostic underestimation at stereotactic percutaneous core needle biopsies (CNB) and vacuum-assisted biopsies (VABB) of nonpalpable breast lesions, with histopathological results of atypical ductal hyperplasia (ADH) or ductal carcinoma in situ (DCIS) subsequently submitted to surgical excision. As a secondary objective, the frequency of ADH and DCIS was determined for the cases submitted to biopsy. Materials and Methods: Retrospective review of 40 cases with diagnosis of ADH or DCIS on the basis of biopsies performed between February 2011 and July 2013, subsequently submitted to surgery, whose histopathological reports were available in the internal information system. Biopsy results were compared with those observed at surgery and the underestimation rate was calculated by means of specific mathematical equations. Results: The underestimation rate at CNB was 50% for ADH and 28.57% for DCIS, and at VABB it was 25% for ADH and 14.28% for DCIS. ADH represented 10.25% of all cases undergoing biopsy, whereas DCIS accounted for 23.91%. Conclusion: The diagnostic underestimation rate at CNB is two times the rate at VABB. Certainty that the target has been achieved is not the sole determining factor for a reliable diagnosis. Removal of more than 50% of the target lesion should further reduce the risk of underestimation.

Enhancement of human adipose-derived stem cell expansion and stability for clinical use

Co-culture techniques associating both dermal fibroblasts and epidermal keratinocytes have shown to have better clinical outcome than keratinocyte culture alone for the treatment of severe burns. Since fat grafting has been shown to improve scar remodelling, new techniques such as cell-therapy-assisted surgical reconstruction with isolated and expanded autologous adipose-derived stem cells (ASCs) would be of benefit to increase graft acceptation. Therefore, integrating ASCs into standardized procedures for cultured skin grafting could be of benefit for the patient if cell quality and quantity could be maintained. The purpose of this study was to evaluate ASC processing from adult tissue with simple isolation (without enzymatic steps), expansion (low density of 325-3,000 cells/cm2) and storage conditions to assure methods to enhance the cellular resistance when transferred back to the patient. Co-culture with cell-banked skin progenitor cells (FE002-SK2) showed an increase of 40-50% ASCs yield at high passages alongside with a better preservation of morphology, proper adipogenic and osteogenic differentiation and efficient biocompatibility with 3D collagen scaffolds. ASCs can be considered as a valuable additional cell source to be delivered in biological bandages to the patient in a need of tissue reconstruction such as burn patients.