Stratégies d'optimisation des traitements à base de capécitabine au moyen des dosages pharmacologiques ou de la modulation de la Thymidine Phosphorylase

BESANCON-BU Médecine pharmacie (250562102) / SudocSudocFranceF

Impact of ticagrelor on P2Y1 and P2Y12 localization and on cholesterol levels in platelet plasma membrane

Ticagrelor is an antiplatelet agent that inhibits platelet activation via P2Y12 antagonism. There are several studies showing that P2Y12 needs lipid rafts to be activated, but there are few data about how ticagrelor impacts lipid raft organization. Therefore, we aimed to investigate how ticagrelor could impact the distribution of cholesterol and consequently alter the organization of lipid rafts on platelet plasma membranes. We identified cholesterol-enriched raft fractions in platelet membranes by quantification of their cholesterol levels. Modifications in cholesterol and protein profiles (Flotillin 1, Flotillin 2, CD36, P2Y1, and P2Y12) were studied in platelets stimulated by ADP, treated by ticagrelor, or both. In ADP-stimulated and ticagrelor-treated groups, we found a decreased level of cholesterol in raft fractions of platelet plasma membrane compared to the control group. In addition, the peak of cholesterol in different experimental groups changed its localization on membrane fractions. In the control group, it was situated on fraction 2, while in ADP-stimulated platelets, it was located in fractions 3 to 5, and in fraction 4 in ticagrelor-treated group. The proteins studied also showed changes in their level of expression and localization in fractions of plasma membrane. Cholesterol levels of plasma membranes have a direct role in the organization of platelet membranes and could be modified by stimulation or drug treatment. Since ticagrelor and ADP both changed lipid composition and protein profile, investigating the lipid and protein composition of platelet membranes is of considerable importance as a focus for further research in anti-platelet management

Comparisons of detergents for purification of platelet membrane lipid rafts: a lipidomics and proteomics analysis focusing on P2XR localization

Introduction: Platelet membrane contains cholesterol and sphingomyelin-rich microdomains also known as lipid rafts (LR). LRs have been considered as pharmacological target of anti-aggregation drugs as they suggested providing a platform for physiological activity of target proteins such as purinergic receptors (P2XR, P2YR) activated by nucleotides (ATP and ADP respectively). Localization of P2XR in these microdomains remains mostly unknown. The aim of this study was to compare the effectivity of different conventional detergents for lipid rafts isolation and investigation on the lipidomics and proteomics features of these microdomains focusing their potentiality of containing P2XR. Obviously, given the lack of well-established methods for platelet membrane studies, we also aimed at comparing the effectivity of different conventional detergents studies of plasma membrane studies. Material and methods: Platelets were obtained from healthy donors from Etablissement Franc_ais du Sang. Platelets were lysed with three different detergents (Brij 35, lubrol and TritonX100) in low and high doses (0.05% and 1%). After density separation of platelets lysate using ultracentrifugation with sucrose gradient (5, 35 and 45%), 12 equal sucrose fractions were collected and concentration of cholesterol (Ch), sphingomyelin (SM) and phosphatidyl-choline (PC) were quantified. Based on these lipidomics results, fractions containing raft and nonraft domains were subjected to proteomics analysis. Results: LRs mainly were observed in fractions 1–4 in all detergents except Triton 0.05% that was not being able to extract lipid rafts. Ch and SM enrichment analysis were performed by Ch/PC and SM/PC ratio in each fraction. It showed that Lubrol 1% and Triton 1% were more suitable detergents as they were able to isolate raft fractions enriched in Ch and SM about 1.5 times more than other detergents (Lubrol 1%: Ch/PC = 1.52 and SM/PC= 1.44; Triton 1%: Ch/PC = 1.77 and SM/PC= 1.48). By proteomics analysis; overall 822 proteins were identified in platelet membrane. Each detergent revealed a different profile of protein’s set. P2XR was found only in fractions contain lipid rafts by Brij 1% and Lubrol 0.05%. Discussion / Conclusion: Investigating on P2XR on the membrane of platelets, Brij 1% and Lubrol 0.05% would be suitable detergents. Our results suggest that depending on the focal objective of study, adopting a compatible detergent has a great importance

Identification of functional proteins related to Factor XIII in platelet lipid rafts

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Comparative lipidomics and proteomics analysis of platelet lipid rafts using different detergents

