Sviluppo di un metodo per il dosaggio plasmatico del busulfan mediante HPLC-ESI-MS/MS in studi di farmacocinetica su pazienti talassemici

L'ottimizzazione del dosaggio busulfan in pazienti sottoposti a trapianto di midollo osseo si raccomanda al fine di ridurre gli effetti tossici associati all’elevata esposizione al farmaco. Una variabilità dell'area sotto la curva di concentrazione / tempo (AUC) comporta il rischio di sovra o sotto dosaggio con il conseguente aumento del rischio di tossicità o di recidiva. E’ stato sviluppato un metodo di analisi rapido, sensibile e specifico per il rilevamento del busulfan nel plasma umano. Il test si basa su una estrazione liquido-liquido con acetato di etile e di rilevamento mediante cromatografia liquida, con ionizzazione electrospray e spettrometria tandem massa (HPLC-ESI-MS/MS). Il busulfan deuterato è stato utilizzato come standard interno. Il metodo è lineare nel range 39-2500 ng / mL, con r2> 0,99 e una durata della corsa cromatografica di soli 5 minuti (con un tempo di ritenzione del busulfan di 1,62 minuti). La precisione inter-day and intra-day è nel range di 1,01 - 3,72% e di 0,35-4,32%, rispettivamente. Il recupero è > 70%. Il limite di rivelabilità e di quantificazione sono di 6 e 10 ng/mL, rispettivamente. Il metodo di analisi riportato, richiede soltanto 200 μL di plasma per l'analisi. Il metodo convalidato è stato applicato con successo per l’analisi di campioni di plasma ottenuti da bambini talassemici sottoposti a un regime di condizionamento con busulfan e sottoposti a trapianto di midollo osseo. Questo metodo permette la correzione della dose consentendo un dosaggio adeguato al fine di ottenere la concentrazione ottimale di busulfan. Questo metodo è attualmente utilizzato per analizzare le concentrazioni plasmatiche di busulfan dopo somministrazione per via endovenosa ed è applicato nel monitoraggio terapeutico dei farmaci.Optimisation of busulfan dosage in patients undergoing bone marrow transplantation is recommended in order to reduce toxic effects associated with high drug exposure. Variation in the area under the concentration /time curve (AUC) results in risk of over or under treatment with excess risk of toxicity or relapse. A rapid, sensitive and specific assay for detection of busulfan in human plasma was developed. The assay is based on a liquid-liquid extraction with ethyl acetate and detection by high performance liquid chromatography with electrospray ionization and tandem mass spectrometry (HPLC-ESI-MS/MS). Deuterated busulfan was used as internal standard. The method was linear over the range 39-2500 ng/mL, with r2 > 0.99 and a run time of only 5 minutes (busulfan retention time of 1.62 minutes). The inter-day and intra-day precision were in the range 1,01- 3,72 % and 0,35-4,32 %, respectively. The recovery was >70%. The limits of detection and quantification were 6 and 10 ng/mL, respectively. The reported assay required only 200 µL of plasma for the analysis. The validated method was successfully applied to analyze plasma samples from children with thalassemia undergoing a conditioning regimen with busulfan and submitted to bone marrow transplantation. This method permits dose correction allowing a better dosing adjustment towards the target level of busulfan. This method is currently used to analyze the plasma concentrations of busulfan after intravenous administration and it is applied in therapeutic drug monitoring

The enzymatic processing of α-dystroglycan by MMP-2 is controlled by two anchoring sites distinct from the active site

Dystroglycan (DG) is a membrane receptor, belonging to the dystrophin-glycoprotein complex (DGC) and formed by two subunits, α-dystroglycan (α-DG) and β-dystroglycan (β -DG). The C-terminal domain of α-DG and the N-terminal extracellular domain of β -DG are connected, providing a link between the extracellular matrix and the cytosol. Under pathological conditions, such as cancer and muscular dystrophies, DG may be the target of metalloproteinases MMP-2 and MMP-9, contributing to disease progression. Previously, we reported that the C-terminal domain α-DG (483-628) domain is particularly susceptible to the catalytic activity of MMP-2; here we show that the α-DG 621-628 region is required to carry out its complete digestion, suggesting that this portion may represent a MMP-2 anchoring site. Following this observation, we synthesized an α-DG based-peptide, spanning the (613-651) C-terminal region. The analysis of the kinetic and thermodynamic parameters of the whole and the isolated catalytic domain of MMP-2 (cdMMP-2) has shown its inhibitory properties, indicating the presence of (at least) two binding sites for the peptide, both located within the catalytic domain, only one of the two being topologically distinct from the catalytic active groove. However, the different behavior between whole MMP-2 and cdMMP-2 envisages the occurrence of an additional binding site for the peptide on the hemopexin-like domain of MMP-2. Interestingly, mass spectrometry analysis has shown that α-DG (613-651) peptide is cleavable even though it is a very poor substrate of MMP-2, a feature that renders this molecule a promising template for developing a selective MMP-2 inhibitor

