Desenvolvimento e validação de método analítico para determinação simultânea de ácido micofenólico e seu metabolito glicuronídeo por CL/EM em plasma e fluído oral de pacientes transplantados renais

Com o envelhecimento da população brasileira, o número de doenças crônicas vem aumentando, entre as quais se destaca a insuficiência renal. O tratamento recomendado é o transplante renal. Uma das principais preocupações do transplante é o alto risco de rejeição, por isso é necessário o uso de imunossupressores que diminuem a atividade do sistema imunológico. Um dos principais fármacos utilizado na terapia de imunossupressão é o micofenolato de mofetila (MMF), um potente inibidor seletivo, não competitivo e reversível da desidrogenase inosina-monofosfato (IMPDH) que atua inibindo a síntese de guanina, provocando uma diminuição da taxa de proliferação. Os imunossupressores estão relacionados a muitos efeitos secundários, sendo necessário um monitoramento terapêutico, a principal matriz utilizada é o plasma. Porém, o uso do fluido oral vem aumentando, por não necessitar de coleta invasiva e pessoal especializado para tal. Um método de cromatografia líquida acoplado ao espectrômetro de massas (LC-MS) foi desenvolvido e validado para a determinação do ácido micofenólico (MPA) e seu metabólito glicuronideo (MPAG), em plasma e fluido oral, com precipitação de proteínas seguido de centrifugação e utilizando cetoprofeno (KET) como padrão interno (PI). Os picos foram separados em condição gradiente, com um tempo de corrida de 16 min, através de uma coluna Agilent Zorbax Eclipse Plus (4.6 x 150 mm, 3.5 μm tamanho de partícula) a 35 °C. Um íon foi utilizado para a quantificação e três íons para a confirmação de cada analito. O método foi linear para todos os analitos no intervalo de concentração de 10,0 – 500,0 ng/mL para o MPA e 30,0 – 500,0 ng/mL para MPAG, com coeficientes de correlação (r) entre 0,9925 – 0,9937 para as amostras de plasma e com r entre 0,9952 – 0,9973 para as amostras de fluido oral. O limite inferior de quantificação foi de 10,0 ng/mL para o MPA e 30,0 ng/mL para o MPAG, com parâmetros de validação dentro do preconizado. O efeito matriz foi avaliado e apresentou resultados adequados, demonstrando que ambos os procedimentos de limpeza das amostras são rápidos e confiáveis, exigindo pequenas quantidades. O método foi aplicado em amostras de pacientes transplantados renais de fluido oral e plasma usando menos pontos que usualmente. Após análise farmacocinética foi encontrado que o fluido oral pode ser uma possível matriz para ser usando no monitoramento terapêutico sendo necessário ainda mais estudos para ser aplicado na clínica. Estudos in vitro para medir e tentar estabelecer uma relação de permeação do MPA dentro do linfócito foram realizados afim de correlacionar com achados nas amostras coletadas de pacientes. Após analisar as amostras dos pacientes foi encontrado que o estudo in vitro utilizando sangue total é que mais se aproxima dos resultados encontrados em amostras reais. Além de MPA dentro do linfócito foi também detectado MPAG, que ainda não havia sido descrito na literatura. A relação da quantidade dos dois metabólitos dentro do linfócito foi menor que 4%.With the aging of the population, the number of chronic diseases is increasing, standing out kidney disease. The recommended treatment for this disease is renal transplantation, which increases the patient’s quality of life. The major concern with transplantation is the high risk of rejection and that is why it is necessary the use of immunosuppressive drugs to decrease the activity of the immune system. One of the main drugs used in immunosuppressive therapy is mycophenolate mofetil (MMF), a potent noncompetitive and reversible selective inhibitor of inosine monophosphate dehydrogenase (IMPDH), which acts inhibiting the synthesis of guanine, producing a decrease in the proliferation rate. Immunosuppressors are related with many side effects, and therapeutic monitoring is required, the main matrix being used is plasma. However, the use of oral fluid is increasing because it does not require invasive collection or specialized personnel to perform it. A liquid chromatography coupled to mass spectrometry (LC-MS) method was developed and validated for the determination of mycophenolic acid (MPA) and its glucuronide metabolite (MPAG) in plasma and oral fluid. Samples were analysed after a simple protein precipitation procedure followed by centrifugation using ketoprofen (KET) as internal standard (PI). The peaks were separated under gradient condition, run time of 16 min, using an Agilent Zorbax Eclipse Plus column (4.6 x 150 mm, 3.5 μm particle size) at 35 °C. One ion was used for quantification and three more for confirmation of each analyte. The method was linear for all analytes in the concentration range of 10.0 – 500.0 ng / mL for MPA and 30.0 – 500.0 ng/mL for MPAG, with correlation coefficients (r) between 0.9925 - 0.9937 for plasma samples and 0.9952 - 0.9973 for oral fluid samples. The lower limit of quantification was 10 ng / mL for MPA and 30 ng / mL for MPAG, with validation parameters within the recommended range. Matrix effect was evaluated and showed adequate results, demonstrating that sample cleaning procedure was fast and reliable, requiring small amounts of organic solvent. The method was applied to samples of oral fluid and plasma from renal transplant patients using limited sample collection. After pharmacokinetic analysis, it was found that the oral fluid might be a possible matrix to be used in therapeutic monitoring and further studies are needed to be applied in the clinic. In vitro studies to measure and try to establish a permeation rate of MPA lymphocyte concentration were performed in order to correlate with findings in the samples collected from patients. After analyzing patients samples, it was found that the in vitro study using total blood is the one that more correlates with what was found on real samples. In addition to MPA it was also detected MPAG inside the lymphocytes, which had not yet been described yet. The quantity relation of the two metabolites inside the lymphocyte was less than 4%

