One and Two-Step In Vitro-In Vivo Correlations Based on USP IV Dynamic Dissolution Applied to Four Sodium Montelukast Products

Montelukast is a weak acid drug characterized by its low solubility in the range of pH 1.2 to 4.5, which may lead to dissolution-limited absorption. The aim of this paper is to develop an in vivo predictive dissolution method for montelukast and to check its performance by establishing a level-A in vitro-in vivo correlation (IVIVC). During the development of a generic film-coated tablet formulation, two clinical trials were done with three different experimental formulations to achieve a similar formulation to the reference one. A dissolution test procedure with a flow-through cell (USP IV) was used to predict the in vivo absorption behavior. The method proposed is based on a flow rate of 5 mL/min and changes of pH mediums from 1.2 to 4.5 and then to 6.8 with standard pharmacopoeia buffers. In order to improve the dissolution of montelukast, sodium dodecyl sulfate was added to the 4.5 and 6.8 pH mediums. Dissolution profiles in from the new method were used to develop a level-A IVIVC. One-step level-A IVIVC was developed from dissolution profiles and fractions absorbed obtained by the Loo–Riegelman method. Time scaling with Levy’s plot was necessary to achieve a linear IVIVC. One-step differential equation-based IVIVC was also developed with a time-scaling function. The developed method showed similar results to a previously proposed biopredictive method for montelukast, and the added value showed the ability to discriminate among different release rates in vitro, matching the in vivo clinical bioequivalence results

Mecanismos implicados en las respuestas vasodilatadoras del nitrocompuesto SIN-1

Centro de Informacion y Documentacion Cientifica (CINDOC). C/Joaquin Costa, 22. 28002 Madrid. SPAIN / CINDOC - Centro de Informaciòn y Documentaciòn CientìficaSIGLEESSpai

La respuesta estricta media la dispersión del biofilm en Pseudomonas putida

Resumen del trabajo presentado a la XI Reunión del Grupo de Microbiología Molecular, celebrada en Sevilla del 6 al 8 de septiembre de 2016.La respuesta estricta es una respuesta a estrés conservada en bacterias. En condiciones de limitación de nutrientes, se activa la síntesis de las alarmonas guanosina 5’-difosfato 3’-difosfato (ppGpp), y guanosina 5’-trifosfato 3’-difosfato (pppGpp) [conjuntamente denominadas (p)ppGpp]. El (p)ppGpp regula directa e indirectamente la expresión de numerosos genes, en ocasiones con el concurso de la proteína auxiliar DksA. En la naturaleza las bacterias alternan una vida planctónica con la formación de comunidades sésiles rodeadas de una matriz extracelular polimérica, denominadas biofilms. Los fenómenos de dispersión programada promueven la transición del biofilm al estado planctónico en respuesta a señales fisiológicas o ambientales. Los biofilms de la bacteria Gram-negativa de suelo Pseudomonas putida experimentan una rápida dispersión en condiciones de estrés nutricional. La dispersión es el resultado de la proteolisis de la adhesina de alto peso molecular LapA por la proteasa periplásmica LapG. La actividad de LapG está regulada por la proteína transmembrana LapD en respuesta a cambios en los niveles del segundo mensajero 3',5'-diguanilato cíclico (di-GMPc). Recientemente hemos demostramos que la fosfodiesterasa BifA es responsable de la hidrólisis de di-GMPc que dispara la respuesta de dispersión. En esta comunicación mostramos que la respuesta estricta media la inducción de la dispersión del biofilm en P. putida. Mediante el análisis fenotípico de mutantes con defectos en respuesta estricta y análisis de expresión génica in vivo e in vitro hemos elucidado los mecanismos implicados, y hemos completando el circuito de transducción de señales que conecta la señal inductora (limitación de nutrientes) con la respuesta (proteolisis de LapA) responsable de la dispersión del biofilm en P. putida.Este trabajo ha sido financiado por los proyectos BIO2010-17853 (MICINN) y BIO2013-42073-P (MINECO).Peer Reviewe

Alternative Pharmacokinetic Metrics in Single-Dose Studies to Ensure Bioequivalence of Prolonged-Release Products at Steady State—A Case Study

(1) Background: this article investigates which PK metrics in a single-dose study (concentration at the end of posology interval, Cτ, partial areas under the curve, pAUCs, or half-value duration, HVD) are more sensitive and less variable for predicting the failure of a prolonged-release product at steady-state that was the bioequivalent for Cmax, AUC0-t and AUC0-inf, in the single-dose study; (2) Methods: a cross-over study was performed in 36 subjects receiving desvenlafaxine 100 mg prolonged-release tablets. Conventional (Cmax, AUC0-t and AUC0-inf) and additional (Cτ, pAUCs and HVD) PK metrics were considered after single-dose conditions. Predicted PK metrics at steady state (AUC0-τ, Cmax,ss, and Cτ,ss) were derived using a population PK model approach; (3) Results: the existing differences in the shape of the concentration–time curves precluded to show equivalence for Cτ,ss in the simulated study at steady state. This failure to show equivalence at steady state was predicted by Cτ, pAUCs and HVD in the single-dose study. Cτ was the most sensitive metric for detecting the different shape, with a lower intra-subject variability than HVD; (4) Conclusions: conventional PK metrics for single-dose studies (Cmax, AUC0-t and AUC0-inf) are not enough to guarantee bioequivalence at steady state for prolonged-release products

The stringent response promotes biofilm dispersal in Pseudomonas putida

Biofilm dispersal is a genetically programmed response enabling bacterial cells to exit the biofilm in response to particular physiological or environmental conditions. In Pseudomonas putida biofilms, nutrient starvation triggers c-di-GMP hydrolysis by phosphodiesterase BifA, releasing inhibition of protease LapG by the c-di-GMP effector protein LapD, and resulting in proteolysis of the adhesin LapA and the subsequent release of biofilm cells. Here we demonstrate that the stringent response, a ubiquitous bacterial stress response, is accountable for relaying the nutrient stress signal to the biofilm dispersal machinery. Mutants lacking elements of the stringent response – (p)ppGpp sythetases [RelA and SpoT] and/or DksA – were defective in biofilm dispersal. Ectopic (p)ppGpp synthesis restored biofilm dispersal in a ∆relA ∆spoT mutant. In vivo gene expression analysis showed that (p)ppGpp positively regulates transcription of bifA, and negatively regulates transcription of lapA and the lapBC, and lapE operons, encoding a LapA-specific secretion system. Further in vivo and in vitro characterization revealed that the PbifA promoter is dependent on the flagellar σ factor FliA, and positively regulated by ppGpp and DksA. Our results indicate that the stringent response stimulates biofilm dispersal under nutrient limitation by coordinately promoting LapA proteolysis and preventing de novo LapA synthesis and secretion.Originally published in thesis in manuscript form.</p