Isabelle Lefebvre

Intracellular delivery of nucleoside monophosphates through a reductase-mediated activation process

On the basis of three different models (namely: ddU, AZT and PMEA), mononucleotide phosphotriester derivatives were designed to be able to liberate the corresponding monophosphate (or phosphonate) inside the cell through a reductase-mediated activation process. It was demonstrated that the use of bis[S-(2-hydroxyethylsulfidyl)-2-thioethyl] esters of ddUMP (11), AZTMP (12) and PMEA (17) resulted in intracellular delivery of the parent monophosphate (or phosphonate). This point was corroborated by observation of an anti-HIV effect of, 11 in various cell lines, for 12 in CEM TK- cells and by the enhanced activity observed for 17. Furthermore, the reported decomposition data in cell extracts fully confirm the validity of this approach and show unambiguously the potential for intracellular reductase-mediated activation of the starting drug.

Rational design for cytosolic delivery of nucleoside monphosphates : “SATE” and “DTE” as enzyme-labile transient phosphate protecting groups

Abstract It was demonstrated that the use of neutral 2′,3′-dideoxyuridine phosphotriesters which incorporate enzyme mediated bioreversible protection such as S-acetylthioethanol (SATE) or dithiodiethanol (DTE) resulted in intracellular delivery of the parent mononucleotide. This point was corroborated by observation of an anti-HIV effect in various cell lines and decomposition data in cell extracts.

Simultaneous analysis of eight nucleoside triphosphates in cell lines by liquid chromatography coupled with tandem mass spectrometry.

In this study, we developed a new method for the simultaneous determination of eight endogenous ribonucleoside triphosphates and deoxyribonucleoside triphosphates based on a combination of a selective sample preparation and an ion-pair liquid chromatography-electrospray tandem mass spectrometry. The sample preparation was based on a protein precipitation coupled with a solid phase extraction using a weak-anion-exchange cartridge. The analytical separation of the nucleotides was achieved on a porous graphitic carbon stationary phase with a binary elution gradient program employing ion-pairing reagents (diethylamine and hexylamine) and organic eluent (methanol). The triple quadrupole mass spectrometer operated in both negative and positive multiple reaction monitoring modes. The calibration assay used the stable isotope labelled analogs of each compounds as standard. Standard calibrations were from 0.25 to 10pmol injected according to deoxyribonucleotides and from 12.5 to 3000pmol injected according to ribonucleotides. The within-run precision of the assay was less than 14.5% and the between-run precision was less than 12.4% for each analytes. Assay accuracy was in the range of 92.3-107.6%. This method allows the determination of NTP and dNTP pools from lysats of several cell lines or peripheral blood mononuclear cell from patient. Assays were performed with different preparation of cells to confirm the quality and the relevance of the described method.

Decomposition Pathways of the Mono- and Bis(Pivaloyloxymethyl) Esters of Azidothymidine 5′-Monophosphate in Cell Extract and in Tissue Culture Medium: An Application of the ‘on-line ISRP-Cleaning’ HPLC Technique

Bis(pivaloyloxymethyl) azidothymidine 5′-monophosphate (piv2-AZTMP) was designed as a cell membrane-permeable precursor of AZTMP. We have reported previously that when incubated with CEM cells deficient in thymidine kinase, piv2-AZTMP gives rise to intracellular AZTMP and the corresponding diphosphate (AZTDP) and triphosphate (AZTTP). Under similar conditions, no intracellular nucleotides were formed with AZT. However, the mechanism by which piv2-AZTMP is converted to AZTMP has not been established. To address this question, we have used the recently developed ‘on-line ISRP-cleaning’ HPLC technique to investigate the stability and metabolic fate of piv2-AZTMP (1) in RPMI 1640 medium, (2) in RPMI containing 10% heat-inactivated fetal calf serum, and (3) in CEM cell extracts. Similar studies were conducted starting with mono(pivaloyloxymethyl) azidothymidine 5′-monophosphate (piv2 AZTMP). From the kinetics of these reactions, it appears that piv2-AZTMP is slowly hydrolyzed to piv1-AZTMP in RPMI and that the metabolic sequence in cell extract and in tissue culture medium is clearly: piv2-AZTMP→ piv1AZTMP→ AZTMP→ AZT. The rate constants are quite different in these three media. Although it is evident that the first step in the metabolism of piv2-AZTMP is catalysed by carboxylate esterase, the enzyme(s) responsible for the second step, piv1-AZTMP→ AZTMP, is less apparent, as carboxylate esterases and/or phosphodiesterases can be taken in account. However, analysis of the kinetic data strongly suggests that carboxylate esterase does not play a significant role and that this second step is mediated by phosphodiesterases. Collectively, these studies demonstrate that piv2-AZTMP is an effective prodrug of AZTMP. They also establish that prv1-AZTMP is an intermediate in this process, and define the sequence of the overall metabolic reaction. With this increased understanding of the metabolism of piv2-AZTMP, it should be possible rationally to design analogues with optimal structural and pharmacological properties for use in vivo.

