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Microglial activation is an early phenomenon in Alzheimer’s disease (AD) that may occur prior to and independently of amyloid-β (Aβ) aggregation. Recent studies in transgenic animal models suggest that the apolipoprotein E ε4 (APOEε4) allele may be a culprit of early microglial activation in AD. However, it is unclear whether the APOEε4 genotype is associated with microglial reactivity in the living human brain. Here, we tested whether APOEε4 carriership is associated with microglial activation in individuals across the aging and AD spectrum. We studied 118 individuals who had positron emission tomography (PET) for Aβ ([18F]AZD4694), tau ([18F]MK6240), and microglial activation ([11C]PBR28), as well as clinical, genetic, and magnetic resonance imaging data. We found that APOEε4 carriership was associated with increased microglial activation mainly in early Braak-staging regions within the medial temporal cortex, and this effect of APOEε4 was independent of Aβ and tau deposition. Furthermore, microglial activation mediated the Aβ-independent effects of APOEε4 on downstream tau accumulation, neurodegeneration, and clinical impairment. Interestingly, the physiological distribution of APOE mRNA expression, obtained from the Allen Human Atlas, predicted the patterns of APOEε4-related microglial activation in our population, suggesting that the deleterious effects of APOEε4 occur at the level of gene expression. These results support a model in which the APOEε4 has Aβ-independent effects on AD pathogenesis by activating microglia in brain regions associated with early tau deposition. Our findings provide a rationale for the development of novel AD therapies targeting the interplay between ApoE and neuroinflammation.

Amyloid-β oligomers (AβOs) toxicity causes mitochondrial dysfunction, leading to synaptic failure in Alzheimer’s disease (AD). Considering presynaptic high energy demand and tight Ca2+ regulation, impairment of mitochondrial function can lead to deteriorated neural activity and cell death. In this study, an AD mouse model induced by ICV (intracerebroventricular) injection of AβOs was used to investigate the toxicity of AβOs on presynaptic function. As a therapeutic approach, GUO (guanosine) was given by oral route to evaluate the neuroprotective effects on this AD model. Following 24 h and 48 h from the model induction, behavioral tasks and biochemical analyses were performed, respectively. AβOs impaired object recognition (OR) short-term memory and reduced glutamate uptake and oxidation in the hippocampus. Moreover, AβOs decreased spare respiratory capacity, reduced ATP levels, impaired Ca2+ handling, and caused mitochondrial swelling in hippocampal synaptosomes. Guanosine crossed the BBB, recovered OR short-term memory, reestablished glutamate uptake, recovered mitochondrial Ca2+ homeostasis, and partially prevented mitochondrial swelling. Therefore, this endogenous purine presented a neuroprotective effect on presynaptic mitochondria and should be considered for further studies in AD models.

Mycophenolic acid is one of the most used immunosuppressive drugs in solid organ transplant treatments in the world. Developing a highly sensitive analytical method to analyse the drug and its metabolites in oral fluid and plasma is important to evaluate the possibility of using oral fluid as a biological matrix in therapeutic drug monitoring, instead of plasma. The liquid chromatography coupled to mass spectrometry (LC-MS) method was developed and validated for determining mycophenolic acid (MPA) and its glucuronide metabolite (MPAG) in oral fluid and plasma, with both matrices presenting a detection limit of 1 ng/mL for MPA and 5 ng/mL for MPAG. Both analytes were analysed after a simple protein precipitation procedure. Transplanted-kidney samples of oral fluid and blood were collected from 13 patients that were hospitalised and kept at − 80 °C until analyses. The proposed method was linear in the concentration range of 5–500 ng/mL for MPA and 10–500 ng/mL for MPAG, with correlation coefficients (r) between 0.9925 and 0.9973. It was then applied to samples collected from kidney-transplanted patients and used for calculation of pharmacokinetics parameters. After comparing plasma and oral fluid concentrations as well as performing a non-compartmental pharmacokinetic analysis of the average curves, it is possible to suggest that oral fluid concentration may be used as an alternative for MPA and MPAG monitoring in kidney transplant patients.

