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Cichlids represent one of the most species-rich families of fishes and have attracted the attention of evolutionary biologists due to the rapid radiation occurring in some groups and the importance of some species in the world aquaculture. Cytogenetic analysis was conducted in 10 cichlid species from the Araguaia River, Amazon Basin, Brazil. The chromosome number was 2n=48 for all analyzed species except for Laetacara araguaiae Ottoni et Costa, 2009 (2n=44). Chromosomal polymorphism was detected only in Geophagus proximus (Castelnau, 1855), which exhibits an extra large submetacentric and and a dot-like chromosomes. Moreover, the C-banding revealed a general pericentromeric heterochromatic pattern and some additional blocks for some species. The heterochromatic blocks corresponding to AgNOR bearing regions were observed in all species and also corresponded to CMA3 positive blocks, which were observed in terminal regions. Besides the general conserved chromosomal and heterochromatin patterns for South American cichlids, the presence of GC-rich heterochromatin was quite different in the species Biotodoma cupido (Heckel, 1840), Geophagus proximus, Retroculus lapidifer (Castelnau, 1855), Crenicichla strigata Günther, 1862 and Heros efasciatus Heckel, 1840. The results suggest that independent events of heterochromatin modification occurred during chromosome evolution in the group, regardless of the conservation of macro-chromosomal structure. [ABSTRACT FROM AUTHOR]

Pitavastatin is commonly prescribed to treat hypercholesterolemia through the regulation of cholesterol biosynthesis. Interestingly, it has also demonstrated a great potential for treating brain tumors, although the detailed cytotoxic mechanism, particularly in glioblastoma, remains incompletely understood. This work explores the activity of pitavastatin in 2D and 3D glioblastoma models, in an attempt to provide a more representative and robust overview of its anticancer potential in glioblastoma. The results show that not only is pitavastatin 10-1000 times-fold more effective in reducing tumoral metabolic activity than temozolomide, but also demonstrate a synergistic activity with this alkylating drug. In addition, low micromolar concentrations of this statin strongly impair the growth and the invasion ability of multicellular tumor spheroids. The obtained qRT-PCR and proteomics data highlight the modulation of cell death via apoptosis (BAX/BCL2, CASP9) and autophagy (BECN1, BNIP3, BNIP3L and LC3B), as well as an epithelial to mesenchymal transition blockage (HTRA1, SERPINE1, WNT5A, ALDH3B1 and EPHA2) and remodeling of the extracellular matrix (VCAN, SERPINE1 and TGFBI). Overall, these results lay the foundation for further investigations on the potential combinatory clinical treatment with temozolomide., Competing Interests: Declaration of Competing Interest The authors declare that there is no financial/personal interest or belief that could affect the manuscript objectivity., (Copyright © 2024. Published by Elsevier B.V.)

Glioblastoma (GBM) stands for the most common and aggressive type of brain tumour in adults. It is highly invasive, which explains its short rate of survival. Little is known about its risk factors, and current therapy is still ineffective. Hence, efforts are underway to develop novel and effective treatment approaches against this type of cancer. Exosomes are being explored as a promising strategy for conveying and delivering therapeutic cargo to GBM cells. They can fuse with the GBM cell membrane and, consequently, serve as delivery systems in this context. Due to their nanoscale size, exosomes can cross the blood-brain barrier (BBB), which constitutes a significant hurdle to most chemotherapeutic drugs used against GBM. They can subsequently inhibit oncogenes, activate tumour suppressor genes, induce immune responses, and control cell growth. However, despite representing a promising tool for the treatment of GBM, further research and clinical studies regarding exosome biology, engineering, and clinical applications still need to be completed. Here, we sought to review the application of exosomes in the treatment of GBM through an in-depth analysis of the scientific and clinical studies on the entire process, from the isolation and purification of exosomes to their design and transformation into anti-oncogenic drug delivery systems. Surface modification of exosomes to enhance BBB penetration and GBM-cell targeting is also a topic of discussion., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

