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Mitochondrial cytopathies can have kidney involvement in up to half of cases. Their diagnosis is challenging due to phenotypic variability, lack of noninvasive tests to assess mitochondrial dysfunction and genetic heterogeneity. We report on a young adult male with hypertrophic cardiomyopathy (HCM) and chronic kidney disease (CKD) with sub-nephrotic proteinuria, who presented to the emergency department with kidney failure and hypervolemia requiring dialysis. A kidney biopsy showed focal segmental and global glomerulosclerosis, extensive foot process effacement, and abnormal mitochondria in podocytes and tubular epithelial cells; the genetic workup identified a rare FASTKD2 exon 2 variant, c.29G>C p.(Ser10Thr), in homozygosity; and functional mitochondrial assays in cultured skin fibroblasts showed reduction in FASTKD2 protein expression and moderate combined impairment in mitochondrial respiratory chain (MRC) assembly and function. This is the first report of a FASTKD2-associated cardiorenal mitochondrial cytopathy, characterized by young adult-onset proteinuric CKD and dilated HCM, in the absence of the severe neurologic manifestations described in patients with biallelic FASTKD2 mutations. We hypothesize that the increased production of reactive oxygen species associated with moderate MRC impairment could result in a smoldering podocytopathy with progressive proteinuric CKD, without overt tubulopathy or encephalomyopathy, which are, instead, pathogenically related to adenosine triphosphate deficiency.
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Concrete is the main ingredient in construction, but it inevitably fractures during its service life, requiring a large amount of cement and aggregate for maintenance. Concrete healing through biomineralization can repair cracks and improve the durability of concrete, which is conducive to saving raw materials and reducing carbon emissions. This paper reviews the biodiversity of microorganisms capable of precipitating mineralization to repair the concrete and their mineralization ability under different conditions. To better understand the mass transfer process of precipitates, two biomineralization mechanisms, microbially-controlled mineralization and microbially-induced mineralization, have been briefly described. The application of microorganisms in the field of healing concrete, comprising passive healing and intrinsic healing, is discussed. The key insight on the interaction between cementitious materials and microorganisms is the main approach for developing novel self-healing concrete in the future to improve the corrosion resistance of concrete. At the same time, the limitations and challenges are also pointed out.
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Biomineralización , Carbonato de Calcio , Materiales de Construcción , Carbono , BiodiversidadRESUMEN
INTRODUCTION: Mitochondrial oxidative phosphorylation (OXPHOS) diseases are challenging both from clinical and therapeutic perspectives. The advent of next-generation sequencing (NGS) boosted the discovery of new genetic defects affecting OXPHOS, with pathogenic variants identified in >350 genes to date [1]. However, in many patients, novel variants of unknown clinical significance are found. Subsequent functional studies may clarify its pathogenic consequences and modify the variant's classification, establishing a genetic diagnosis [2, 3]. METHODS: Analysis of data obtained from patients (P1-P5) with novel genetic causes and functional genomics' studies performed, namely OXPHOS respiratory/glycolytic rates (Seahorse XF), enzymatic activity and assembly (BN-page), protein levels (SDS-WB), single muscle fiber assay, NGS and bioinformatics. RESULTS/CASE REPORT: P1-Leigh syndrome (40y, male); Complex IV activity deficiency (full assembly absent), homozygous deletion (c.-11_13del, SURF1), not detected by NGS[2]. P2- Epileptic encephalopathy (8y, male); homozygous c.882-1G>A, FASTKD2; OXPHOS decrease; reduced FASTKD2 expression and abnormal respiratory/glycolytic rates. P3-Cardiomyopathy/ nephropathy (39y, male); c.29G>C, FASTKD2; OXPHOS decrease; reduced FASTKD2 levels. P4-CPEO (62y, female); multiple OXPHOS deficiency; mtDNA alterations (m.7486G>A, MTTS1; 4,977bp del); higher levels of mutant mtDNA alterations in COX-deficient fibers [3]. P5- Polyneuropathy (15y, female); heterozygous c.1437C>A, POLG; combined def. or normal OXPHOS activity/respiratory capacity (tissue variable), raised CI assembly; normal POLG levels. Also, proteins' expression levels were reduced (P1-4), confirming pathogenicity. In P5, data do not support pathogenicity. CONCLUSION: If specific functional results are similar to controls, one might inquire about the pathogenicity of the studied variant and more genetic or bioinformatics analyses and family investigations are needed. There are also limitations of NGS in mutation detection that Sanger sequencing can overcome (P1). When performed first, the OXPHOS activity may guide to genetic screening or interpretation, concordant to later assembly results. All cases were solved and data may be crucial for genetic counseling.