Lipid rafts play a pivotal role in physiological functions of platelets. Their isolation using nonionic mild detergents is considered as the gold standard method, but there is no consensual detergent for lipid raft studies. We aimed to investigate which detergent is the most suitable for lipid raft isolation from platelet membrane, based on lipidomics and proteomics analysis. Platelets were obtained from healthy donors. Twelve sucrose fractions were extracted by three different detergents, namely Brij 35, Lubrol WX, and Triton X100, at 0.05% and 1%. After lipidomics analysis and determination of fractions enriched in cholesterol (Ch) and sphingomyelin (SM), proteomics analysis was performed. Lipid rafts were mainly observed in 1-4 fractions, and non-rafts were distributed on 5-12 fractions. Considering the concentration of Ch and SM, Lubrol WX 1% and Triton X100 1% were more suitable detergents as they were able to isolate lipid raft fractions that were more enriched than non-raft fractions. By proteomics analysis, overall, 822 proteins were identified in platelet membrane. Lipid raft fractions isolated with Lubrol WX 0.05% and Triton X100 1% contained mainly plasma membrane proteins. However, only Lubrol WX 0.05 and 1% and Triton X100 1% were able to extract non-denaturing proteins with more than 10 transmembrane domains. Our results suggest that Triton X100 1% is the most suitable detergent for global lipid and protein studies on platelet plasma membrane. However, the detergent should be adapted if investigation of an association between specific proteins and lipid rafts is planned

Daptomycin Physiology-Based Pharmacokinetic Modeling to Predict Drug Exposure and Pharmacodynamics in Skin and Bone Tissues

International audienceBackground and objective: Daptomycin has been recommended in the treatment of bone and joint infection. Previous work showed that the approved dosage of daptomycin may be insufficient to achieve optimal exposure in patients with bone and joint infection. However, those studies assumed that bone exposure was similar to steady-state daptomycin-free plasma concentrations. We sought to establish a physiologically based pharmacokinetic (PBPK) model of daptomycin to describe the dynamics of daptomycin disposition in bone and skin tissue.Methods: a PBPK model of daptomycin was built using PK-Sim®. Daptomycin concentrations in plasma and bone were obtained from three previously published studies. Physicochemical drug characteristics, mass balance, anthropometrics, and experimental data were used to build and refine the PBPK model. Internal validation of the PBPK model was performed using the usual diagnostic plots. The final PBPK model was then used to run simulations with doses of 6, 8, 10, and 12 mg/kg/24 h. Pharmacokinetic profiles were simulated in 1.000 subjects and the probabilities of target attainment for the area under the concentration-time curve over the bacterial minimum inhibitory concentration were computed in blood, skin, and bone compartments.Results: the final model showed a good fit of all datasets with an absolute average fold error between 0.5 and 2 for all pharmacokinetic quantities in blood, skin and bone tissues. Results of dosing simulations showed that doses ≥10 mg/kg should be used in the case of bacteremia caused by Staphylococcus aureus with a minimum inhibitory concentration >0.5 mg/L or Enterococcus faecalis with a minimum inhibitory concentration >1 mg/L, while doses ≥12 mg/kg should be used in the case of bone and joint infection or complicated skin infection. When considering a lower minimum inhibitory concentration, doses of 6-8 mg/kg would likely achieve a sufficient success rate. However, in the case of infections caused by E. faecalis with a minimum inhibitory concentration >2 mg/L, a higher dosage and combination therapy would be necessary to maximize efficacy.Conclusions: we developed the first daptomycin PBPK/pharmacodynamic model for bone and joint infection, which confirmed that a higher daptomycin dosage is needed to optimize exposure in bone tissue. However, such higher dosages raise safety concerns. In this setting, therapeutic drug monitoring and model-informed precision dosing appear necessary to ensure the right exposure on an individual basis

Investigating the impact of posaconazole prophylaxis on systematic fungal screening using galactomannan antigen, Aspergillus fumigatus qPCR, and Mucorales qPCR

International audienc

An APCI LC-MS/MS method for routine determination of capecitabine and its metabolites in human plasma.