This study focuses on the preparation of layered bacterial nanocellulose (BNC) patches for drug delivery and wound healing in the context of herpes labialis. Nanostructured patches were prepared by selective aqueous diffusion of acyclovir (ACV, antiviral drug), hyaluronic acid (HA, skin healing promoter), and glycerol (GLY, plasticizer and humectant) in the BNC network, followed by assembly into trilayered patches with ACV on the central layer of the patch (ACV T ) or divided between two layers (ACV H ), to modulate drug release. Both patches showed good layers' adhesion and thermal stability (125 °C), UV barrier properties, good static (Young's modulus up to 0.9 GPa (dry) and 0.7 GPa (wet)) and dynamic mechanical performance, and adhesion strength (21 kPa) comparable to or higher than other materials and commercial adhesives for wound healing. In vitro drug dissolution showed faster ACV release from the ACV H patch (77 ± 5 %, 10 min) than from the ACV T one (50 ± 7 %), suggesting efficient drug delivery. ACVH closely resembled a commercial cream formulation in terms of release and permeation profiles. The patches were non-cytotoxic toward L929 fibroblasts, promoting cell adhesion and wound closure (in vitro). These results underscore the dual-action potential of the layered patches for managing herpetic lesions., Competing Interests: Declaration of competing interest The authors proclaim that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this manuscript., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Glioblastoma (GB) is the most common and lethal primary form of malignant brain cancers. Its intrinsic aggressiveness and the blood-brain barrier (BBB) are two major factors that limit the efficacy of standard therapies. In recent years, nanostructured lipid carriers (NLCs) have established themselves as a promising avenue for improving drug delivery to the brain, overcoming the challenges associated with the low drug permeability of the BBB. This work delves into the systematic development of a novel carrier for pitavastatin delivery by establishing a "get it right at the first time" quality by design perspective, supported by multivariate analysis, computational modelling, and molecular docking. The manufacturing process was comprehensively evaluated at each step, from raw material selection to NLC purification, thus leading to a carrier with optimal colloidal, encapsulation and stability properties. The cytotoxic behaviour of the carrier was assessed in a panel of GB cell lines, which includes a low, a medium and a highly sensitive cell line to pitavastatin, providing a better discriminatory power and addressing the different potential in the therapeutic responses. The results obtained reflect a strong antiglioblastoma activity in concentrations where the standard of care lacks activity, as well as a swift and prominent internalization by GB cells. Overall, this work positions these long-term stable parenteral systems in line with the hypothesis of providing more effective surrogate therapeutics in the field of GB., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: João Basso reports financial support was provided by Foundation for Science and Technology. The remaining authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Glioblastoma (GB) is one of the most lethal types of neoplasms with unique anatomic, physiologic, and pathologic features that usually persist after exposure to standard therapeutic modalities. It is biologically aggressive, and the existence of the blood-brain barrier (BBB) limits the efficacy of standard therapies. In this work, we hypothesize the potential of surface-functionalized ultra-small nanostructured lipid carriers (usNLCs) with charge-switchable cell-penetrating peptides (CPPs) to overcome this biological barrier and improve targeted delivery to brain tumor tissues. The big question is: what is the potential of CPPs in directing nanoparticles toward brain tumor tissue? To answer this question, the usNLCs were functionalized with distinct biomolecules [five CPPs, c(RGDfK) and transferrin, Tf] through electrostatic interaction and its ability as a targeting approach to BBB (HBMEC) and glioma cells (U87 cells) evaluated in terms of physicochemical properties, cellular uptake, permeability in a 2D-BBB model, and tumor growth inhibition. Monte Carlo simulations elucidated CPP adsorption patterns. The permeability studies revealed that targeted usNLCs, especially usNLCs Tf and usNLCs CPP4 , exhibited an increased permeability coefficient compared to the non-targeted usNLCs. Functionalized usNLCs evidenced enhanced uptake in BBB cells, with smaller CPPs showing higher internalization (CPP1 and CPP2). Similarly, functionalized usNLCs exhibited more significant cytotoxicity in glioma cells, with specific CPPs promoting favorable internalization. Analysis of the endocytic pathway indicated that usNLCs CPPs were mainly internalized by direct translocation and caveolae-mediated endocytosis. Optimal usNLCs with dual targeting capabilities to both BBB and GB cells provide a promising therapeutic strategy for GB., Competing Interests: Declaration of conflict of interests The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Despite the continuous research on understanding how topical drugs and the skin interact, the development of a topical generic product remains a challenge. Due to their local action effect rather than systemic, establishing suitable frameworks for documenting bioequivalence between reference and test formulations is anything but straightforward. In previous years, clinical endpoint trials were considered the gold standard method to demonstrate bioequivalence between topical products. Nevertheless, significant financial and time resources were required to be allocated owing to the inherent complexity of these studies. To address this problem, regulatory authorities have begun to accept alternative approaches that could lead to a biowaiver, avoiding the need for clinical endpoint trials. These alternatives encompass various in vitro and/or in vivo techniques that have been analysed and the benefits and drawbacks of each method have been considered. Furthermore, other factors like the integration of a quality by design framework to ensure a comprehensive understanding of the product and process quality attributes have also been taken into account. This review delves into international regulatory recommendations for semisolid topical products, with a focus on those established by the European Medicines Agency, as well as the Food and Drug Administration. Both approaches were carefully examined, discussing aspects such as acceptance criteria, sample size, and microstructure evaluation. Additionally, novel and innovative therapeutic-driven approaches based on in vitro disease models for the rapid and effective development of topical generic products are presented., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Glioblastoma (GBM) remains the epitome of aggressiveness and lethality in the spectrum of brain tumors, primarily due to the blood-brain barrier (BBB) that hinders effective treatment delivery, tumor heterogeneity, and the presence of treatment-resistant stem cells that contribute to tumor recurrence. Nanoparticles (NPs) have been used to overcome these obstacles by attaching targeting ligands to enhance therapeutic efficacy. Among these ligands, peptides stand out due to their ease of synthesis and high selectivity. This article aims to review single and multiligand strategies critically. In addition, it highlights other strategies that integrate the effects of external stimuli, biomimetic approaches, and chemical approaches as nanocatalytic medicine, revealing their significant potential in treating GBM with peptide-functionalized NPs. Alternative routes of parenteral administration, specifically nose-to-brain delivery and local treatment within the resected tumor cavity, are also discussed. Finally, an overview of the significant obstacles and potential strategies to overcome them are discussed to provide a perspective on this promising field of GBM therapy.