Race-related population attributable fraction of preventable risk factors of dementia : a latino population-based study

Background: risk factors for dementia have distinct frequency and impact in relation to race. Our aim was to identify differences in modifiable risk factors of dementia related to races and estimate their population attributable fraction (PAF). Methods: an epidemiological cohort was used to estimate the prevalence of 10 modifiable risk factors for dementia among five races—White, Black, Brown, Asian, and Indigenous. Sample weighting was used to estimate the prevalence and PAF of each risk factor in each race. Results:a total of 9070 individuals were included. Overall adjusted PAF was the lowest in Indigenous (38.9%), and Asian individuals (41.2%). Race-related prevalence of individual risk factors was widely variable in our population, but hearing loss was the most important contributor to the overall PAF in all races. Conclusions Public policies aiming to reduce preventable risk factors for dementia should take into consideration the race of the target populations

Functional recovery caused by human adipose tissue mesenchymal stem cell-derived extracellular vesicles administered 24 h after stroke in rats

Ischemic stroke is a major cause of death and disability, intensely demanding innovative and accessible therapeutic strategies. Approaches presenting a prolonged period for therapeutic intervention and new treatment administration routes are promising tools for stroke treatment. Here, we evaluated the potential neuroprotective properties of nasally administered human adipose tissue mesenchymal stem cell (hAT-MSC)-derived extracellular vesicles (EVs) obtained from healthy individuals who underwent liposuction. After a single intranasal EV (200 µg/kg) administered 24 h after a focal permanent ischemic stroke in rats, a higher number of EVs, improvement of the blood–brain barrier, and re-stabilization of vascularization were observed in the recoverable peri-infarct zone, as well as a significant decrease in infarct volume. In addition, EV treatment recovered long-term motor (front paws symmetry) and behavioral impairment (short- and long-term memory and anxiety-like behavior) induced by ischemic stroke. In line with these findings, our work highlights hAT-MSC-derived EVs as a promising therapeutic strategy for stroke