Amyloid-β (Aβ) dysmetabolism is thought to be the main trigger for neurodegenerative events in Alzheimer's disease (AD). In particular, soluble Aβ oligomers (AβOs) are proposed as key mediators of synaptic and cognitive dysfunction in AD. Over the past few decades, AβOs prepared from synthetic Aβ have been widely applied in vitro and in vivo, the so-called chemical models of AD, uncovering their multiple neurotoxic mechanisms. However, the lack of a reliable quality control (QC) for synthetic AβOs may reflect poor experimental reproducibility. In keeping with this, we optimized and validated a rapid and reproducible SECHPLC method using fluorescence detection for the QC of synthetic AβOs. Our analytical method offers an unprecedent alternative to improve the reproducibility of AD chemical models.

Immunosuppressive therapy is used in solid organ transplant treatment, and mycophenolic acid (MPA) is one of the immunosuppressive drugs most used worldwide. It is a potent, selective, non-competitive, and reversible inosine monophosphate dehydrogenase (IMPDH) inhibitor that acts to inhibit guanine synthesis. To improve solubility, MPA is used as the prodrug mycophenolate mofetil (MMF) or as an enteric-coated mycophenolate sodium salt (EC-MPS). It is metabolized into mycophenolic acid phenyl glucuronide (MPAG), the inactive and major metabolite, and into acyl glucuronide (AcMPAG), pharmacologically active. In kidney transplantation, combined immunosuppressive therapy with cyclosporine (CsA) and tacrolimus (Tac) is widely used, showing beneficial effects. This paper aimed to review papers published in the last two decades and discuss factors that can interfere with the pharmacokinetics of MPA. Data collected confirm that MPA plasma levels should be monitored to evaluate immunosuppressive therapy since pharmacokinetics can be influenced by factors such as interpatient variability, coadministration of other immunosuppressive agents, post-transplant period, renal function, and dose. However, to perform drug monitoring, costs and facility may be limitations. Monitoring MPAG together with MPA would be a great improvement in therapy as it represents a big part of MPA levels and can be related to the increase of adverse effects.

Sepsis is characterized by a severe and disseminated inflammation. In the central nervous system, sepsis promotes synaptic dysfunction and permanent cognitive impairment. Besides sepsis-induced neuronal dysfunction, glial cell response has been gaining considerable attention with microglial activation as a key player. By contrast, astrocytes' role during acute sepsis is still underexplored. Astrocytes are specialized immunocompetent cells involved in brain surveillance. In this context, the potential communication between the peripheral immune system and astrocytes during acute sepsis still remains unclear. We hypothesized that peripheral blood mononuclear cell (PBMC) mediators are able to affect the brain during an episode of acute sepsis. With this in mind, we first performed a data-driven transcriptome analysis of blood from septic patients to identify common features among independent clinical studies. Our findings evidenced pronounced impairment in energy-related signaling pathways in the blood of septic patients. Since astrocytes are key for brain energy homeostasis, we decided to investigate the communication between PBMC mediators and astrocytes in a rat model of acute sepsis, induced by cecal ligation and perforation (CLP). In the CLP animals, we identified widespread in vivo brain glucose hypometabolism. Ex vivo analyses demonstrated astrocyte reactivity along with reduced glutamate uptake capacity during sepsis. Also, by exposing cultured astrocytes to mediators released by PBMCs from CLP animals, we reproduced the energetic failure observed in vivo. Finally, by pharmacologically inhibiting phosphoinositide 3-kinase (PI3K), a central metabolic pathway downregulated in the blood of septic patients and reduced in the CLP rat brain, we mimicked the PBMC mediators effect on glutamate uptake but not on glucose metabolism. These results suggest that PBMC mediators are capable of directly mediating astrocyte reactivity and contribute to the brain energetic failure observed in acute sepsis. Moreover, the evidence of PI3K participation in this process indicates a potential target for therapeutic modulation.

