Effect of a short-term HAART on SIV load in macaque tissues is dependent on time of initiation and antiviral diffusion

<p>Abstract</p> <p>Background</p> <p>HIV reservoirs are rapidly established after infection, and the effect of HAART initiated very early during acute infection on HIV reservoirs remains poorly documented, particularly in tissue known to actively replicate the virus. In this context, we used the model of experimental infection of macaques with pathogenic SIV to assess in different tissues: (i) the effect of a short term HAART initiated at different stages during acute infection on viral dissemination and replication, and (ii) the local concentration of antiviral drugs.</p> <p>Results</p> <p>Here, we show that early treatment with AZT/3TC/IDV initiated either within 4 hours after intravenous infection of macaques with SIVmac251 (as a post exposure prophylaxis) or before viremia peak (7 days post-infection [pi]), had a strong impact on SIV production and dissemination in all tissues but did not prevent infection. When treatment was initiated after the viremia peak (14 days pi) or during early chronic infection (150 days pi), significant viral replication persists in the peripheral lymph nodes and the spleen of treated macaques despite a strong effect of treatment on viremia and gut associated lymphoid tissues. In these animals, the level of virus persistence in tissues was inversely correlated with local concentrations of 3TC: high concentrations of 3TC were measured in the gut whereas low concentrations were observed in the secondary lymphoid tissues. IDV, like 3TC, showed much higher concentration in the colon than in the spleen. AZT concentration was below the quantification threshold in all tissues studied.</p> <p>Conclusions</p> <p>Our results suggest that limited antiviral drug diffusion in secondary lymphoid tissues may allow persistent viral replication in these tissues and could represent an obstacle to HIV prevention and eradication.</p

Dodecyl creatine ester improves cognitive function and identifies key protein drivers including KIF1A and PLCB1 in a mouse model of creatine transporter deficiency

Creatine transporter deficiency (CTD), a leading cause of intellectual disability is a result of the mutation in the gene encoding the creatine transporter SLC6A8, which prevents creatine uptake into the brain, causing mental retardation, expressive speech and language delay, autistic-like behavior and epilepsy. Preclinical in vitro and in vivo data indicate that dodecyl creatine ester (DCE) which increases the creatine brain content, might be a therapeutic option for CTD patients. To gain a better understanding of the pathophysiology and DCE treatment efficacy in CTD, this study focuses on the identification of biomarkers related to cognitive improvement in a Slc6a8 knockout mouse model (Slc6a8−/y) engineered to mimic the clinical features of CTD patients which have low brain creatine content. Shotgun proteomics analysis of 4,035 proteins in four different brain regions; the cerebellum, cortex, hippocampus (associated with cognitive functions) and brain stem, and muscle as a control, was performed in 24 mice. Comparison of the protein abundance in the four brain regions between DCE-treated intranasally Slc6a8−/y mice and wild type and DCE-treated Slc6a8−/y and vehicle group identified 14 biomarkers, shedding light on the mechanism of action of DCE. Integrative bioinformatics and statistical modeling identified key proteins in CTD, including KIF1A and PLCB1. The abundance of these proteins in the four brain regions was significantly correlated with both the object recognition and the Y-maze tests. Our findings suggest a major role for PLCB1, KIF1A, and associated molecules in the pathogenesis of CTD

Mise au point d'un cocktail de substrats phénotypant les principales enzymes responsables de la pharmacocinétique des médicaments

Ce travail de thèse avait pour but de développer un cocktail de substrats sondes permettant l établissement d un phénotype basé sur l activité des protéines responsables de la pharmacocinétique des médicaments. Mon travail a consisté, d une part à la sélection des enzymes ciblées et des substrats sondes ; d autre part au développement d une méthode de dosage des substrats et des métabolites et à la validation de ce cocktail CIME à l aide de modèles in vitro et in vivo et enfin, aux réflexions nécessaires à la rédaction du protocole de la première étude clinique utilisant ce cocktail chez le volontaire.This PhD thesis was aimed to develop a cocktail of probe substrates allowing the establishment of a human phenotype based on the activity of proteins responsible for drugs pharmacokinetics. My work consisted in the targeted proteins and the probe substrates selection, in the assay method development for the substrates and metabolites, in the validation of this CIME cocktail by means of in vitro and in vivo experiments and finally, in the necessary considerations on the protocol writing of the first clinical study using this cocktail in volunteers.CHATENAY M.-PARIS 11-BU Pharma. (920192101) / SudocSudocFranceF

Pharmacologie intracellulaire des antirétroviraux utilisés contre le VIH (développement et méthodes analytiques et intérêt de la mesure des concentrations intracellulaires pour la compréhension des phénomènes d'efficacité et d'échappement thérapeutique)