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INTRODUCTION: Leigh syndrome (LS) is clinically and genetically heterogeneous and presents defective mitochondrial bioenergetics. Patients present neurological symptoms and imagiological features that may result in early death [1]. The LS has been associated with mitochondrial DNA (mtDNA) variants, e.g., m.8993T>G (L156R) and m.8993T>C (L156P), in the MT-ATP6 gene. They lead to the substitution of a highly conserved amino acid in subunit 6 of ATP synthase, affecting the F0 domain and ATP synthesis [1-3]. We present five cases with m.8993T>G and a family harbouring m.8993T>C+m.1555A>G (proband and four relatives). METHODS: Our laboratory received 48 samples from LS-suspected patients. The samples (various tissues) were assessed for bioenergetics (activity of mitochondrial respiratory chain (MRC) complexes, ubiquinone content) and genetic analyses (mtDNA copy number, Sequencing and PCR-RFLP) by established protocols. RESULTS/CASE REPORT: Bioenergetics were assessed in 5 patients (various tissues) with varying levels of MRC/ATP synthase impairment. Six cases had a mtDNA pathogenic variant in the 8993 nucleotide associated with LS. Five cases presented the m.8993T>G variant, one of which (P5) possibly de novo. This variant was homoplasmy (P1-3) or very high heteroplasmy (P4/5, 90-95%). Of the four patients with bioenergetics assessment, three (P1/3/4) had deficiencies of MRC complexes, and P5 had small deficits. The other case (familial, proband and 4 relatives) presented a combination of m.1555A>G (homoplasmy) and m.8993T>C (heteroplasmy) variants. The proband presents m.8993T>C in 95% heteroplasmy and 85-35% in three relatives. All have m.1555A>G in homoplasmy, including the fourth relative without m.8993T>C. A deficiency (31%) was found in complex V activity in muscle for proband. CONCLUSION: We present a case series of patients harbouring pathogenic variants in the 8993 nucleotide of mtDNA, which have been associated with LS and impairment of MRC's complex V. These cases highlight the variability in clinical symptoms and their severity, as well as genetic heterogeneity within LS. Many patients will not present a classic pathogenic variant and there are many cases of asymptomatic relatives (carriers). It is important to get a broader view of the cases - classical methods and multiple tissue analysis are still valuable tools for the comprehensive characterization of patients.
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INTRODUCTION: Mitochondrial diseases are rare, heterogeneous, incurable and complex to diagnose. Probably due to their rareness, there is still a lack of literacy in this area, especially in society, but also in schools and in general, health care services. Accordingly, tools that may bring advancement in science and health literacy are needed. Mit.OnOff is a science communication project based on a bilateral partnership between the University of Coimbra (Portugal) and the University of Bergen (Norway). It aims to inform society about rare diseases related to mitochondrial cytopathies with an emphasis on LHON. METHODS: The initiative focuses on the creation of an illustrated book explaining the diseases caused by the failure of energy production in simple and accessible language. The aim is to raise awareness (particularly in Portugal and Norway) and provide in-depth knowledge to people suffering from these diseases. RESULTS/CASE REPORT: This project involves expert scientists in the field of mitochondrial disease, science communicators and artists in alignment with the United Nations SDGs, Agenda 2030. Mit.OnOff is a bilateral partnership (Portugal and Norway) established to address the lack of knowledge and health literacy on the subject of mitochondrial disease. The book will be distributed in both countries, creating a sense of inclusion and visibility and influencing decisions regarding these diseases. It is a relevant educational medium (e.g., schools, health care provision). The distribution of the book is complemented with other communication materials. Oral communications are made, together with public involvement, in which special glasses will be distributed to simulate a mitochondrial disease that leads to blindness (LHON) for the public to experience what it is like living with a rare disease. CONCLUSION: It is hoped that the production of this book will give patients a sense of inclusion and representation in the media. This, in turn, will contribute to achieving the SDG targets (3,4,5,8,10,12), i.e., ensuring people live healthy lives, reducing child mortality, and increasing life expectancy, ensuring access to inclusive, equitable and quality education for all, ensuring gender equality, and contributing to a peaceful and prosperous world.