Introduction Even after removal of some stimulants, like fenproporex, amfepramone and mazindol, from Brazilian market, the use of these substances is still high, especially by drivers. Mazindol is the second most used anorectic agent in the world acting as an indirect sympathomimetic agonist, having stimulatory action on central nervous system. Plasma is a good matrix to monitor since it reflects the psychomotor effects of these drugs, but unlike urine has an invasive collection; drug levels and detection time are quite low. Method The method involved a liquid–liquid extraction of the samples and a LC–MS analysis was fully validated. Method was used to analyze samples of urine and plasma collected from health volunteers in a period of 24 h. Metabolite of mazindol was synthesized using alkaline conditions. Results After validation the method proved to be adequate to analyze samples collected from health volunteers. Method was linear in the concentration range of 0.1–10 ng/mL (r = 0.9982) for plasma and 5–50 ng/mL (r = 0.9973) for urine. Discussion Analysis of the samples showed that mazindol can be detected after 1 h of administration and that concentration levels in urine were always higher than in plasma. Mazindol metabolite was detected only in urine.

There are no pharmacokinetics studies in oral fluid reported in the literature, as well as there are no data on correlation of drug levels in plasma, urine, and oral fluid in order to propose alternative matrices to monitor the use of mazindol by drivers. The present work aimed to study, preliminarily, mazindol’s pharmacokinetics in plasma and oral fluid, as well as investigate the correlation of drug levels in urine, plasma, and oral fluid. Blood, urine, and oral fluid samples from seven healthy male volunteers were collected at 0, 1, 2, 4, 5, 6, 8, 10, and 24 h after administration of tablets of 2 mg mazindol and analyzed by a previously validated method by LC-MS with liquid-liquid extraction. Levels of the drug found were higher in plasma when compared with oral fluid and higher in urine in relation to plasma. The study of the mazindol’s pharmacokinetics showed that the most suitable model to describe the variation of the concentration over time is the compartment open model with absorption and elimination following the first-order kinetics, and confirming literature data, drug is metabolized, being the major metabolite detected, but not quantified. It was not found a good correlation between the concentrations of mazindol in urine and plasma, but between plasma and oral fluid, there was a good correlation, suggesting this as an alternative matrix to plasma. However, studies involving more subjects are needed.

Fetal exposure to illicit drugs is a worldwide problem, since many addicted women do not stop using it during pregnancy. Cocaine consumed in powdered (snorted or injected) or smoked (crack cocaine) form are harmful for the baby and its side effects are not completely known. Meconium, the first stool of a newborn, is a precious matrix usually discarded, that may contain amounts of substances consumed in the last two trimesters of pregnancy. Analyzing this biological matrix it is possible to detect the unaltered molecule of cocaine (COC) or its metabolite benzoylecgonine (BZE) and pyrolytic products anhydroecgonine methyl ester (AEME) and anhydroecgonine (AEC). A liquid chromatography mass spectrometry (LC-MS) method was validated for meconium samples after solvent extraction, followed by direct injection of 10μL. Linearity covered a concentration range of 15 to 500ng/mg with a lower limit of quantification (LLOQ) of 15ng/mg for all analytes. Matrix effect was evaluated and showed adequate results. Detection of illicit substances usage can be crucial for the baby, since knowing that can help provide medical care as fast as possible. The method proved to be simple and fast, and was applied to 17 real meconium samples.