Durant ce travail de thèse, plusieurs méthodes analytiques ont été validées pour quantifier par couplage LC-MS/Ms et Lc-UV dans le plasma et le milieu intracellulaire les différents antirétroviraux utilisés contre le VIH. Ces outils analytiques ont permis d'étudier la transformation de l'AZT conduisant à la formation de d4T-TP dont l'identité a été confirmée. Des informations sur l'étape de la transformation et le profil cinétique de la d4T-TP issue d'AZT ont été apportées. Il a été montré que l'utilisation des concentrations intracellulaires pouvait être un outil prédictif de l'efficacité et permettrait de comprendre les phénomènes d'échappement thérapeutique. Ainsi, la d4TP-TP ne participerait pas à l'efficacité antivirale de l'AZT mais au contraire la diminuerait. La mesure des concentrations plasmatiques et intracellulaires constitue donc un outil indispensable à la pharmacologie des antirétroviraux utilisés contre le VIH et devrait permettre de mettre en place des traitements efficaces.Several analytical methods were developed using LC/MS/MS and LC/UV for the determination of the antiretroviral drugs used against HIV in plasma and intracellular medium. In particular, these analytical lethods enabled to study the transformation of AZT that leads to the formation of d4TP-TP, compound thaty was formally identified. Some information concerning the step of this transformation, and the intracellular pharmacocinetik profile of d4TP-TP from AZT was given. We showed that intracellular concentrations could be a predictive tool for efficient treatment and allowed understanding treatment failure phenomena. Thus d4TP-TP from AZT did not participate to effectiveness of AZT and in contrary d4TP-TP decreased its. The use of plasmatic and intracellular concentrations is an essential tool for the pharmacology of antiviral drugs used against HIV and will allow setting up an effective treatment.CHATENAY M.-PARIS 11-BU Pharma. (920192101) / SudocSudocFranceF

Dosage des métabolites phosphorylés actifs intracellulaires des inhibiteurs nucléosidiques de la transcriptase inverse (INTIs) du VIH (développements analytiques et applications pharmacologiques)

PARIS-BIUP (751062107) / SudocSudocFranceF

P-Glycoprotein, breast cancer resistance protein, organic anion transporter 3, and transporting peptide 1a4 during blood-brain barrier maturation : involvement of Wnt/beta-Catenin and Endothelin-1 signaling

International audienceOur current knowledge about drug transporters in the maturational brain is very limited. In this study, we provide a comprehensive overview of the expression and activity profile of P-glycoprotein (P-gp), Breast Cancer Resistance Protein (bcrp), Organic Anion Transporter 3 (oat3), and Transporting Peptide 1a4 (oatp1a4) transporters during blood-brain barrier (BBB) maturation. Gene and protein expressions of the analyzed transporters increase as the brain matures, with no variation in their activity for P-gp and bcrp, while the transport activity of oat3 and oatpla4 increases during brain maturation from preterm up to adulthood. For the first time, we illustrate a downregulation of nuclear beta-catenin expression in brain capillaries when bcrp, P-gp, oat3, and oatpla4 transporters are at their highest expression levels. In vivo activation of beta-catenin in rat brains, by intracerebroventricular (ICV) injection of a GSK-3 inhibitor, enhances the activity of P-gp, bcrp, oat3, and oatp1a4. Interestingly, in an in vitro BBB model consisting of a coculture of primary endothelial brain cells with astrocytes or in vivo, activation of beta-catenin enhances the mRNA expression of ET -1. Interestingly, blocking the ETA receptor for endothelin-1 in vivo by ICV injection of a ETA antagonist decreases transporter activity mediated by the activation of beta-catenin. These findings shed light on the role of an interaction between beta-catenin and endothelin-1 signaling in the regulation of these transporters at the BBB

Human Immunodeficiency Virus Type 1: Resistance to Nucleoside Analogues and Replicative Capacity in Primary Human Macrophages▿

Antiretroviral treatment failure is associated with the emergence of resistant human immunodeficiency virus type 1 (HIV-1) populations which often express altered replicative capacity (RC). The resistance and RC of clinical HIV-1 strains, however, are generally assayed using activated peripheral blood mononuclear cells (PBMC) or tumor cell lines. Because of their high proliferation rate and concurrent high deoxynucleoside triphosphate (dNTP) content, both resistance and RC alterations might be misestimated in these cell systems. We have evaluated the resistance of HIV-1 clones expressing a variety of RT resistance mutations in primary human macrophages using a single cycle system. Our experiments indicate that d4T, ddI, and 3TC are more potent in macrophages than in HeLa-derived P4 tumor cells. Mutant viruses bearing thymidine analogue mutations (TAMs) or the K65R mutation had similar resistance levels in the two cell types. Strikingly, however, the M184V mutant, although fully resistant to 3TC in P4 cells, maintained some susceptibility to 3TC in macrophages from 8 of 11 donors. Using the same system, we found that the impact of resistance mutations on HIV RC was minimal in activated PBMC and in P4 cells. In contrast, mutant viruses exhibited strongly impaired RC relative to the wild type (WT) in macrophages, with the following RC order: WT > two TAMs > four TAMs = M184V > K65R. In undifferentiated monocytes, WT virus replication could be detected in three of six donors, but replication of all mutant viruses remained undetectable. Altogether, our results confirm that nucleoside reverse transcriptase inhibitors (NRTIs) are powerful antiviral agents in differentiated macrophages, reveal that HIV resistance to some NRTIs may be less efficient in these cells, and indicate that resistance-associated loss of RC is more pronounced in macrophages than in high-dNTP content cell systems