Development of a sensitive and selective LC/MS/MS method for the simultaneous determination of intracellular 1-beta-d-arabinofuranosylcytosine triphosphate (araCTP), cytidine triphosphate (CTP) and deoxycytidine triphosphate (dCTP) in a human follicular lymphoma cell line

A method was developed for the quantification of araCTP, CTP and dCTP in a human follicular lymphoma cell line. This method involves solid phase extraction (SPE) using a weak anion-exchanger (WAX) cartridge, a porous graphitic carbon high-performance liquid chromatography (HPLC) column separation, and tandem mass spectrometry (MS/MS) detection. By using a triple quadrupole mass spectrometer operating in negative ion multiple reaction monitoring (MRM) mode, the method was able to achieve a lower limit of quantification (LLOQ) of 0.1 microg mL(-1) for araCTP and of 0.01 microg mL(-1) for both CTP and dCTP. The method was validated and used to determine the amount of araCTP, CTP and dCTP formed after incubation of araC and an araCMP prodrug in the human follicular lymphoma cell line RL.

Comparison of cytotoxicity of mononucleoside phosphotriester derivatives bearing biolabile phosphate protecting groups in normal human bone marrow progenitor cells

The effects of three mononucleoside phosphotriester derivatives of 3′-azido-2′,3′-dideoxythymidine (AZT) which incorporate biolabile phosphate protecting groups, namely S-acetyl-2-thioethyl (MeSATE), S-(2-hydroxyethylsulfidyl)-2-thioethyl (DTE), and pivaloyloxymethyl (POM) were studied and compared to their nucleoside parent in human myeloid colony-forming cells. Moreover, the relative antiviral potency of these three pronucleotides were determined in primary human peripheral blood mononuclear cells infected with human immunodeficiency virus type 1. The results indicate that the SATE and DTE pro-moieties, as well as their degradation products, do not induce additional toxicity. The bis(MeSATE) phosphotriester derivative of AZT emerged as the most selective inhibitor with an in-vitro therapeutic index of the same order of magnitude as observed for AZT. This study has been extended to the corresponding bis(MeSATE) and bis(DTE) phosphotriester derivatives of 2′,3′-dideoxyuridine (ddU).

Kinetics study of the biotransformation of an oligonucleotide prodrug in cells extract by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry

The fate of a dodecathymidine prodrug in cell extract was monitored by MALDI-TOF MS. This technique allows a facile identification and a relative quantification of metabolites produced. We showed that the relative peak intensities were similar to the relative metabolite proportions that permitted the determination of their half-lives. The oligonucleotide prodrug was fully metabolized to yield the T12 phosphorothioate likely through a carboxyesterase mediated mechanism.

DESIGN OF NEW MONONUCLEOTIDE PRODRUGS: ARYL (SATE) PHOSPHOTRIESTER DERIVATIVES

Synthesis, biological activities and decomposition kinetics of novel phosphotriester derivatives of 3′-azido-2′,3′-dideoxythymidine (AZT) bearing a S-tButyl-2-thioethyl (tBuSATE) group and L-tyrosinyl residues are reported. All the derivatives appeared to be potent inhibitors of HIV-1 replication in various cell culture experiments. The proposed decomposition process of these mixed phosphotriesters may involve successively an esterase and then a phosphodiesterase activation.

Anti-HIV Pronucleotides: SATE Versus Phenyl as a Protecting Group of AZT Phosphoramidate Derivatives

We comparatively studied the decomposition pathways in CEM cell extract of several PHENYL phosphoramidate diesters of AZT. A correlation between anti-HIV activities in TK- cell lines and pharmacokinetic data has been observed. This study would help to design corresponding SATE phosphoramidate diesters which revealed potent anti-HIV properties.

Synthesis, Decomposition Pathways and “In Vitro” Evaluation of Bioreversible Phosphotriesters of Azt

Abstract The synthesis, pharmacokinetic data and biological evaluation of a series of phosphotriesters containing S-acyl-2-thioethyl groups as enzyme-labile phosphate protecting groups and AZT as a model are described. A comparison of pharmacokinetic data and “in vitro” experiments show that such bioreversible phosphotriesters of AZT are able to cross cell membranes and deliver the corresponding nucleoside monophosphate inside the cell. Moreover, kinetic data show that modification of the protecting groups can allow to modulate both the extracellular stability of the parent compond and the delivery of nucleoside monophosphate inside the cell.