Soluble amyloid-beta isoforms predict downstream Alzheimer’s disease pathology

Background: Changes in soluble amyloid-beta (Aβ) levels in cerebrospinal fluid (CSF) are detectable at early preclinical stages of Alzheimer’s disease (AD). However, whether Aβ levels can predict downstream AD pathological features in cognitively unimpaired (CU) individuals remains unclear. With this in mind, we aimed at investigating whether a combination of soluble Aβ isoforms can predict tau pathology (T+) and neurodegeneration (N+) positivity. Methods: We used CSF measurements of three soluble Aβ peptides (Aβ1–38, Aβ1–40 and Aβ1–42) in CU individuals (n = 318) as input features in machine learning (ML) models aiming at predicting T+ and N+. Input data was used for building 2046 tuned predictive ML models with a nested cross-validation technique. Additionally, proteomics data was employed to investigate the functional enrichment of biological processes altered in T+ and N+ individuals. Results: Our findings indicate that Aβ isoforms can predict T+ and N+ with an area under the curve (AUC) of 0.929 and 0.936, respectively. Additionally, proteomics analysis identified 17 differentially expressed proteins (DEPs) in individuals wrongly classified by our ML model. More specifically, enrichment analysis of gene ontology biological processes revealed an upregulation in myelinization and glucose metabolism-related processes in CU individuals wrongly predicted as T+. A significant enrichment of DEPs in pathways including biosynthesis of amino acids, glycolysis/gluconeogenesis, carbon metabolism, cell adhesion molecules and prion disease was also observed. Conclusions: Our results demonstrate that, by applying a refined ML analysis, a combination of Aβ isoforms can predict T+ and N+ with a high AUC. CSF proteomics analysis highlighted a promising group of proteins that can be further explored for improving T+ and N+ prediction

Development of a cell-targeted liposome formulation for small cell lung cancer

Lung cancer is one of the most common cancers with small cell lung cancer (SCLC) representing 18% of the total lung cancer diagnoses. Conventional treatments include the administration of high dosages of cisplatin, but as most anti-cancer agents, this drug is not targeted which causes toxicity and undesirable severe side-effects which limit its clinical application. Liposome-based formulations have been recently exploited for improving the delivery and therapeutic effect of cisplatin (Lipoplatin®). In this study, we sought to generate a targeted liposomal formulation. The gastrin releasing peptide (GRP) receptor is over-expressed on many cancer cells including SCLC. A conventional liposomal cisplatin formulation was modified by adding a GRP receptor agonist to a pegylated lipid which was then inserted into pre-formed liposomes. However, the biological activity of peptide-modified liposomal cisplatin was comparable to the control liposomes when tested on GRP receptor positive SCLC cells (NCI-H345), and GRP receptor negative control cells (A549), using the MTS assay. ICP-AES analysis of cisplatin encapsulation efficiency showed encapsulation to be decreased in the targeted formulation. Fluorescence liposomes were also prepared to evaluate the uptake of the cells, using carboxyfluorescein as fluorescence agent. Results shows higher uptake by target- liposomes

Desenvolvimento e validação de método analítico para determinação simultânea de ácido micofenólico e seu metabolito glicuronídeo por CL/EM em plasma e fluído oral de pacientes transplantados renais