Sviluppo di un metodo per il dosaggio plasmatico del busulfan mediante HPLC-ESI-MS/MS in studi di farmacocinetica su pazienti talassemici

L'ottimizzazione del dosaggio busulfan in pazienti sottoposti a trapianto di midollo osseo si raccomanda al fine di ridurre gli effetti tossici associati all’elevata esposizione al farmaco. Una variabilità dell'area sotto la curva di concentrazione / tempo (AUC) comporta il rischio di sovra o sotto dosaggio con il conseguente aumento del rischio di tossicità o di recidiva. E’ stato sviluppato un metodo di analisi rapido, sensibile e specifico per il rilevamento del busulfan nel plasma umano. Il test si basa su una estrazione liquido-liquido con acetato di etile e di rilevamento mediante cromatografia liquida, con ionizzazione electrospray e spettrometria tandem massa (HPLC-ESI-MS/MS). Il busulfan deuterato è stato utilizzato come standard interno. Il metodo è lineare nel range 39-2500 ng / mL, con r2> 0,99 e una durata della corsa cromatografica di soli 5 minuti (con un tempo di ritenzione del busulfan di 1,62 minuti). La precisione inter-day and intra-day è nel range di 1,01 - 3,72% e di 0,35-4,32%, rispettivamente. Il recupero è > 70%. Il limite di rivelabilità e di quantificazione sono di 6 e 10 ng/mL, rispettivamente. Il metodo di analisi riportato, richiede soltanto 200 μL di plasma per l'analisi. Il metodo convalidato è stato applicato con successo per l’analisi di campioni di plasma ottenuti da bambini talassemici sottoposti a un regime di condizionamento con busulfan e sottoposti a trapianto di midollo osseo. Questo metodo permette la correzione della dose consentendo un dosaggio adeguato al fine di ottenere la concentrazione ottimale di busulfan. Questo metodo è attualmente utilizzato per analizzare le concentrazioni plasmatiche di busulfan dopo somministrazione per via endovenosa ed è applicato nel monitoraggio terapeutico dei farmaci.Optimisation of busulfan dosage in patients undergoing bone marrow transplantation is recommended in order to reduce toxic effects associated with high drug exposure. Variation in the area under the concentration /time curve (AUC) results in risk of over or under treatment with excess risk of toxicity or relapse. A rapid, sensitive and specific assay for detection of busulfan in human plasma was developed. The assay is based on a liquid-liquid extraction with ethyl acetate and detection by high performance liquid chromatography with electrospray ionization and tandem mass spectrometry (HPLC-ESI-MS/MS). Deuterated busulfan was used as internal standard. The method was linear over the range 39-2500 ng/mL, with r2 > 0.99 and a run time of only 5 minutes (busulfan retention time of 1.62 minutes). The inter-day and intra-day precision were in the range 1,01- 3,72 % and 0,35-4,32 %, respectively. The recovery was >70%. The limits of detection and quantification were 6 and 10 ng/mL, respectively. The reported assay required only 200 µL of plasma for the analysis. The validated method was successfully applied to analyze plasma samples from children with thalassemia undergoing a conditioning regimen with busulfan and submitted to bone marrow transplantation. This method permits dose correction allowing a better dosing adjustment towards the target level of busulfan. This method is currently used to analyze the plasma concentrations of busulfan after intravenous administration and it is applied in therapeutic drug monitoring

BDE-47, -99, -209 and Their Ternary Mixture Disrupt Glucose and Lipid Metabolism of Hepg2 Cells at Dietary Relevant Concentrations: Mechanistic Insight through Integrated Transcriptomics and Proteomics Analysis

Polybrominated diphenyl ethers (PBDEs) are persistent organic chemicals implied as flame retardants. Humans are mainly exposed to BDE-47, -99, and -209 congeners by diet. PBDEs are metabolic disruptors with the liver as the main target organ. To investigate their mode of action at a human-relevant concentration, we exposed HepG2 cells to these congeners and their mixture at 1 nM, analyzing their transcriptomic and proteomic profiles. KEGG pathways and GSEA Hallmarks enrichment analyses evidenced that BDE-47 disrupted the glucose metabolism and hypoxia pathway; all the congeners and the MIX affected lipid metabolism and signaling Hallmarks regulating metabolism as mTORC1 and PI3K/AKT/MTOR. These results were confirmed by glucose secretion depletion and increased lipid accumulation, especially in BDE-47 and -209 treated cells. These congeners also affected the EGFR/MAPK signaling; further, BDE-47 enriched the estrogen pathway. Interestingly, BDE-209 and the MIX increased ERα gene expression, whereas all the congeners and the MIX induced ERβ and PPARα. We also found that PBDEs modulated several lncRNAs and that HNRNAP1 represented a central hub in all the four interaction networks. Overall, the PBDEs investigated affected glucose and lipid metabolism with different underlying modes of action, as highlighted by the integrated omics analysis, at a dietary relevant concentration. These results may support the mechanism-based risk assessment of these compounds in relation to liver metabolism disruption