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Water bodies on Mars and the icy moons of the outer solar system are now recognized as likely being associated with high levels of salt. Therefore, the study of high salinity environments and their inhabitants has become increasingly relevant for Astrobiology. Members of the archaeal class Halobacteria are the most successful microbial group living in hypersaline conditions and are recognized as key model organisms for exposure experiments. Despite this, data for the class is uneven across taxa and widely dispersed across the literature, which has made it difficult to properly assess the potential for species of Halobacteria to survive under the polyextreme conditions found beyond Earth. Here we provide an overview of published data on astrobiology-linked exposure experiments performed with members of the Halobacteria, identifying clear knowledge gaps and research opportunities.
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Biogenic silver nanoparticles (AgNPs) are considered a promising alternative to their synthetic versions. However, the environmental impact of such nanomaterials is still scarcely understood. Thus, the present study aims at assessing the antimicrobial action and ecotoxicity of AgNPs biosynthesized by the fungus Aspergillus niger IBCLP20 towards three freshwater organisms: Chlorella vulgaris, Daphnia similis, and Danio rerio (zebrafish). AgNPs IBCLP20 showed antibacterial action against Klebsiella pneumoniae between 5 and 100 µg mL-1, and antifungal action against Trichophyton mentagrophytes in concentrations ranging from 20 to 100 µg mL-1. The cell density of the microalgae Chlorella vulgaris decreased 40% after 96 h of exposure to AgNPs IBCLP20, at the highest concentration analysed (100 µg L-1). The 48 h median lethal concentration for Daphnia similis was estimated as 4.06 µg L-1 (2.29-6.42 µg L-1). AgNPs IBCLP20 and silver nitrate (AgNO3) caused no acute toxicity on adult zebrafish, although they did induce several physiological changes. Mycosynthetized AgNPs caused a significant increase (p < 0.05) in oxygen consumption at the highest concentration studied (75 µg L-1) and an increase in the excretion of ammonia at the lower concentrations, followed by a reduction at the higher concentrations. Such findings are comparable with AgNO3, which increased the oxygen consumption on low exposure concentrations, followed by a decrease at the high tested concentrations, while impairing the excretion of ammonia in all tested concentrations. The present results show that AgNPs IBCLP20 have biocidal properties. Mycogenic AgNPs induce adverse effects on organisms of different trophic levels and understanding their impact is detrimental to developing countermeasures aimed at preventing any negative environmental effects of such novel materials.
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Chlorella vulgaris , Nanopartículas del Metal , Amoníaco , Animales , Antibacterianos/farmacología , Antifúngicos/farmacología , Daphnia , Nanopartículas del Metal/toxicidad , Plata/toxicidad , Nitrato de Plata/toxicidad , Pez CebraRESUMEN
This paper investigates labour market resilience for seven Portuguese NUTS-2 regions over the period 1995-2018 detailing its relationship with levels of education and highlighting the period following the 2007-08 financial and economic crisis. We define resilience as the ability of regional employment to recover from a recessionary shock over an entire business cycle. Our results point to the existence of labour market resilience to the different business cycles for the different regions in terms of total hours worked. The same conclusion applies to employment of workers with different levels of educational attainment, low, medium and high, defined according to the highest level of education completed by employees. Investigating in more detail the potential differentiated impact of the Portuguese Great Recession (PGR), covering the period after the 2007-08 crisis, our findings suggest however no resilience in terms of total hours worked and employment of workers with low levels of education, corresponding so far to a situation of jobless economic recovery. The conclusions are mixed for employment of workers with medium levels of education, while we found evidence of labour market resilience to the PGR for employment of workers with high levels of education. The strong negative impact of the PGR at the economic level thus seems to have hindered labour market resilience for employees and regions less endowed with human capital.
