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The Human Immodeficiency Viruses type 1 and 2 (HIV-1 and HIV-2) are both causing AIDS (Acquired ImmunoDeficiency Syndrome). However, HIV-2 has several discrepancies compared to HIV-1 : the disease progression is slower, the viremia is weaker, and the efficiency of transmission is lower. This explains in part why that virus is mainly present in West Africa and did not caused a pandemic as HIV-1. Genetic differences are responsible for structural variations in the proteins that are targeted by the antiretroviral drugs, which were developed against HIV-1. Therefore HIV-2 is naturally resistant to some of these molecules. The analysis of the available data in the literature, as well as the observations made form the patients of Belgian and Luxembourg, allowed us to propose guidelines for the treatment of HIV-2 infection. The antiretroviral combination should include protease inhibitors (PI) and nucleosidic reverse transcriptase inhibitors (NRTI). The most appropriated PIs seem to be lopinavir, saquinavir and darunavir Les virus de l’immunodéficience humaine de type 1 et 2 (VIH-1 et VIH-2) sont deux virus responsables du développement du SIDA (Syndrome d’ImmunoDéficience Acquise). Cependant, le VIH-2 présente plusieurs particularités qui le différencient du virus de type 1 : l’évolution de l’infection est en général plus lente, la virémie est plus faible, et l’efficacité de transmission est moindre. Ceci montre en partie pourquoi ce virus est présent majoritairement en Afrique de l’Ouest et n’a pas causé de pandémie comme le VIH-1. Des différences génétiques sont responsables de variations structurelles des protéines visées par les thérapies antirétrovirales développées contre le VIH-1. Dès lors, le VIH-2 est naturellement résistant à certaines molécules. L’analyse des données de la littérature, ainsi que les observations menées chez les patients suivis en Belgique et au Luxembourg, ont permis d’élaborer des recommandations pour le traitement de l’infection VIH-2. L’association de molécules antirétrovirales devrait inclure des inhibiteurs de protéase (IP) et des inhibiteurs nucléosidiques de la transcriptase inverse. Les IP les plus appropriés semblent être le lopinavir, le saquinavir et la darunavir
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Protease inhibitors (PIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs) belong to two classes of drugs which have, since their introduction on the market, significantly reduced the morbidity and mortality related to the HIV infection. Nevertheless, the rate of therapeutic failure remains high, and the use of these drugs is still associated with important toxicity episodes. The use of therapeutic drug monitoring, combined with a pharmacogenetic approach, could considerably improve the efficacy of antiretroviral treatments (ARV). However, this approach requires the use of reliable dosage methods in biological media. Therefore, we first developed and validated two analytical methods for the simultaneous quantification of 10 ARV (8 PIs [IDV, SQV, NFV, APV, ATZ, RTV, LPV, TPV] and 2 NNRTIs [NVP and EFV]) in plasma and lymphocytes, the site of therapeutic action of these drugs. A method using liquid chromatography coupled with an UV detector (UPLC-DAD) was developed and covered plasmatic concentrations ranging from 25 to 10.000 µg/L for all ARV, except the TPV (1.875 to 75.000 µg/L). For ARV intralymphocytic dosages for concentrations ranging from 0,5 to 100 µg/L of cell extract, a liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method was validated. However, this method must still be improved for some compounds before being used in daily clinical practice. Pharmacogenetics could also play an essential role in the individualization of treatments. The second objective of this work was to investigate the impact of genetic polymorphisms of the P-glycoprotein (P-gp), protein involved in the transport of PIs, as well as biotransformation enzymes (CYP3A5 and CYP2B6) on the intracellular accumulation of some PIs and NNRTIs. First, it appears that the ABCB1 1199G>A polymorphism affects the affinity of the P-gp for its substrates and is associated with an enhanced cellular efflux of some IPs (ATZ, RTV). In addition, our results suggest that the activity of the P-gp towards NFV and Rh123 seems to be affected by the ABCB1 3435C>T polymorphism. Also, we showed that the polymorphism CYP3A5*3 could be important to explain a part of the interindividual variability observed in the intralymphocytic metabolism of SQV. Finally, we observed a variation in the efllux and the accumulation of some ARV (ATZ, RTV and NVP) among two cell lines (H9 and MT4) characterized by differences in their P-gp activity. In conclusion, this work provides the possibility to quantify PIs and NNRTIs in plasma and lymphocytes ; and highlights some genetic polymorphisms likely to explain the interindividual variability observed in the pharmacokinetics of ARV. Les inhibiteurs de la protéase (IPs) et les inhibiteurs non-nucléosidiques de la transcriptase inverse (NNRTIs) appartiennent à deux classes de médicaments qui ont, depuis leur introduction sur le marché, permis une réduction significative de la morbidité de la mortalité liées au VIH. Cependant, le taux d’échec thérapeutique demeure élevé, et l’usage de ces médicaments reste associé à d’importants phénomènes de toxicité. L’utilisation du monitoring thérapeutique, combinée à une approche pharmacogénomique, pourrait considérablement améliorer l’efficacité des traitements antirétroviraux (ARV). Cette approche nécessite toutefois l’utilisation de méthodes de dosage fiables dans les milieux biologiques. Ainsi, dans un premier temps, nous avons développé et validé deux méthodes analytiques pour le dosage simultané de 10 ARV (8 IPs [IDV, SQV, NFV, APV, ATZ, RTV, LPV, TPV] et 2 NNRTIs [NVP et EFV]) dans le plasma et dans les lymphocytes, sites d’action de ces médicaments. Une méthode de chromatographie liquide couplée à un détecteur UV (UPLC-DAD) a été développée pour les dosages plasmatiques sur une gamme de concentrations de 25 à 10.000µg/L pour tous les ARV, excepté le TPV (1.875 à 75.000 µg/L). Pour les dosages intralymphocytaires des ARV sur une gamme de concentrations de 0,5 à 100 µg/L, une méthode de chromatographie liquide couplée à un spectromètre de masse en tandem (LC-MS/MS) a été validée. Cette méthode doit cependant être améliorée pour certains des composés avant de pouvoir être utilisée en routine clinique. La pharmacogénomique pourrait également jouer un rôle essentiel dans l’individualisation des traitements. Le second objectif de ce travail était d’investiguer l’impact de polymorphismes génétiques de la P-gp, protéine impliquée dans le transport des IPs, ainsi que d’enzymes de biotransformation (CYP3A5 et CYP2B6) sur l’accumulation intracellulaire de certains IPs et NNRTIs. Il apparaît tout d’abord que le polymorphisme ABCB1 1199G>A affecte l’affinité de la P-gp pour ses substrats et est associé à un efflux cellulaire accru de certains IPs (ATZ, RTV). Par ailleurs, nos résultats suggèrent que l’activité de la P-gp vis-à-vis du NFV et de la Rh123 semble être affectée négativement par le polymorphisme ABCB1 3435C>T. Aussi, nous avons montré que le polymorphisme CYP3A5*3 pourrait être important pour expliquer la variabilité interindividuelle observée dans le métabolisme intralymphocytaire du SQV. Enfin, nous avons observé une variation dans l’efflux et l’accumulation de certains ARV (ATZ, RTV et NVP) au sein de deux lignées cellulaires (H9 et MT4) présentant une activité de la P-gp différente. Ce mémoire offre donc la possibilité de doser les IPs et les NNRTIs dans le plasma et les lymphocytes ; et met en évidence certains polymorphismes génétiques susceptibles d’expliquer la variabilité interindividuelle observée dans la pharmacocinétique des ARV.
Protease Inhibitors --- P-Glycoprotein --- Cytochrome P-450 --- Lymphocytes --- Plasma --- HIV Reverse Transcriptase
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Acquired Immunodeficiency Syndrome --- HIV Protease Inhibitors --- HIV Infections --- Anti-HIV Agents --- HIV-1 --- HIV Reverse Transcriptase --- drug therapy --- therapeutic use --- drug therapy --- pharmacology --- genetics
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The Reverse Transcriptase (RT) of Human Immunodeficiency Virus Type 1 (HIV-1) arguably ranks amongst one of the most extensively studied retroviral enzymes. Heterologous expression and purification of HIV-1 RT in the early eighties, approval of the first nucleoside analogue RT inhibitor (NRTI) in 1987, discovery of resistance to RT inhibitors, approval of the first non-nucleoside analogue RT inhibitor (NNRTI) in 1996 and the various crystal structures of RT with and without bound substrate(s) and/or inhibitors represent only a few of the important milestones that describe the a bench-to-bedside success in the continuing effort to combat HIV-1 infection and its consequences. Nucleoside and nonnucleoside RT inhibitors remain important components in frequently used drug regimens to treat the infection. RT inhibitors also play important roles in recently validated strategies to prevent transmission of the virus. The relevance of HIV-1 RT as a drug target has simultaneously triggered interest in basic research studies aimed at providing a more detailed understanding of interactions between proteins, nucleic acids, and small molecule ligands in general terms. In light of the ever-growing knowledge on structure and function of HIV-1 RT, this enzyme serves as a valuable “model system” in efforts to develop novel experimental tools and to explain biochemical processes. This monograph is designed to provide an overview of important aspects in past and current HIV-1 RT research, with focus on mechanistic aspects and translation of knowledge into drug discovery and development. The first section includes chapters with emphasis placed on the coordination of the RT-associated DNA polymerase and ribonuclease H (RNase H) activities. The second covers mechanisms of action and future perspectives associated with NRTIs and NNRTIs, while the third section includes chapters focusing on novel strategies to target the RT enzyme. Chapters of the final part are intended to discuss mechanisms involved in HIV variability and the development of drug resistance. We hope that these contributions will stimulate interest, and encourage research aimed at the development of novel RT inhibitors. The lack of bona fide RNase H inhibitors with potent antiviral activity provides an example for challenges and opportunities in the field.
HIV (Viruses) --- Reverse transcriptase --- RNA-Directed DNA Polymerase --- DNA-Directed DNA Polymerase --- DNA Nucleotidyltransferases --- Nucleotidyltransferases --- Phosphotransferases --- Transferases --- Enzymes --- Enzymes and Coenzymes --- Chemicals and Drugs --- HIV Reverse Transcriptase --- Biology --- Health & Biological Sciences --- Microbiology & Immunology --- Immunology. --- HIV infections --- Research --- Methodology. --- HIV (Viruses) infections --- HTLV-III infections --- HTLV-III-LAV infections --- Human T-lymphotropic virus III infections --- Immunobiology --- Medicine. --- Virology. --- Infectious diseases. --- Biomedicine. --- Infectious Diseases. --- Life sciences --- Serology --- Lentivirus infections --- Sexually transmitted diseases --- Medical virology. --- Emerging infectious diseases. --- Medical microbiology --- Virology --- Virus diseases --- Emerging infections --- New infectious diseases --- Re-emerging infectious diseases --- Reemerging infectious diseases --- Communicable diseases --- Microbiology
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