Protocol to study in vitro drug metabolism and identify montelukast metabolites from purified enzymes and primary cell cultures by mass spectrometry.

We present an optimized protocol set to study the production of drug metabolites in different in vitro systems. We detail the necessary steps to identify the metabolites of xenobiotics produced in different metabolic-competent systems, from purified enzymes to primary cell cultures. It is coupled to a high-resolution mass spectrometry analytical approach and can be adapted to study any xenobiotic. This protocol was optimized using montelukast, an antagonist of the cysteinyl leukotriene receptor 1, widely used for asthma management. For complete details on the use and execution of this protocol, please refer to Marques et al. (2022).1.

Optimized protocol for obtaining and characterizing primary neuron-enriched cultures from embryonic chicken brains.

We present here an optimized protocol to obtain primary neuron-enriched cultures from embryonic chicken brains with no need for an animal facility. The protocol details the steps to isolate a neuron-enriched cell fraction from chicken embryos, followed by characterization of the chicken neurons with mass spectrometry proteomics and cell staining. Because of the high homology between chicken and human amyloid precursor protein processing machinery, these chicken neurons can be used as an alternative to rodent models for studying Alzheimer disease.

The mechanisms underlying montelukast's neuropsychiatric effects - new insights from a combined metabolic and multiomics approach.

AIMS: Montelukast (MTK) is an antagonist of the cysteinyl leukotrienes receptor 1 widely used to manage asthma symptoms among adults and children. However, it has been associated with an increasing number of neuropsychiatric adverse drug reactions (ADRs), particularly among children, including depression, sleep disturbance, and suicidal ideation. The aims of this work were to characterize MTK metabolism in vitro and in vivo and to identify its effects at the metabolome and proteome levels in order to explain its toxicity. MAIN METHODS: An extensive study of montelukast metabolism was carried out using in vitro systems, an embryonic neuron-enriched cell model, and a mouse model. Metabolites were identified by high-resolution mass spectrometry, and a combined mass spectrometry-based metabolomics and proteomics approach was employed to assess the effect of MTK on mice and isolated chicken neurons. KEY FINDINGS: Eighteen new MTK metabolites were identified. MTK's ability to react with glutathione was confirmed. The multi-omics approach employed confirmed that montelukast interferes with the glutathione detoxification system in the brain. Moreover, montelukast is also able to dysregulate various neurotransmitter and neurosteroid pathways, particularly those involved in regulation of the hypothalamic-pituitary-adrenal axis, also interfering with mitochondrial function in neuronal cells. SIGNIFICANCE: Results clearly indicate that montelukast therapeutic effects are accompanied by a strong modulation of specific processes in the central nervous system that may explain the observed neuropsychiatric reactions. Moreover, the results also suggest that adverse drug reactions are more likely to occur in children, due to the early maturation stage of their brains.

Leukotrienes vs. Montelukast-Activity, Metabolism, and Toxicity Hints for Repurposing.

Increasing environmental distress is associated with a growing asthma incidence; no treatments are available but montelukast (MTK)-an antagonist of the cysteinyl leukotrienes receptor 1-is widely used in the management of symptoms among adults and children. Recently, new molecular targets have been identified and MTK has been proposed for repurposing in other therapeutic applications, with several ongoing clinical trials. The proposed applications include neuroinflammation control, which could be explored in some neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases (AD and PD). However, this drug has been associated with an increasing number of reported neuropsychiatric adverse drug reactions (ADRs). Besides, and despite being on the market since 1998, MTK metabolism is still poorly understood and the mechanisms underlying neuropsychiatric ADRs remain unknown. We review the role of MTK as a modulator of leukotriene pathways and systematize the current knowledge about MTK metabolism. Known toxic effects of MTK are discussed, and repurposing applications are presented comprehensively, with a focus on AD and PD.

Flexible ZnO-mAb nanoplatforms for selective peripheral blood mononuclear cell immobilization.

Cancer is the second cause of death worldwide. This devastating disease requires specific, fast, and affordable solutions to mitigate and reverse this trend. A step towards cancer-fighting lies in the isolation of natural killer (NK) cells, a set of innate immune cells, that can either be used as biomarkers of tumorigenesis or, after autologous transplantation, to fight aggressive metastatic cells. In order to specifically isolate NK cells (which express the surface NKp30 receptor) from peripheral blood mononuclear cells, a ZnO immunoaffinity-based platform was developed by electrodeposition of the metal oxide on a flexible indium tin oxide (ITO)-coated polyethylene terephthalate (PET) substrate. The resulting crystalline and well-aligned ZnO nanorods (NRs) proved their efficiency in immobilizing monoclonal anti-human NKp30 antibodies (mAb), obviating the need for additional procedures for mAb immobilization. The presence of NK cells on the peripheral blood mononuclear cell (PBMCs) fraction was evaluated by the response to their natural ligand (B7-H6) using an acridine orange (AO)-based assay. The successful selection of NK cells from PBMCs by our nanoplatform was assessed by the photoluminescent properties of AO. This easy and straightforward ZnO-mAb nanoplatform paves the way for the design of biosensors for clinic diagnosis, and, due to its inherent biocompatibility, for the initial selection of NK cells for autotransplantation immunotherapies.