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Habitability has been generally defined as the capability of an environment to support life. Ecologists have been using Habitat Suitability Models (HSMs) for more than four decades to study the habitability of Earth from local to global scales. Astrobiologists have been proposing different habitability models for some time, with little integration and consistency among them, being different in function to those used by ecologists. Habitability models are not only used to determine whether environments are habitable, but they also are used to characterize what key factors are responsible for the gradual transition from low to high habitability states. Here we review and compare some of the different models used by ecologists and astrobiologists and suggest how they could be integrated into new habitability standards. Such standards will help improve the comparison and characterization of potentially habitable environments, prioritize target selections, and study correlations between habitability and biosignatures. Habitability models are the foundation of planetary habitability science, and the synergy between ecologists and astrobiologists is necessary to expand our understanding of the habitability of Earth, the Solar System, and extrasolar planets.
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Exobiología , Medio Ambiente Extraterrestre , Planeta Tierra , PlanetasRESUMEN
Hot-melt extrusion has found extensive application as a feasible pharmaceutical technological option over recent years. HME applications include solubility enhancement, taste masking, and sustained drug release. As bioavailability enhancement is a hot topic of today's science, one of the main applications of HME is centered on amorphous solid dispersions. This review describes the most significant aspects of HME technology and its use to prepare solid dispersions as a drug formulation strategy to enhance the solubility of poorly soluble drugs. It also addresses molecular and thermodynamic features critical for the physicochemical properties of these systems, mainly in what concerns miscibility and physical stability. Moreover, the importance of applying the Quality by Design philosophy in drug development is also discussed, as well as process analytical technologies in pharmaceutical HME monitoring, under the current standards of product development and regulatory guidance. Graphical Abstract.
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Química Farmacéutica/métodos , Portadores de Fármacos/síntesis química , Desarrollo de Medicamentos/métodos , Tecnología de Extrusión de Fusión en Caliente/métodos , Disponibilidad Biológica , Composición de Medicamentos/métodos , Composición de Medicamentos/tendencias , Tecnología de Extrusión de Fusión en Caliente/tendencias , Calor , Solubilidad , Tecnología Farmacéutica/métodos , Tecnología Farmacéutica/tendencias , TermodinámicaRESUMEN
After a less dynamic period, space exploration is now booming. There has been a sharp increase in the number of current missions and also of those being planned for the near future. Microorganisms will be an inevitable component of these missions, mostly because they hitchhike, either attached to space technology, like spaceships or spacesuits, to organic matter and even to us (human microbiome), or to other life forms we carry on our missions. Basically, we never travel alone. Therefore, we need to have a clear understanding of how dangerous our "travel buddies" can be; given that, during space missions, our access to medical assistance and medical drugs will be very limited. Do we explore space together with pathogenic microorganisms? Do our hitchhikers adapt to the space conditions, as well as we do? Do they become pathogenic during that adaptation process? The current review intends to better clarify these questions in order to facilitate future activities in space. More technological advances are needed to guarantee the success of all missions and assure the reduction of any possible health and environmental risks for the astronauts and for the locations being explored.
