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Quantitative metabolism of natural and antiviral nucleosides and nucleotides in human cells by LC-MS/MS

Abstract : Our laboratory is specialized in drug discovery and development, more specifically in nucleoside analogue research as potential antiviral agents. The laboratory is organized in several teams, including, chemists, virologists, molecular biologists and pharmacologists. The pharmacologists study the metabolism of both FDA approved and new drugs in vitro and in humans. amdoxovir TM is in development in our laboratory as anti-human immunodeficiency virus (HIV) and is in phase I/II clinical trial. The analytical team studies antiviral agents from cell culture work to clinical trials. To accomplish this goal, we used high performance liquid chromatography tandem mass spectrometry, which is sensitive and specific enough to detect analytes in the ppb range, in a complex biological matrix. Since only the triphosphate forms of the nucleoside analogues are active intracellularly, it was necessary to develop a method to analyze these polar compounds. The method, presented in the first chapter, was successfully used for the simultaneous quantification of the nucleotide (phosphate forms) metabolites of approved nucleoside, amdoxovir as well as for endogenous natural nucleotides in human lymphocytes and macrophages. The limits of quantification were low enough to measure nucleotide levels in the ppb range. In the second chapter, we extended our knowledge on amdoxovir metabolism in primary human lymphocytes cells. To do so, we incubated amdoxovir with nucleosides susceptible of inhibiting its phosphorylation. Then, we established the lack of interaction between amdoxovir and three other nucleosides analogues. Finally, deeper studies on endogenous natural nucleotides provided an understanding of the antiviral synergistic effects between amdoxovir and zidovudine that was demonstrated in vitro and in humans. The development and validation of a methodology to quantify amdoxovir, its metabolite and the approved drug zidovudine in plasma is presented in the first chapter. This method was applied to a proof-of-concept study conducted in 24 HIV-infected individuals.
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Submitted on : Monday, July 4, 2011 - 5:56:47 PM
Last modification on : Saturday, June 6, 2020 - 3:56:49 AM
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  • HAL Id : pastel-00605911, version 1
  • PRODINRA : 246184

Citation

Emilie Fromentin. Quantitative metabolism of natural and antiviral nucleosides and nucleotides in human cells by LC-MS/MS. Pharmacology. AgroParisTech, 2010. English. ⟨NNT : 2010AGPT0040⟩. ⟨pastel-00605911⟩

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