Projet ANR-06-BLAN-0157 – AnaTools : Analytical Tools for Cancer Chemotherapy Improvement
Programme : Programme blanc (BLANC) 2006
Année de financement : 2006
Coordinateur du projet : CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE LANGUEDOC-ROUSSILLON (CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE LANGUEDOC-ROUSSILLON)
Partenaires : Centre national de la recherche scientifique; Centre national de la recherche scientifique; Centre national de la recherche scientifique; Institut national de la santé et de la recherche médicale
Montant : 401000.0 euros
Date de début : 2006
Durée : 48 mois
Résumé : Scientific background and objectives Quantification of intracellular physiological and cytotoxic nucleoside/nucleotide pools by on-line SSPE LC-MS/MS methods The major obstacles of cancer chemotherapy are the severe side effects and the development of drug resistance. Due to the modest tumor specificity of many anticancer drugs, normal tissues are also damaged. This prevents the application of high sufficient doses to eradicate less sensitive tumor cell populations. Thereby, tumors develop drug resistance that leads to treatment failure and fatal consequences for patients. In addition, it is a well-known clinical observation that the same doses of medication cause considerable heterogeneity in efficacy and toxicity across human populations. This heterogeneity can lead to unpredictable life-threatening or even lethal adverse effects in small groups of patients. In this context, the current project focuses on cytotoxic nucleoside analogues which are commonly used in the treatment of solid tumors and hematological malignancies. Based on preliminary results, we propose to develop new analytical methods leading (i) to examine changes in endogenous nucleoside/nucleotide pools in cancer cells and how imbalances may lead to genetic mutations and a more aggressive cancer phenotype; (ii) to apply the method to the pharmacology of a nucleoside model (araC) in order to quantify its intracellular concentration as well as those of its phosphorylated metabolites. Initially developed using biological models (cell extracts, cell lines), the method will be applied to clinical samples. In this case, data will be correlated to the expression level (determined by rt-PCR) of selected cellular enzymes and results will be analyzed with mathematical, statistical and informatic tools in order to study the relationships between the different nucleoside and nucleotide pools, the influence of the different protein expression levels on these pools, and correlations between the pools and protein expression levels with sensitivity to cytotoxic compounds.  The global objectives of this project are (i) to study the cellular impact induced by deregulation of physiological nucleoside/nucleotide pools, (ii) to identify best-tolerated and most effective treatment regimen using cytotoxic nucleosides and, (iii) to predict the emergence of resistance to this therapeutic class.   Methodology The quantification of nucleosides, as well as their nucleotides, constitutes a challenge since the method requires both highest sensitivity and selectivity in order to detect, in a complex biological matrix, smallest amounts of analytes present among the myriad of other ribonucleotides and deoxyribonucleotides. Current approaches consist in the extraction and concentration of target analytes using solid phase extraction (SPE) sorbent followed by their analysis by liquid-chromatography (LC). However, as retention on classical SPE sorbents applied to samples is mainly based on non selective hydrophobic interactions, many other matrix components are co-extracted thus rendering difficult the detection and the quantification of target analytes in LC. Mass spectrometry (MS) seems to appear as a technique of choice to circumvent such a problem of identification of target analytes from complex matrices. However, it has been now largely reported that direct coupling of LC and MS suffers from suppression ionisation effect due to matrix components that may induce a high variability in the ionisation process of the target analytes, thus decreasing the method reliability in term of quantification.  In this project, we propose to develop a selective solid-phase extraction (SSPE) procedure based on a sorbent developing a molecular recognition retention mechanism to isolate the target analytes from the complex matrices increasing by the way specificity and sensitivity of the method. This sorbent could be an immunosorbent produced using immobilised antibodies specific of the target analytes or a m