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Background: Somatosensation depends on primary sensory neurons of the trigeminal and dorsal root ganglia (DRG). Transcriptional profiling of mouse DRG sensory neurons has defined at least 18 distinct neuronal cell types. Using an advillin promoter, we have generated a transgenic mouse line that only expresses diphtheria toxin A (DTA) in sensory neurons in the presence of Cre recombinase. This has allowed us to ablate specific neuronal subsets within the DRG using a range of established and novel Cre lines that encompass all sets of sensory neurons. Methods: A floxed-tdTomato-stop-DTA bacterial artificial chromosome (BAC) transgenic reporter line (AdvDTA) under the control of the mouse advillin DRG promoter was generated. The line was first validated using a Na v1.8 Cre and then crossed to CGRP CreER (Calca), Th CreERT2, Tmem45b Cre, Tmem233 Cre, Ntng1 Cre and TrkB CreER (Ntrk2) lines. Pain behavioural assays included Hargreaves', hot plate, Randall-Selitto, cold plantar, partial sciatic nerve ligation and formalin tests. Results: Motor activity, as assessed by the rotarod test, was normal for all lines tested. Noxious mechanosensation was significantly reduced when either Na v1.8 positive neurons or Tmem45b positive neurons were ablated whilst acute heat pain was unaffected. In contrast, noxious mechanosensation was normal following ablation of CGRP-positive neurons but acute heat pain thresholds were significantly elevated and a reduction in nocifensive responses was observed in the second phase of the formalin test. Ablation of TrkB-positive neurons led to significant deficits in mechanical hypersensitivity in the partial sciatic nerve ligation neuropathic pain model. Conclusions: Ablation of specific DRG neuronal subsets using the AdvDTA line will be a useful resource for further functional characterization of somatosensory processing, neuro-immune interactions and chronic pain disorders.
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Mycobacterial infections are a resurgent and increasingly relevant problem. Within these, tuberculosis (TB) is particularly worrying as it is one of the top ten causes of death in the world and is the infectious disease that causes the highest number of deaths. A further concern is the on-going emergence of antimicrobial resistance, which seriously limits treatment. The COVID-19 pandemic has worsened current circumstances and future infections will be more incident. It is urgent to plan, draw solutions, and act to mitigate these issues, namely by exploring new approaches. The aims of this review are to showcase the extensive research and application of silver nanoparticles (AgNPs) and other metal nanoparticles (MNPs) as antimicrobial agents. We highlight the advantages of mycogenic synthesis, and report on their underexplored potential as agents in the fight against all mycobacterioses (non-tuberculous mycobacterial infections as well as TB). We propose further exploration of this field.
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Quality-by-Design (QbD) is a methodology used to build quality into products and is characterized by a well-defined roadmap. In this study, the application of Artificial Neural Networks (ANNs) in the QbD-based development of a test drug product is presented, where material specifications are defined and correlated with its performance in vivo. Along with other process parameters, drug particle size distribution (PSD) was identified as a critical material attribute and a three-tier specification was needed. An ANN was built with only five hidden nodes in one hidden layer, using hyperbolic tangent functions, and was validated using a random holdback of 33% of the dataset. The model led to significant and valid prediction formulas for the three responses, with R2 values higher than 0.94 for all responses, both for the training and the validation datasets. The prediction formulas were applied to contour plots and tight limits were set based on the design space and feasible working area for the drug PSD, as well as for process parameters. The manufacturing process was validated through the production of three exhibit batches of 180,000 tablets in the industrial GMP facility, and the ANN model was applied to successfully predict the in vitro dissolution, with a bias of approximately 5%. The product was then tested on two clinical studies (under fasting and fed conditions) and the criteria to demonstrate bioequivalence to the Reference Listed Drug were met. In this study, ANNs were successfully applied to support the establishment of drug specifications and limits for process parameters, bridging the formulation development with in vitro performance and the positive clinical results obtained in the bioequivalence studies.
