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1.
Pharm Res ; 40(5): 1115-1140, 2023 May.
Artigo em Inglês | MEDLINE | ID: mdl-36456666

RESUMO

Biopharmaceuticals have established an indisputable presence in the pharmaceutical pipeline, enabling highly specific new therapies. However, manufacturing, isolating, and delivering these highly complex molecules to patients present multiple challenges, including the short shelf-life of biologically derived products. Administration of biopharmaceuticals through inhalation has been gaining attention as an alternative to overcome the burdens associated with intravenous administration. Although most of the inhaled biopharmaceuticals in clinical trials are being administered through nebulization, dry powder inhalers (DPIs) are considered a viable alternative to liquid solutions due to enhanced stability. While freeze drying (FD) and spray drying (SD) are currently seen as the most viable solutions for drying biopharmaceuticals, spray freeze drying (SFD) has recently started gaining attention as an alternative to these technologies as it enables unique powder properties which favor this family of drug products. The present review focus on the application of SFD to produce dry powders of biopharmaceuticals, with special focus on inhalation delivery. Thus, it provides an overview of the critical quality attributes (CQAs) of these dry powders. Then, a detailed explanation of the SFD fundamental principles as well as the different existing variants is presented, together with a discussion regarding the opportunities and challenges of SFD as an enabling technology for inhalation-based biopharmaceuticals. Finally, a review of the main formulation strategies and their impact on the stability and performance of inhalable biopharmaceuticals produced via SDF is performed. Overall, this review presents a comprehensive assessment of the current and future applications of SFD in biopharmaceuticals for inhalation delivery.


Assuntos
Produtos Biológicos , Secagem por Atomização , Humanos , Administração por Inalação , Liofilização , Inaladores de Pó Seco , Pós , Tamanho da Partícula , Aerossóis
2.
Stem Cells ; 34(12): 2861-2874, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27603577

RESUMO

Conversion of astrocytes to neurons, via de-differentiation to neural stem cells (NSC), may be a new approach to treat neurodegenerative diseases and brain injuries. The signaling factors affecting such a cell conversion are poorly understood, and they are hard to identify in complex disease models or conventional cell cultures. To address this question, we developed a serum-free, strictly controlled culture system of pure and homogeneous "astrocytes generated from murine embryonic stem cells (ESC)." These stem cell derived astrocytes (mAGES), as well as standard primary astrocytes resumed proliferation upon addition of FGF. The signaling of FGF receptor tyrosine kinase converted GFAP-positive mAGES to nestin-positive NSC. ERK phosphorylation was necessary, but not sufficient, for cell cycle re-entry, as EGF triggered no de-differentiation. The NSC obtained by de-differentiation of mAGES were similar to those obtained directly by differentiation of ESC, as evidenced by standard phenotyping, and also by transcriptome mapping, metabolic profiling, and by differentiation to neurons or astrocytes. The de-differentiation was negatively affected by inflammatory mediators, and in particular, interferon-γ strongly impaired the formation of NSC from mAGES by a pathway involving phosphorylation of STAT1, but not the generation of nitric oxide. Thus, two antagonistic signaling pathways were identified here that affect fate conversion of astrocytes independent of genetic manipulation. The complex interplay of the respective signaling molecules that promote/inhibit astrocyte de-differentiation may explain why astrocytes do not readily form neural stem cells in most diseases. Increased knowledge of such factors may provide therapeutic opportunities to favor such conversions. Stem Cells 2016;34:2861-2874.