The fundamental idea underlying the use of amorphous solid dispersions (ASDs) is to make the most of the solubility advantage of the amorphous form of a drug. However, the drug stability becomes compromised due to the higher free energy and disorder of molecular packing in the amorphous phase, leading to crystallization. Polymers are used as a matrix to form a stable homogeneous amorphous system to overcome the stability concern. The present work aims to design ASD-based formulations under the umbrella of quality by design principles for improving oral drug bioavailability, using celecoxib (CXB) as a model drug. ASDs were prepared from selected polymers and tested both individually and in combinations, using various manufacturing techniques: high-shear homogenization, high-pressure homogenization, microfluidics-on-a-chip, and spray drying. The resulting dispersions were further optimized, resorting to a 3 2 full-factorial design, considering the drug:polymers ratio and the total solid content as variables. The formulated products were evaluated regarding analytical centrifugation and the influence of the different polymers on the intrinsic dissolution rate of the CXB-ASDs. Microfluidics-on-a-chip led to the amorphous status of the formulation. The in vitro evaluation demonstrated a remarkable 26-fold enhancement in the intrinsic dissolution rate, and the translation of this formulation into tablets as the final dosage form is consistent with the observed performance enhancement. These findings are supported by ex vivo assays, which exhibited a two-fold increase in permeability compared to pure CXB. This study tackles the bioavailability hurdles encountered with diverse active compounds, offering insights into the development of more effective drug delivery platforms., (© 2024. The Author(s).)

Olive anthracnose, caused by Colletotrichum fungi, and the olive fruit fly Bactrocera olea are, respectively, the most important fungal disease and pest affecting olive fruits worldwide, leading to detrimental effects on the yield and quality of fruits and olive oil. This study focuses on the content of hydroxytyrosol (HYT) and its derivatives (the "olive oil polyphenols" health claim) in olive oils extracted from fruits of 'Galega Vulgar' and 'Cobrançosa' cultivars, naturally affected by olive anthracnose and olive fly. The olives, with different damage levels, were harvested from organic rainfed orchards, located in the center of Portugal, at four harvest times over three years. Galega oils extracted from olives with a higher anthracnose and olive fly incidence showed no conformity for the extra virgin olive oil (EVOO) and virgin olive oil (VOO) categories, presenting high acidity and negative sensory notes accompanied by the disappearance of oleacein. Conversely, no sensory defects were observed in Cobrançosa oils, regardless of disease and pest incidence levels, and quality criteria were still in accordance with the EVOO category. The total HYT and tyrosol (TYR) content (>5 mg/20 g) allows for the use of the "olive oil polyphenols" health claim on the label of all the analyzed Cobrançosa olive oils.