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Química Farmacéutica/métodos , Composición de Medicamentos/métodos , Redes Neurales de la Computación , Tamaño de la Partícula , Control de Calidad , Solubilidad , Comprimidos/química , Equivalencia TerapéuticaRESUMEN
Metal nanoparticles (MNPs) have been extensively studied. They can be produced via different methods (physical, chemical, or biogenic), but biogenic synthesis has become more relevant, mainly for being referred by many as eco-friendly and more advantageous than others. Biogenic MNPs have been largely used in a wide variety of applications, from industry, to agriculture, to health sectors, among others. Even though they are increasingly researched and used, there is still space for exploring further applications and increasing their functionality and our understanding of their synthesis process. Here, we provide an overview of MNPs and biogenic MNPs, and we analyze the potential application of their formation process to astrobiology and the detection of life on Mars and other worlds. According to current knowledge, we suggest that they can be used as potential biosignatures in extra-terrestrial samples. We present the advantages and disadvantages of this approach, suggest further research, and propose its potential use for the search for life in future space exploration.
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One of the applications of Hot-Melt Extrusion (HME) is the stabilization of amorphous drugs through its incorporation into polymeric blends in the form of Amorphous Solid Dispersions (ASDs). In this study, HME was applied to solve a real problem in the development of an ibrutinib product, stabilizing the amorphous form. A systematic approach was followed by combining theoretical calculations, high-throughput screening (HTS) focused on physical stability and Principal Components Analysis (PCA). The HTS enabled the evaluation of 33 formulations for physical stability and the PCA was key to select four promising systems. The low relevance of drug loading on the drug crystallization supported the HME tests with a very high drug load of 50%. Milled extrudates were characterized and demonstrated to be fully amorphous. The thermal analysis detected a glass transition temperature much higher than the predicted values. Along with several weak intermolecular interactions detected in Raman spectroscopy, a dipolar interaction involving the α, ß unsaturated ketone function of ibrutinib was also noticed. The additive effect of these intermolecular interactions changed markedly the performance of the ASDs. The physical strength of the prepared systems was corroborated by stability studies until 6 months at long-term and accelerated conditions.
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Adenina/análogos & derivados , Composición de Medicamentos/métodos , Estabilidad de Medicamentos , Piperidinas/química , Adenina/química , Cristalización , Calor , Polímeros/química , Espectrometría Raman/métodos , Temperatura de TransiciónRESUMEN
The Earth's atmosphere is an extremely large and sparse environment which is quite challenging for the survival of microorganisms. We have long wondered about the limits to life in the atmosphere, starting with Leeuwenhoek's observation of "animalcules" collected from the air. In the past century, significant progress has been made to capture and identify biological material from varying elevations, from a few meters above ground level, to the clouds near mountaintops, and the jet streams, the ozone layer, and even higher up in the stratosphere. Collection and detection techniques have been developed and advanced in order to assess the potential diversity of life from very high altitudes. Studies of microbial life in the stratosphere with its multiple stressors (cold, dry, irradiated, with low pressure and limited nutrients), have recently garnered considerable attention. Here, we review studies of Earth's atmosphere, with emphasis on the stratosphere, addressing implications for astrobiology, the dispersal of microbes around our planet, planetary protection, and climate change.