A method to assay cocaine (COC), its metabolites benzoylecgonine (BZE), ecgonine (ECG), ecgonine methyl esther (EME), and benzoylnorecgonine (BNE), pyrolytic products anhydroecgonine (AEC) and anhydroecgonine methyl ester (AEME) and adulterant levamisole (LEV) was developed and validated by liquid chromatography-mass spectrometry (LC-MS) using a chemometric approach including a two-level factorial design in the screening step and face-centered central composite design (FCCCD) to achieve the optimization. The method was carried out on positive electrospray ionization (ESI+) with a flow of 1 mL min−1 in isocratic mode consisting of 53% methanol and 47% ammonium acetate 10 mmol L−1 pH 6.3. The chromatographic separation was obtained with a Phenomenex Luna C18(2) column (250 mm × 4.6 mm, particle size 5 μm), with the temperature set at 31 °C. Validation parameters such as specificity, linearity, precision and accuracy were evaluated. The method was linear over the concentration range of 1–100 ng mL−1 for COC, AEME, EME, LEV, BZE and ECG and 5–100 ng mL−1 for AEC and BNE. The method was successfully applied to identify and quantify the analytes.

Drug abuse by nursing mothers is an ongoing concern because it may cause many adverse effects to the newborns. The development of analytical methods to analyze drugs of abuse in colostrum (first milk produced after birth) has a huge importance, because it enables the monitoring and the correct follow-up to users and newborns. A liquid chromatography mass spectrometry (LC–MS) method was developed and validated for the determination of cocaine and smoked cocaine (crack) biomarkers in colostrum. Cocaine (COC) and its major metabolite benzoylecgonine (BZE), the pyrolytic products anhydroecgonine methyl ester (AEME) and anhydroecgonine (AEC) were analyzed after a simple protein precipitation procedure using atropine (ATP) as internal standard (IS). Applying a chemometric approach study, all peaks were chromatographically separated at isocratic condition with a Kinetex HILIC column for polar compounds, at 30 °C in 12 min. One ion was detected for the quantification and three ions for confirmation of each analyte. The method was linear for all analytes in the concentration range of 5–300 ng/mL with correlation coefficients ( r ) between 0.9983 and 0.9996. The lower limit of quantification (LLOQ) was 5 ng/mL with acceptable validation parameters. Matrix effect was assessed by post-extraction addition approach and showed good results, demonstrating that protein precipitation cleaning procedure is fast, reliable and demand small quantities of organic solvent. The LC–MS method is fast and cheap compared to other equipments and was also successfully applied to assess real samples of colostrum from nursing mothers who were suspect of cocaine/crack abuse.

A simple capillary zone electrophoresis (CZE) method was developed and validated for the analysis of lodenafil carbonate in tablets. Response surface methodology was used for optimization of the pH and concentration of the buffer, applied voltage and temperature. The method employed 50 mmol L-1 borate buffer at pH 10 as background electrolyte with an applied voltage of 15 kV. The separation was carried out in a fused-silica capillary maintained at 32.5 ºC and the detection wavelength was 214 nm. The method was validated showing specificity, linearity (r = 0.9995), precision (relative standard deviation less than 2%) and accuracy (99.95%). The method proved to be robust by a fractional factorial design evaluation. The proposed CZE method was successfully applied for the quantitative analysis of lodenafil carbonate in tablets and the results compared to the high performance liquid chromatography and ultraviolet spectrophotometric methods, showed non-significant differences. Um método simples de eletroforese capilar de zona (CZE) foi desenvolvido e validado para análise de carbonato de lodenafila em comprimidos. A metodologia de superfície de resposta foi utilizada para a otimização do pH, concentração do tampão, voltagem e temperatura. O método utilizou como eletrólito, tampão borato 50 mmol L-1, pH 10, voltagem de 15 kV. A separação foi efetuada em capilar de sílica fundida mantida a 32,5 ºC e o comprimento de onda de detecção foi de 214 nm. O método foi validado, demonstrando especificidade, linearidade (r = 0,9995), precisão (desvio padrão relativo inferior a 2%) e exatidão (99,95%). O método demonstrou ser robusto, através de avaliação por planejamento fatorial fracionário. O método CZE proposto foi aplicado com sucesso para a análise quantitativa de carbonato de lodenafila em comprimidos e os resultados, comparados com os métodos por cromatografia líquida de alta eficiência e espectrofotometria no ultravioleta, não apresentaram diferença significativa.