This study focuses on the characterisation and incorporation of Moringa oleifera leaf powder (MOP) from Luanda (Angola) and its extract (MOE) in fortified chocolate mousse. Dark green (DG) leaves presented superior nutritional values compared to other leaves. DG contained a higher concentration of mineral salts (10 ± 1 mg/100 g of dry leaves), phenolic compounds (267 ± 4 mg GAE/g), vitamins (1.9 ± 0.2 mg/g of dry extract) and strong antioxidant capacity (IC 50 , 115 ± 8 µg/mL). Therefore, DG leaves were used to fortify the chocolate mousse. The leaves were prepared in three samples: control, 2 % MOP (w/w) and 2 % MOE (v/v). Textural and rheological analysis of chocolate mousse samples revealed a pseudoplastic profile for all samples, with decreased texture attributes and viscosity due to the incorporation. The sensory evaluation demonstrated that MOP and MOE samples presented 93 % and 88 % resemblance to the original product regarding general acceptance, respectively., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Over the past decade, topically applied drug products have experienced extraordinary price increases, due to the shortage of multisource generic drug products. This occurrence is mainly related to the underlying challenges evolved in topical bioequivalence documentation. Although there has been continuing regulatory efforts to present surrogate in vitro methods to clinical endpoint studies, there is still a continued need for cost- and time-efficient alternatives that account for product specificities. Hence, this work intended to expose bioequivalence assessment issues for complex topical formulations, and more specifically those related with product efficacy guidance. As a model drug and product, a bifonazole 10 mg/g cream formulation was selected and two different batches of the commercially available Reference Product (RP) were used: RP1 that displayed lower viscosity and RP4 which presented high, but not the highest, viscosity. In vitro human skin permeation testing (IVPT) was carried out and the results were evaluated by means of the traditional bioequivalence assessment approach proposed by the EMA, as well as by the Scaled Average Bioequivalence assessment approach proposed by the FDA. Based on previous experience, there was an expectation of a high level of variability in the results, thus alternative methods to evaluate local drug skin availability were developed. More specifically, an infected skin disease model, where ex vivo human skin was infected and ATP levels were used as a biological marker for monitoring antifungal activity after product application. The results showed that permeation equivalence could not be supported between the different RP batches. In contrast, this statistical difference between the formulation batches was not indicated in the disease model. Nevertheless, in pivotal IVPT studies, the lowest permeant formulation (RP4) evidenced a higher antifungal in vitro activity as reported by the lower levels of ATP. A critical appraisal of the results is likewise presented, focusing on an outlook of the real applicability of the regulatory guidances on this subject., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

This review outlines the evolutionary journey from traditional two-dimensional (2D) cell culture to the revolutionary field of organ-on-a-chip technology. Organ-on-a-chip technology integrates microfluidic systems to mimic the complex physiological environments of human organs, surpassing the limitations of conventional 2D cultures. This evolution has opened new possibilities for understanding cell-cell interactions, cellular responses, drug screening, and disease modeling. However, the design and manufacture of microchips significantly influence their functionality, reliability, and applicability to different biomedical applications. Therefore, it is important to carefully consider design parameters, including the number of channels (single, double, or multi-channels), the channel shape, and the biological context. Simultaneously, the selection of appropriate materials compatible with the cells and fabrication methods optimize the chips' capabilities for specific applications, mitigating some disadvantages associated with these systems. Furthermore, the success of organ-on-a-chip platforms greatly depends on the careful selection and utilization of cell resources. Advances in stem cell technology and tissue engineering have contributed to the availability of diverse cell sources, facilitating the development of more accurate and reliable organ-on-a-chip models. In conclusion, a holistic perspective of in vitro cellular modeling is provided, highlighting the integration of microfluidic technology and meticulous chip design, which play a pivotal role in replicating organ-specific microenvironments. At the same time, the sensible use of cell resources ensures the fidelity and applicability of these innovative platforms in several biomedical applications.