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Atmósfera , Bacterias/aislamiento & purificación , Planeta Tierra , Ambientes Extremos , Presión Atmosférica , Bacterias/crecimiento & desarrollo , Bacterias/efectos de la radiación , Respuesta al Choque por Frío , Radiación Cósmica , Desecación , Polvo , Exobiología , Nutrientes , Rayos UltravioletaRESUMEN
The aim of this study was to develop a fast, effective, and material sparing screening method to design amorphous solid dispersions (ASDs) of etravirine to drive more effectively the development process, leading to improved bioavailability (BA) and stability. A systematic step-by-step approach was followed by combining theoretical calculations with high-throughput screening (HTS) and software-assisted multivariate statistical analysis. The thermodynamic miscibility and interaction of the drug in several polymers were predicted using Hansen solubility parameters (δ). The selected polymers were evaluated by HTS, using solvent evaporation. Binary compositions were evaluated by their solubilization capacity and physical stability over 2 months. JMP 14.0 was used for multivariate statistical analysis using principal components analysis. Extrusion was performed in Thermo Scientific HAAKE MiniLab II, and extrudates were characterized by assay, related substances, dissolution, and physical state (polarized light microscopy (PLM), Raman spectroscopy, and X-ray powder diffraction (XRPD)). A short stability study was performed where milled extrudates were exposed to 25 °C/60%RH and 40 °C/75%RH for 3 months. Through thermodynamic predictions, five main polymers were selected. The HTS enabled the evaluation of 42 formulations for solubilization capacity and physical stability. The three most promising compositions were selected for hot-melt extrusion (HME) tests. In general, a good correlation was found among the results of theoretical predictions, HTS, and HME. Poly(vinylpyrrolidone) (PVP)-based formulations were shown to be easily extrudable, with low degradation and complete amorphicity, whereas in Soluplus, the drug was not miscible, leading to a high crystalline content. The drug release rate was improved more than two times with PVP, and the manufactured ASD was demonstrated to be stable physically and chemically. A fast and effective screening technique to develop stable ASDs for a poorly soluble drug was successfully developed as applied to etravirine. The given method is easy to use, requires a low amount of drug, and is fairly accurate in predicting the amorphization of the drug when formulated. The success of HME formulation development of etravirine was undoubtedly enhanced with this high-throughput tool, which led to the identification of extrudates with improved biopharmaceutical properties. The structural characterization performed by PLM, XRPD, and Raman spectroscopy demonstrated that the HME prototype was essentially amorphous. The unexpected stability at 40 °C/75%RH was correlated with the presence of molecular interaction characterized by Raman spectroscopy.
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Portadores de Fármacos/química , Composición de Medicamentos/métodos , Evaluación Preclínica de Medicamentos/métodos , Tecnología de Extrusión de Fusión en Caliente/métodos , Nitrilos/química , Nitrilos/farmacocinética , Pirimidinas/química , Pirimidinas/farmacocinética , Disponibilidad Biológica , Química Farmacéutica/métodos , Liberación de Fármacos , Estabilidad de Medicamentos , Excipientes/química , Microscopía de Polarización , Polietilenglicoles/química , Polivinilos/química , Povidona/química , Solubilidad , Espectrometría Raman , Difracción de Rayos XRESUMEN
This study aims to evaluate the effect of freeze-drying and long-term storage on the biotechnological potential of Aspergillus section Nigri strains. Twelve selected strains were freeze-dried and aged by accelerated storage, at 37 °C in the dark, for 2 and 4 weeks. To assess possible changes as a consequence of the ageing in the freeze-drying ampoules, morphological characteristics, mycotoxins and enzymes production, matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALTI-TOF MS) spectra, and M13 phage probe fingerprinting were used as part of a polyphasic approach. Phenotypical changes were observed; nevertheless, they did not substantially affect the potential biotechnological use of these strains. The activity of hydrolytic enzymes (protease, carboxymethylcellulase, xylanase, pectinase and mannanase) was maintained or increased after freeze-drying. MALDI-TOF MS data originated spectra that grouped, for the majority of samples, according to strain independently of preservation time point. M13 profiles revealed the presence of some genetic polymorphisms after preservation. However, the three studied times still clustered for more than 50% of strains. Our results show that the studied strains maintain their biotechnological potential after preservation, with minimal phenotypic alterations. These findings provide evidence that freeze-drying preservation is a suitable option to preserve biotechnologically relevant aspergilli strains from section Nigri, and one should consider that the observed effects might be species/strain-dependent.
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The pharmaceutical development of amorphous solid dispersions (ASDs) by hot-melt extrusion (HME) is briefly reviewed. A systematic step-by-step approach is presented, where thermodynamics, polymer screening, multivariate statistics and process optimization are combined, to increase the success of HME-based drug product development. The quality by design (QbD) concept is introduced and applied to HME. Steps and tools for its effective implementation are provided, including risk assessment highlighting crucial points. The technical and scientific specificities of HME-based ASDs are discussed in light of the current paradigm of drug development and in-line with regulatory guidelines from the ICH regions. Case studies of recently approved HME products are presented.