International audienceThe anticancer drug capecitabine and its metabolites [including the active metabolite 5-fluorouracil (5-FU)] display high pharmacokinetic inter-patient variability. Such variability, which may lead to treatment failure or toxicity, could need drug concentration measurement to individualize dosing regimen. However, usual assay methods are often long and fastidious. A simultaneous and cost-effective method was thus developed for the determination of the concentrations of these compounds in human plasma. Compounds were extracted via a classic liquid-liquid extraction. Chromatographic analysis was performed on a C18 reverse phase column with detection by atmosphere pressure chemical ionization LC-MS/MS. Our method allows a good chromatographic separation of the compounds and was fully validated following Food and Drug Administration (FDA) recommendations (good selectivity, no carry-over, linearity of the calibration curves without weighting, deviations from nominal concentrations of standard samples lower than 15%, intra- and inter-assay precision and accuracy lower than 15%). Recovery and stability were also acceptable following the FDA guidelines. A matrix effect impairing the determination of 5-FU was avoided by using a stable isotopic derivative of 5-FU as internal standard. Interestingly, this method allows detection of TetraHydroUridine, an inhibitor of ex vivo degradation of metabolites, which is essential for the stability, the adequate conditioning of blood samples and for good laboratory practice, essential in routine determination. This method seems usable to routinely determine concentrations of capecitabine and its metabolites in blood and may be helpful in further studies aiming at performing therapeutic drug monitoring

Surveillance and management of Echinococcus multilocularis in a wildlife park.

International audienceThe fox tapeworm Echinococcus multilocularis is the causative agent of alveolar echinococcosis, a severe zoonotic disease that may be fatal if untreated. A broad spectrum of mammalian species may be accidentally infected even in captivity. In April 2011, liver lesions due to E. multilocularis were observed during the necropsy of a captive-born nutria (Myocastor coypus) in a French wildlife park, leading to initiation of a study to survey the parasite's presence in the park. A comparable environmental contamination with fox's feces infected by E. multilocularis was reported inside (17.8%) and outside (20.6%) the park. E. multilocularis worms were found in the intestines of three of the five roaming foxes shot in the park. Coprological analyses of potential definitive hosts in captivity (fox, lynx, wildcat, genet, wolf, bear and raccoon) revealed infection in one Eurasian wolf. Voles trapped inside the park also had a high prevalence of 5.3%. After diagnosis of alveolar echinococcosis in a Lemur catta during necropsy, four other cases in L. catta were detected by a combination of ultrasound and serology. These animals were treated twice daily with albendazole. The systematic massive metacestode development and numerous protoscoleces in L. catta confirmed their particular sensitivity to E. multilocularis infection. The autochthonous origin of the infection in all the captive animals infected was genetically confirmed by EmsB microsatellite analysis. Preventive measures were implemented to avoid the presence of roaming foxes, contact with potential definitive hosts and contaminated food sources for potential intermediate hosts

An Immunodominant and Conserved B-Cell Epitope in the Envelope of Simian Foamy Virus Recognized by Humans Infected with Zoonotic Strains from Apes

International audienceCross-species transmission of simian foamy viruses (SFVs) from nonhu-man primates (NHPs) to humans is currently ongoing. These zoonotic retroviruses establish lifelong persistent infection in their human hosts. SFV are apparently non-pathogenic in vivo, with ubiquitous in vitro tropism. Here, we aimed to identify envelope B-cell epitopes that are recognized following a zoonotic SFV infection. We screened a library of 169 peptides covering the external portion of the envelope from the prototype foamy virus (SFVpsc_huHSRV.13) for recognition by samples from 52 Central African hunters (16 uninfected and 36 infected with chimpanzee, gorilla, or Cercopithecus SFV). We demonstrate the specific recognition of peptide N 96-V 110 located in the leader peptide, gp18 LP. Forty-three variant peptides with truncations, alanine substitutions, or amino acid changes found in other SFV species were tested. We mapped the epitope between positions 98 and 108 and defined six amino acids essential for recognition. Most plasma samples from SFV-infected humans cross-reacted with sequences from apes and Old World monkey SFV species. The magnitude of binding to peptide N 96-V 110 was significantly higher for samples of individuals infected with a chimpanzee or gorilla SFV than those infected with a Cercopithecus SFV. In conclusion, we have been the first to define an immunodomi-nant B-cell epitope recognized by humans following zoonotic SFV infection. IMPORTANCE Foamy viruses are the oldest known retroviruses and have been mostly described to be nonpathogenic in their natural animal hosts. SFVs can be transmitted to humans, in whom they establish persistent infection, like the simian lenti-and deltaviruses that led to the emergence of two major human pathogens, human immunodeficiency virus type 1 and human T-lymphotropic virus type 1. This is the first identification of an SFV-specific B-cell epitope recognized by human plasma samples. The immunodominant epitope lies in gp18 LP , probably at the base of the envelope trimers. The NHP species the most genetically related to humans transmitted SFV strains that induced the strongest antibody responses. Importantly, this epitope is well conserved across SFV species that infect African and Asian NHPs