The COVID-19 pandemic prompted a social, economic and health crisis that had a major impact on the mental health of the global community, particularly nurses. The objective of the current study is to conduct a longitudinal evaluation of the trajectory of depressive, anxiety, trauma, and fear of COVID-19 symptoms, comparing self-reports of nurses and the general population over a six-month period. Self-report questionnaires were administered online to a sample of 180 nurses and 158 individuals from the general population for the baseline assessment (T1) and follow-up at 6 months (T2). Levels of symptoms reported by nurses were generally greater and tended to worsen over time, as opposed to the levels of symptoms reported by the general population that tended to improve. Levels of depressive, anxiety, and trauma symptoms were significantly different between nurses and the general population over time. Levels of fear of COVID-19 declined significantly from T1 to T2 in both groups. These results suggest that it is crucial to monitor the longer-term effects of COVID-19 and to develop resilience-promoting interventions tailored to the unique needs of this vulnerable group., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Vitorino et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Hydrogels are polymeric materials that possess a set of characteristics meeting various requirements of an ideal wound dressing, making them promising for wound care. These features include, among others, the ability to absorb and retain large amounts of water and the capacity to closely mimic native structures, such as the extracellular matrix, facilitating various cellular processes like proliferation and differentiation. The polymers used in hydrogel formulations exhibit a broad spectrum of properties, allowing them to be classified into two main categories: natural polymers like collagen and chitosan, and synthetic polymers such as polyurethane and polyethylene glycol. This review offers a comprehensive overview and critical analysis of the key polymers that can constitute hydrogels, beginning with a brief contextualization of the polymers. It delves into their function, origin, and chemical structure, highlighting key sources of extraction and obtaining. Additionally, this review encompasses the main intrinsic properties of these polymers and their roles in the wound healing process, accompanied, whenever available, by explanations of the underlying mechanisms of action. It also addresses limitations and describes some studies on the effectiveness of isolated polymers in promoting skin regeneration and wound healing. Subsequently, we briefly discuss some application strategies of hydrogels derived from their intrinsic potential to promote the wound healing process. This can be achieved due to their role in the stimulation of angiogenesis, for example, or through the incorporation of substances like growth factors or drugs, such as antimicrobials, imparting new properties to the hydrogels. In addition to substance incorporation, the potential of hydrogels is also related to their ability to serve as a three-dimensional matrix for cell culture, whether it involves loading cells into the hydrogel or recruiting cells to the wound site, where they proliferate on the scaffold to form new tissue. The latter strategy presupposes the incorporation of biosensors into the hydrogel for real-time monitoring of wound conditions, such as temperature and pH. Future prospects are then ultimately addressed. As far as we are aware, this manuscript represents the first comprehensive approach that brings together and critically analyzes fundamental aspects of both natural and synthetic polymers constituting hydrogels in the context of cutaneous wound healing. It will serve as a foundational point for future studies, aiming to contribute to the development of an effective and environmentally friendly dressing for wounds.

By definition, biosimilar medicinal products are biological medicinal products that are similar to other biological medicinal products that are already on the market-the reference medicinal products. Access to biosimilar medicines is a current reality. However, to achieve this goal, it is extremely important to consistently and scientifically substantiate the regulatory requirements necessary for biosimilar medicines when accessing the market. Based on an analysis of the raw materials and the type of methods used in the manufacturing processes of biological medicines, it is known that this tends to be more complex for the quality of the finished product than the manufacture of molecules obtained through a chemical process. It is then relevant to highlight the main differences between both products: biological medicines manufactured using biotechnology and the current generics containing active pharmaceutical ingredients (APIs) obtained from synthetic processes. Once arriving at the approval process of these medicinal products, it is imperative to analyse the guidance documents and the regulatory framework that create the rules that allow these biosimilar medicinal products to come to the market. The present review aimed at documenting comparatively the specific provisions of European legislation, through the European Medicines Agency (EMA), as well as the legislation of the United States of America, through the Food and Drug Administration (FDA). This was then translated into a critical appraisal of what concerns the specific criteria that determine the favourable evaluation of a biosimilar when an application for marketing authorisation is submitted to different regulatory agencies. The gathered evidence suggests that the key to the success of biosimilar medicines lies in a more rigorous and universal regulation as well as a greater knowledge, acceptance, and awareness of health professionals to enable more patients to be treated with biological strategies at an earlier stage of the disease and with more affordable medicines, ensuring always the safety and efficacy of those medicines.