Com o envelhecimento da população brasileira, o número de doenças crônicas vem aumentando, entre as quais se destaca a insuficiência renal. O tratamento recomendado é o transplante renal. Uma das principais preocupações do transplante é o alto risco de rejeição, por isso é necessário o uso de imunossupressores que diminuem a atividade do sistema imunológico. Um dos principais fármacos utilizado na terapia de imunossupressão é o micofenolato de mofetila (MMF), um potente inibidor seletivo, não competitivo e reversível da desidrogenase inosina-monofosfato (IMPDH) que atua inibindo a síntese de guanina, provocando uma diminuição da taxa de proliferação. Os imunossupressores estão relacionados a muitos efeitos secundários, sendo necessário um monitoramento terapêutico, a principal matriz utilizada é o plasma. Porém, o uso do fluido oral vem aumentando, por não necessitar de coleta invasiva e pessoal especializado para tal. Um método de cromatografia líquida acoplado ao espectrômetro de massas (LC-MS) foi desenvolvido e validado para a determinação do ácido micofenólico (MPA) e seu metabólito glicuronideo (MPAG), em plasma e fluido oral, com precipitação de proteínas seguido de centrifugação e utilizando cetoprofeno (KET) como padrão interno (PI). Os picos foram separados em condição gradiente, com um tempo de corrida de 16 min, através de uma coluna Agilent Zorbax Eclipse Plus (4.6 x 150 mm, 3.5 μm tamanho de partícula) a 35 °C. Um íon foi utilizado para a quantificação e três íons para a confirmação de cada analito. O método foi linear para todos os analitos no intervalo de concentração de 10,0 – 500,0 ng/mL para o MPA e 30,0 – 500,0 ng/mL para MPAG, com coeficientes de correlação (r) entre 0,9925 – 0,9937 para as amostras de plasma e com r entre 0,9952 – 0,9973 para as amostras de fluido oral. O limite inferior de quantificação foi de 10,0 ng/mL para o MPA e 30,0 ng/mL para o MPAG, com parâmetros de validação dentro do preconizado. O efeito matriz foi avaliado e apresentou resultados adequados, demonstrando que ambos os procedimentos de limpeza das amostras são rápidos e confiáveis, exigindo pequenas quantidades. O método foi aplicado em amostras de pacientes transplantados renais de fluido oral e plasma usando menos pontos que usualmente. Após análise farmacocinética foi encontrado que o fluido oral pode ser uma possível matriz para ser usando no monitoramento terapêutico sendo necessário ainda mais estudos para ser aplicado na clínica. Estudos in vitro para medir e tentar estabelecer uma relação de permeação do MPA dentro do linfócito foram realizados afim de correlacionar com achados nas amostras coletadas de pacientes. Após analisar as amostras dos pacientes foi encontrado que o estudo in vitro utilizando sangue total é que mais se aproxima dos resultados encontrados em amostras reais. Além de MPA dentro do linfócito foi também detectado MPAG, que ainda não havia sido descrito na literatura. A relação da quantidade dos dois metabólitos dentro do linfócito foi menor que 4%.With the aging of the population, the number of chronic diseases is increasing, standing out kidney disease. The recommended treatment for this disease is renal transplantation, which increases the patient’s quality of life. The major concern with transplantation is the high risk of rejection and that is why it is necessary the use of immunosuppressive drugs to decrease the activity of the immune system. One of the main drugs used in immunosuppressive therapy is mycophenolate mofetil (MMF), a potent noncompetitive and reversible selective inhibitor of inosine monophosphate dehydrogenase (IMPDH), which acts inhibiting the synthesis of guanine, producing a decrease in the proliferation rate. Immunosuppressors are related with many side effects, and therapeutic monitoring is required, the main matrix being used is plasma. However, the use of oral fluid is increasing because it does not require invasive collection or specialized personnel to perform it. A liquid chromatography coupled to mass spectrometry (LC-MS) method was developed and validated for the determination of mycophenolic acid (MPA) and its glucuronide metabolite (MPAG) in plasma and oral fluid. Samples were analysed after a simple protein precipitation procedure followed by centrifugation using ketoprofen (KET) as internal standard (PI). The peaks were separated under gradient condition, run time of 16 min, using an Agilent Zorbax Eclipse Plus column (4.6 x 150 mm, 3.5 μm particle size) at 35 °C. One ion was used for quantification and three more for confirmation of each analyte. The method was linear for all analytes in the concentration range of 10.0 – 500.0 ng / mL for MPA and 30.0 – 500.0 ng/mL for MPAG, with correlation coefficients (r) between 0.9925 - 0.9937 for plasma samples and 0.9952 - 0.9973 for oral fluid samples. The lower limit of quantification was 10 ng / mL for MPA and 30 ng / mL for MPAG, with validation parameters within the recommended range. Matrix effect was evaluated and showed adequate results, demonstrating that sample cleaning procedure was fast and reliable, requiring small amounts of organic solvent. The method was applied to samples of oral fluid and plasma from renal transplant patients using limited sample collection. After pharmacokinetic analysis, it was found that the oral fluid might be a possible matrix to be used in therapeutic monitoring and further studies are needed to be applied in the clinic. In vitro studies to measure and try to establish a permeation rate of MPA lymphocyte concentration were performed in order to correlate with findings in the samples collected from patients. After analyzing patients samples, it was found that the in vitro study using total blood is the one that more correlates with what was found on real samples. In addition to MPA it was also detected MPAG inside the lymphocytes, which had not yet been described yet. The quantity relation of the two metabolites inside the lymphocyte was less than 4%

Desenvolvimento e validação de método para determinação de cocaína seus principais metabólitos e produtos de pirólise por CLAE-UV e CLAE-CAD

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