Hot-Melt Extrusion: a Roadmap for Product Development.

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.

Five-Stage Approach for a Systematic Screening and Development of Etravirine Amorphous Solid Dispersions by Hot-Melt Extrusion.

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.

Application of microbial fuel cell technology for vinasse treatment and bioelectricity generation.

OBJECTIVE: Our study evaluated the performance of different two-chambered microbial fuel cell (MFC) prototypes, operated with variable distance between electrodes and Nafion membrane and specific inoculum concentration, applied for vinasse treatment. RESULTS: The performance of the developed MFC resulted in a maximum current density of 1200 mA m-2 and power density of 800 mW m-2 in a period of 61 days. MFC performed a chemical oxygen demand removal at a rate ranging from 51 to 60%. CONCLUSIONS: Taking our preliminary results into consideration, we concluded that the MFC technology presents itself as highly promising for the treatment of vinasse.

Bioprospecting Red Sea Coastal Ecosystems for Culturable Microorganisms and Their Antimicrobial Potential.

Microorganisms that inhabit unchartered unique soil such as in the highly saline and hot Red Sea lagoons on the Saudi Arabian coastline, represent untapped sources of potentially new bioactive compounds. In this study, a culture-dependent approach was applied to three types of sediments: mangrove mud (MN), microbial mat (MM), and barren soil (BS), collected from Rabigh harbor lagoon (RHL) and Al-Kharrar lagoon (AKL). The isolated bacteria were evaluated for their potential to produce bioactive compounds. The phylogenetic characterization of 251 bacterial isolates based on the 16S rRNA gene sequencing, supported their assignment to five different phyla: Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, and Planctomycetes. Fifteen putative novel species were identified based on a 16S rRNA gene sequence similarity to other strain sequences in the NCBI database, being ≤98%. We demonstrate that 49 of the 251 isolates exhibit the potential to produce antimicrobial compounds. Additionally, at least one type of biosynthetic gene sequence, responsible for the synthesis of secondary metabolites, was recovered from 25 of the 49 isolates. Moreover, 10 of the isolates had a growth inhibition effect towards Staphylococcus aureus, Salmonella typhimurium and Pseudomonas syringae. We report the previously unknown antimicrobial activity of B. borstelensis, P. dendritiformis and M. salipaludis against all three indicator pathogens. Our study demonstrates the evidence of diverse cultured microbes associated with the Red Sea harbor/lagoon environments and their potential to produce antimicrobial compounds.

The archaeal class Halobacteria and astrobiology: Knowledge gaps and research opportunities.

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.

Enhanced solid-state stability of amorphous ibrutinib formulations prepared by hot-melt extrusion.

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.

Artificial neural networks applied to quality-by-design: From formulation development to clinical outcome.

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.

Hot-melt extrusion in the pharmaceutical industry: toward filing a new drug application.

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.

Polyphasic, Including MALDI-TOF MS, Evaluation of Freeze-Drying Long-Term Preservation on Aspergillus (Section Nigri) Strains.

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.

Establishment of a Quality Management System Based on ISO 9001 Standard in a Public Service Fungal Culture Collection.

Collaborations between different Microbiological Resource Centres (mBRCs) and ethical sourcing practices are mandatory to guarantee biodiversity conservation, successful and sustainable preservation and fair share of benefits that arise from the use of genetic resources. Since microbial Culture Collections (CCs) are now engaged in meeting high quality operational standards, they are facing the challenge of establishing quality control criteria to certify their biological materials. The authentication/certification of strains is nowadays a demand from the bioeconomy sector for the global operation of mBRCs. The achievement of consistent quality assurance and trust within the mBRCs and microbial CCs context is a dynamic and never-ending process. A good option to facilitate that process is to implement a Quality Management System (QMS) based on the ISO 9001 standard. Here, we report a detailed description of all the steps taken for the QMS implementation at the Portuguese CC of filamentous fungi: Micoteca da Universidade do Minho (MUM). Our aim is to provide guidelines for the certification of other CCs, so that they can also enhance the search and choice of the most consistent, reliable, and effective operating methods, with assured procedures and validation of preservation; and guarantee trustworthy relations with all stakeholders.

High Laccase Expression by Trametes versicolor in a Simulated Textile Effluent with Different Carbon Sources and PHs.

Textile effluents are highly polluting and have variable and complex compositions. They can be extremely complex, with high salt concentrations and alkaline pHs. A fixed-bed bioreactor was used in the present study to simulate a textile effluent treatment, where the white-rot fungus, Trametes versicolor, efficiently decolourised the azo dye Reactive Black 5 over 28 days. This occurred under high alkaline conditions, which is unusual, but advantageous, for successful decolourisation processes. Active dye decolourisation was maintained by operation in continuous culture. Colour was eliminated during the course of operation and maximum laccase (Lcc) activity (80.2 U∙L(-1)) was detected after glycerol addition to the bioreactor. Lcc2 gene expression was evaluated with different carbon sources and pH values based on reverse transcriptase-PCR (polymerase chain reaction). Glycerol was shown to promote the highest lcc2 expression at pH 5.5, followed by sucrose and then glucose. The highest levels of expression occurred between three and four days, which corroborate the maximum Lcc activity observed for sucrose and glycerol on the bioreactor. These results give new insights into the use of T. versicolor in textile dye wastewater treatment with high pHs.