Over the last years, the pharmaceutical industry has faced real challenges regarding quality assurance. In this context, the establishment of more holistic approaches to the pharmaceutical development has been encouraged. The emergence of the Quality by Design (QbD) paradigm as systematic, scientific and risk-based methodology introduced a new concept of pharmaceutical quality. In essence, QbD can be interpreted as a strategy to maximize time and cost savings. An in-depth understanding of the formulation and manufacturing process is demanded to optimize the safety, efficacy and quality of a drug product at all stages of development. This innovative approach streamlines the pharmaceutical Research and Development (R&D) process, provides greater manufacturing flexibility and reduces regulatory burden. To assist in QbD implementation, International Conference on Harmonisation (ICH), U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) organized and launched QbD principles in their guidance for industry, identifying key concepts and tools to design and develop a high-quality drug product. Despite the undeniable advantages of the QbD approach, and the widespread information on QbD regulatory expectations, its full implementation in the pharmaceutical field is still limited. The present review aims to establish a crosswise overview on the current application status of QbD within the framework of the ICH guidelines (ICH Q8(R2) - Q14 and ICH Q2(R2)). Moreover, it outlines the way information gathered from the QbD methodology is being harmonized in Marketing Authorization Applications (MAAs) for European market approval. This work also highlights the challenges that hinder the deployment of the QbD strategy as a standard practice., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Glioblastoma (GB) is one of the most lethal types of neoplasms. Its biologically aggressive nature and the presence of the blood-brain barrier (BBB) limit the efficacy of standard therapies. Several strategies are currently being developed to both overcome the BBB and deliver drugs site specifically to tumor cells. This work hypothesizes a two-pronged approach to tackle GB: drug repurposing with celecoxib (CXB) and a nanoformulation using ultra-small nanostructured lipid carriers (usNLCs). CXB antitumor druggable activity was inspected bioinformatically and screened in four glioma cell lines aiming at the comparison with temozolomide (TMZ), as standard of care. Delving into formulation design, it was tailored aiming at (i) improving the drug solubility/loading properties, (ii) assigning a thermal-triggerable drug release based on a lipid matrix with a low melting point, and (iii) enhancing the cytotoxic effect by selecting a template targetable to tumor cells. For this purpose, an integrated analysis of the critical material attributes (CMAs), critical process parameters (CPPs), and critical quality attributes (CQAs) was conducted under the umbrella of a quality by design approach. CMAs that demonstrate a high-risk level for the final quality and performance of the usNLCs include the drug solubility in lipids (solid and liquid), the lipid composition (envisioning a thermoresponsive approach), the ratio between lipids (solid vs. liquid), and the surfactant type and concentration. Particle size was shown to be governed by the interaction lipid-surfactant followed by surfactant type. The drug encapsulation did not influence colloidal characteristics, making it a promising carrier for lipophilic drugs. In general, usNLCs exhibited a controlled drug release during the 72 h at 37 °C with a final release of ca. 25%, while at 45 °C this was doubled. The in vitro cellular performance depended on the surfactant type and lipid composition, with the formulations containing a sole solid lipid (Suppocire ® NB) and Kolliphor ® RH40 as surfactant being the most cytotoxic. usNLCs with an average diameter of ca. 70 nm and a narrow size distribution (PdI lower than 0.2) were yielded, exhibiting high stability, drug protection, sustained and thermo-sensitive release properties, and high cytotoxicity to glioma cells, meeting the suitable CQAs for parenteral administration. This formulation may pave the way to a multi-addressable purpose to improve GB treatment., (© 2023. The Author(s).)