Assuntos
Astrócitos/citologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Interferon gama/farmacologia , Células-Tronco Neurais/citologia , Neurogênese , Animais , Astrócitos/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Desdiferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/efeitos dos fármacos , Células-Tronco Multipotentes/metabolismo , Células-Tronco Neurais/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
3.
Neurochem Res ; 42(1): 244-253, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27068034

RESUMO

Proliferation and differentiation of neural stem cells (NSCs) have a crucial role to ensure neurogenesis and gliogenesis in the mammalian brain throughout life. As there is growing evidence for the significance of metabolism in regulating cell fate, knowledge on the metabolic programs in NSCs and how they evolve during differentiation into somatic cells may provide novel therapeutic approaches to address brain diseases. In this work, we applied a quantitative analysis to assess how the central carbon metabolism evolves upon differentiation of NSCs into astrocytes. Murine embryonic stem cell (mESC)-derived NSCs and astrocytes were incubated with labelled [1-13C]glucose and the label incorporation into intracellular metabolites was followed by GC-MS. The obtained 13C labelling patterns, together with uptake/secretion rates determined from supernatant analysis, were integrated into an isotopic non-stationary metabolic flux analysis (13C-MFA) model to estimate intracellular flux maps. Significant metabolic differences between NSCs and astrocytes were identified, with a general downregulation of central carbon metabolism during astrocytic differentiation. While glucose uptake was 1.7-fold higher in NSCs (on a per cell basis), a high lactate-secreting phenotype was common to both cell types. Furthermore, NSCs consumed glutamine from the medium; the highly active reductive carboxylation of alpha-ketoglutarate indicates that this was converted to citrate and used for biosynthetic purposes. In astrocytes, pyruvate entered the TCA cycle mostly through pyruvate carboxylase (81%). This pathway supported glutamine and citrate secretion, recapitulating well described metabolic features of these cells in vivo. Overall, this fluxomics study allowed us to quantify the metabolic rewiring accompanying astrocytic lineage specification from NSCs.


Assuntos
Astrócitos/metabolismo , Diferenciação Celular/fisiologia , Análise do Fluxo Metabólico/métodos , Células-Tronco Neurais/metabolismo , Neurogênese/fisiologia , Animais , Linhagem Celular , Sobrevivência Celular/fisiologia , Células Cultivadas , Camundongos
4.
Glia ; 64(5): 695-715, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26689134

RESUMO

Availability of homogeneous astrocyte populations would facilitate research concerning cell plasticity (metabolic and transcriptional adaptations; innate immune responses) and cell cycle reactivation. Current protocols to prepare astrocyte cultures differ in their final content of immature precursor cells, preactivated cells or entirely different cell types. A new method taking care of all these issues would improve research on astrocyte functions. We found here that the exposure of a defined population of pluripotent stem cell-derived neural stem cells (NSC) to BMP4 results in pure, nonproliferating astrocyte cultures within 24-48 h. These murine astrocytes generated from embryonic stem cells (mAGES) expressed the positive markers GFAP, aquaporin 4 and GLT-1, supported neuronal function, and acquired innate immune functions such as the response to tumor necrosis factor and interleukin 1. The protocol was applicable to several normal or disease-prone pluripotent cell lines, and the corresponding mAGES all exited the cell cycle and lost most of their nestin expression, in contrast to astrocytes generated by serum-addition or obtained as primary cultures. Comparative gene expression analysis of mAGES and NSC allowed quantification of differences between the two cell types and a definition of an improved marker set to define astrocytes. Inclusion of several published data sets in this transcriptome comparison revealed the similarity of mAGES with cortical astrocytes in vivo. Metabolic analysis of homogeneous NSC and astrocyte populations revealed distinct neurochemical features: both cell types synthesized glutamine and citrate, but only mature astrocytes released these metabolites. Thus, the homogeneous cultures allowed an improved definition of NSC and astrocyte features.


Assuntos
Astrócitos/fisiologia , Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/fisiologia , Células-Tronco Neurais/fisiologia , Animais , Aquaporina 4/genética , Aquaporina 4/metabolismo , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/metabolismo , Células Cultivadas , Citocinas/metabolismo , Citocinas/farmacologia , Embrião de Mamíferos , Transportador 2 de Aminoácido Excitatório/genética , Transportador 2 de Aminoácido Excitatório/metabolismo , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Glucose/metabolismo , Ácido Glutâmico/metabolismo , Antígeno Ki-67/metabolismo , Ácido Láctico/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Nestina/metabolismo , Fatores de Tempo , Transcriptoma/fisiologia
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