Identification of the Junctional Plaque Protein Plakophilin 3 in Cytoplasmic Particles Containing RNA-binding Proteins and the Recruitment of Plakophilins 1 and 3 to Stress Granules

Recent studies on the subcellular distribution of cytoplasmic plaque proteins of intercellular junctions have revealed that a number of such proteins can also occur in the cyto- and the nucleoplasm. This occurrence in different, and distant locations suggest that some plaque proteins play roles in cytoplasmic and nuclear processes in addition to their involvement in cell–cell adhesive interactions. Plakophilin (PKP) 3, a member of the arm-repeat family of proteins, occurs, in a diversity of cell types, both as an architectural component in plaques of desmosomes and dispersed in cytoplasmic particles. In immuno-selection experiments using PKP3-specific antibodies, we have identified by mass spectrometric analysis the following RNA-binding proteins: Poly (A) binding protein (PABPC1), fragile-X-related protein (FXR1), and ras-GAP-SH3-binding protein (G3BP). Moreover, the RNA-binding proteins codistributed after sucrose gradient centrifugation in PKP3-containing fractions corresponding to 25–35 S and 45–55 S. When cells are exposed to environmental stress (e.g., heat shock or oxidative stress) proteins FXR1, G3BP, and PABPC1 are found, together with PKP3 or PKP1, in “stress granules” known to accumulate stalled translation initiation complexes. Moreover, the protein eIF-4E and the ribosomal protein S6 are also detected in PKP3 particles. Our results show that cytoplasmic PKP3 is constitutively associated with RNA-binding proteins and indicate an involvement in processes of translation and RNA metabolism

Carbon monoxide signaling in human red blood cells: evidence for pentose phosphate pathway activation and protein deglutathionylation

The biochemistry underlying the physiological, adaptive, and toxic effects of carbon monoxide (CO) is linked to its affinity for reduced transition metals. We investigated CO signaling in the vasculature, where hemoglobin (Hb), the CO most important metal-containing carrier is highly concentrated inside red blood cells (RBCs)

Cerebral Arterial Thrombosis in Ulcerative Colitis

Thrombosis, mainly venous, is a rare and well-recognized extraintestinal manifestation of inflammatory bowel disease (IBD). We describe a 25-year-old Caucasian man affected by ulcerative colitis and sclerosing cholangitis with an episode of right middle cerebral arterial thrombosis resolved by intraarterial thrombolysis. We perform a brief review of the International Literature

Proteomic and functional analyses reveal pleiotropic action of the anti-tumoral compound NBDHEX in Giardia duodenalis

Giardiasis, a parasitic diarrheal disease caused by Giardia duodenalis, affects one billion people worldwide. Treatment relies only on a restricted armamentarium of drugs. The disease burden and the increase in treatment failure highlight the need for novel, safe and well characterized drug options. The antitumoral compound NBDHEX is effective in vitro against Giardia trophozoites and inhibits glycerol-3-phosphate dehydrogenase. Aim of this work was to search for additional NBDHEX protein targets. The intrinsic NBDHEX fluorescence was exploited in a proteomic analysis to select and detect modified proteins in drug treated Giardia. In silico structural analysis, intracellular localization and functional assays were further performed to evaluate drug effects on the identified targets. A small subset of Giardia proteins was covalently bound to the drug at specific cysteine residues. These proteins include metabolic enzymes, e.g. thioredoxin reductase (gTrxR), as well as elongation factor 1B-gamma (gEF1B gamma), and structural proteins, e.g. alpha-tubulin. We showed that NBDHEX in vitro binds to recombinant gEF1B gamma and gTrxR, but only the last one could nitroreduce NBDHEX leading to drug modification of gTrxR catalytic cysteines, with concomitant disulphide reductase activity inhibition and NADPH oxidase activity upsurge. Our results indicate that NBDHEX reacts with multiple targets whose roles and/or functions are specifically hampered. In addition, NBDHEX is in turn converted to reactive intermediates extending its toxicity. The described NBDHEX pleiotropic action accounts for its antigiardial activity and encourages the use of this drug as a promising alternative for the future treatment of giardiasis. (C) 2017 The Authors. Published by Elsevier Ltd on behalf of Australian Society for Parasitology