The present study aims to outline a rational framework for the design and development of a 1.0% (w/v) hydrocortisone nanocrystal-based formulation, resorting to a simple, efficient, and scalable nanonization methodology, based on the high-pressure homogenization (HPH) technique. Accordingly, the innovative product was comprehensively optimized following a Quality by Design (QbD) approach. The thorough selection of formulation composition was driven by a dual purpose: improving skin permeation and stability. In the early stage of development, a Failure Mode, Effects and Criticality Analysis (FMECA) diagram was employed to identify the most impactful variables for the critical quality attributes (CQAs). In this sense, a rotatable, three-factor and five-level circumscribed central composite design (CCCD) was applied to investigate how squalene concentration (x 1 ), soluplus concentration (x 2 ) and HPH-time (x 3 ) influence physicochemical properties, performance and physical stability of the formulation. A robust Design Space (DS) was defined, establishing the optimal settings for the critical variables, whose combination meets the requirements set in the quality target product profile (QTPP). Morphological analysis revealed the cuboidal shape of hydrocortisone nanocrystals. In what concerns colloidal properties, the most promising formulation disclosed a small particle size (Dx(50) = 311.8 ± 1.5 nm), along with narrow size distribution (span value = 1.91 ± 0.17). Zeta potential results (-2.19 ± 0.15 mV--12.1 ± 0.4 mV) suggested a steric hindrance stabilization. FTIR spectra showed no chemical interactions between drug and formulation components. XRD diffractograms confirmed loss of crystallinity during the downsizing process. In vitro studies revealed an improvement on drug release rate (316 ± 21-516 ± 35 μg/cm 2 /√t), compared to the coarse suspension and commercial products, and a straight dependence on the stabilizer concentration and HPH time. The permeation flux across the skin (0.16 ± 0.02-1.2 ± 0.5 μg/cm 2 /h) appeared to be dependent on the drug physicochemical properties, in particular saturation solubility. Further characterization of the experimental formulations pointed out the role of the stabilizing component to prevent against physical instability phenomena. This organic solvent-free, and therefore "green" nanocrystal production technology offers great potential for pharmaceutical R&D and drug delivery by enabling the development of new forms of conventional drugs with optimal physicochemical properties and performance., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Ana Simoes reports financial support was provided by Fundação para a Ciência e a Tecnologia., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

The need for consistency in analytical method development reinforces the dependence of pharmaceutical product development and manufacturing on robust analytical data. The Analytical Quality by Design (AQbD), akin to the product Quality by Design (QbD) endows a high degree of confidence to the method quality developed. AQbD involves the definition of the analytical target profile as starting point, followed by the identification of critical method variables and critical analytical attributes, supported on risk assessment and design of experiment tools for the establishment of a method operable design region and control strategy of the method. This systematic approach moves away from reactive troubleshooting to proactive failure reduction. The objective of this review is to highlight the elements of the AQbD framework and provide an overview of their implementation status in various analytical methods used in the pharmaceutical field. These methodologies include but are not limited to, high-performance liquid chromatography, UV-Vis spectrophotometry, capillary electrophoresis, supercritical fluid chromatography, and high-performance thin-layer chromatography. Finally, a critical appraisal is provided to highlight how regulators have encouraged AQbD principles application to boost the prevention of method failures and a better understanding of the method operable design region (MODR) and control strategy, ultimately resulting in cost-effectiveness and regulatory flexibility.

The buccal mucosa is arising within the pharmaceutical landscape as an attractive option for local and systemic drug delivery, mostly due to its high vascularization, inherent permeability and robustness. Still, one of the major challenges in bringing oromucosal preparations to market remains the accurate evaluation of permeability. During pre-clinical drug development, in vitro permeation assessment is essential, and methodologies, based on the selection of a proper membrane in a diffusion cell, have become appealing alternatives to the conventional cell-based models. The development of such methods is being constrained by the number of variables - related to study conditions, setup and formulation - that need to be optimized to accurately estimate buccal permeation. The gap of knowledge over the mentioned variables may lead to long costly developments and poorly accurate methods, especially if the empirical analytical approach is used. In this paper, a systematic risk-based analytical quality by design approach was applied to the development of a buccal in vitro permeation method, ensuring that all sources of variability affecting permeation process were identified, explained and managed by appropriate measures. Researchers are guided through a step by step model, successfully demonstrating with experimental data the impact of critical variables on method's performance., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Analytical method validation ensures that a method provides trustworthy information about a particular sample when applied in accordance with the predefined protocol. According to regulatory standards, the rheological characteristics of topically applied semisolid formulations are one of the key elements involved in microstructure equivalence documentation. Therefore, for generic drug product manufacturers, it is a dire need to take a step forward in rheology method development and validation procedures. This paper aims to apply Analytical Quality by Design (AQbD) principles towards the development and validation of rheology methods for topical creams, as complex semisolid formulations. Risk assessment was carried out through an Ishikawa diagram and an estimate failure mode, effects, and criticality analysis (FMECA). Sample application, peltier temperature control, and sample rest time were identified as critical method variables (CMVs), and a 2 3 full factorial design was applied to understand their impact on rotational, creep recovery and, oscillatory measurements. The development of the method was carried out as per the ICH Q8-Q10, and Q14 guidelines and validated according to ICH Q2 (R2) guideline. The method demonstrated adequate precision (RSD < 15%), as well as selectivity. AQbD provided a comprehensive framework for developing a reliable and effective rheology method for this type of formulation.

The most aggressive primary malignant brain tumor in adults is glioblastoma (GBM), which has poor overall survival (OS). There is a high relapse rate among patients with GBM despite maximally safe surgery, radiation therapy, temozolomide (TMZ), and aggressive treatment. Hence, there is an urgent and unmet clinical need for new approaches to managing GBM. The current study identified modules (MYC, EGFR, PIK3CA, SUZ12, and SPRK2) involved in GBM disease through the NeDRex plugin. Furthermore, hub genes were identified in a comprehensive interaction network containing 7560 proteins related to GBM disease and 3860 proteins associated with signaling pathways involved in GBM. By integrating the results of the analyses mentioned above and again performing centrality analysis, eleven key genes involved in GBM disease were identified. ProteomicsDB and Gliovis databases were used for determining the gene expression in normal and tumor brain tissue. The NetworkAnalyst and the mGWAS-Explorer tools identified miRNAs, SNPs, and metabolites associated with these 11 genes. Moreover, a literature review of recent studies revealed other lists of metabolites related to GBM disease. The enrichment analysis of identified genes, miRNAs, and metabolites associated with GBM disease was performed using ExpressAnalyst, miEAA, and MetaboAnalyst tools. Further investigation of metabolite roles in GBM was performed using pathway, joint pathway, and network analyses. The results of this study allowed us to identify 11 genes ( UBC , HDAC1 , CTNNB1 , TRIM28 , CSNK2A1 , RBBP4 , TP53 , APP , DAB1 , PINK1 , and RELN ), five miRNAs (hsa-mir-221-3p, hsa-mir-30a-5p, hsa-mir-15a-5p, hsa-mir-130a-3p, and hsa-let-7b-5p), six metabolites (HDL, N6-acetyl-L-lysine, cholesterol, formate, N, N-dimethylglycine/xylose, and X2. piperidinone) and 15 distinct signaling pathways that play an indispensable role in GBM disease development. The identified top genes, miRNAs, and metabolite signatures can be targeted to establish early diagnostic methods and plan personalized GBM treatment strategies.

In recent years, the regulatory mechanisms for topical generic product bioequivalence (BE) assessment have been subjected to noteworthy changes, with the FDA issuing product specific guidances, and the EMA adopting a more universal approach with the quality and equivalence of topical products draft guideline. The agencies advise on a modular strategy for BE documentation. Nevertheless, their scope, data analysis and criteria are rather distinct. This study aims to tackle bioequivalence assessment issues of complex topical formulations starting by statistical implications of the EMA/FDA approaches concerning the documentation of qualitative (Q1), quantitative (Q2), microstructure (Q3) and performance requirements (Q4). As a model drug product, a bifonazole 10 mg/g cream formulation was selected. For this specific formulation, the commercially available Reference Product (RP) was compared with two comparator products, also commercially available, referred to as comparator product A (CPA) and comparator product B (CPB). The former displays Q1 sameness and Q2 differences, whilst CPB is categorically considered as Q1/Q2 different. Furthermore, intending to establish a regulatory rationale for the submission of a generic product according to the updated regulatory requirements, the RP was likewise compared with a Test Product (TP). This formulation was designed to display equal Q1/Q2 profile to that of the RP. Validated rheology and in vitro release test (IVRT) methods were used to infer on Q3/Q4 characteristics. During rheology studies, statistically significant RP batch to batch differences were observed. Therefore, in an attempt to surpass this heterogeneity, the initial pool of RP batches was expanded to include RP product batches at different lifecycle stages. Despite this effort, it was not possible to classify the RP/TP, RP/CPA or RP/CPB as rheologically equivalent products. Nevertheless, product performance results, retrieved from IVRT, were able to sustain equivalence between the RP and the formulations exhibiting Q1 sameness (TP and CPA). It should however be mentioned, that for some RP batch combinations, the IVRT results failed to demonstrate equivalence according to the EMA requirements. Enlarging the RP batch pool was then a critical step in further understanding an optimum statistical approach for establishing equivalence in product performance. This study highlights the need to that a 'one-fits-all approach' may not be an optimum path way for establishing the regulatory strategy and requirements to support generic product bioequivalence., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)