A novel fluorescence-based assay for the rapid detection and quantification of cellular deoxyribonucleoside triphosphates

Current methods for measuring deoxyribonucleoside triphosphates (dNTPs) employ reagent and labor-intensive assays utilizing radioisotopes in DNA polymerase-based assays and/or chromatography-based approaches. We have developed a rapid and sensitive 96-well fluorescence-based assay to quantify cellular dNTPs utilizing a standard real-time PCR thermocycler. This assay relies on the principle that incorporation of a limiting dNTP is required for primer-extension and Taq polymerase-mediated 5–3′ exonuclease hydrolysis of a dual-quenched fluorophore-labeled probe resulting in fluorescence. The concentration of limiting dNTP is directly proportional to the fluorescence generated. The assay demonstrated excellent linearity (R2 > 0.99) and can be modified to detect between ∼0.5 and 100 pmol of dNTP. The limits of detection (LOD) and quantification (LOQ) for all dNTPs were defined as <0.77 and <1.3 pmol, respectively. The intra-assay and inter-assay variation coefficients were determined to be <4.6% and <10%, respectively with an accuracy of 100 ± 15% for all dNTPs. The assay quantified intracellular dNTPs with similar results obtained from a validated LC–MS/MS approach and successfully measured quantitative differences in dNTP pools in human cancer cells treated with inhibitors of thymidylate metabolism. This assay has important application in research that investigates the influence of pathological conditions or pharmacological agents on dNTP biosynthesis and regulation

A new ultrafast and high-throughput mass spectrometric approach for the therapeutic drug monitoring of the multi-targeted anti-folate pemetrexed in plasma from lung cancer patients

An analytical assay has been developed and validated for ultrafast and high-throughput mass spectrometric determination of pemetrexed concentrations in plasma using matrix assisted laser desorption/ionization–triple quadrupole–tandem mass spectrometry. Patient plasma samples spiked with the internal standard methotrexate were measured by multiple reaction monitoring. The detection limit was 0.4 fmol/μL, lower limit of quantification was 0.9 fmol/μL, and upper limit of quantification was 60 fmol/μL, respectively. Overall observed pemetrexed concentrations in patient samples ranged between 8.7 (1.4) and 142.7 (20.3) pmol/μL (SD). The newly developed mass spectrometric assay is applicable for (routine) therapeutic drug monitoring of pemetrexed concentrations in plasma from non-small cell lung cancer patients

Factors for Hematopoietic Toxicity of Carboplatin: Refining the Targeting of Carboplatin Systemic Exposure

Purpose Area under the curve (AUC) dosing is routinely carried out for carboplatin, but the chosen target AUC values remain largely empirical. This multicenter pharmacokinetic-pharmacodynamic (PK-PD) study was performed to determine the covariates involved in the interindividual variability of carboplatin hematotoxicity that should be considered when choosing individual target AUCs.Patients and Methods Three hundred eighty-three patients received carboplatin as part of established regimens. A semi-physiologic population PK-PD model was applied to describe separately the time course of absolute neutrophil and platelet counts using NONMEM software. The plasma ultrafiltrable carboplatin concentration (CCarbo) was assumed to inhibit the proliferation of blood cell precursors through a linear model: drug effect = slope × CCarbo. The slope corresponds to the patients\u27 sensitivity to carboplatin hematotoxicity. The relationships between the patients\u27 sensitivity to the neutropenic or thrombopenic effects of carboplatin and various covariates, including associated chemotherapies, demographic, biologic, and pharmacogenetic data, were studied. Results The sensitivity of carboplatin-induced thrombocytopenia decreased in the case of concomitant paclitaxel chemotherapy (slope decreased by 24%), whereas it increased with coadministration of etoposide and gemcitabine (slope increased by 45% and 133%, respectively). For neutropenia, the sensitivity increased when carboplatin was combined with other cytotoxics (slope increased by 76%). Conclusion This study provides useful information to clinicians to better estimate the hematopoietic toxicity of carboplatin and thus choose more rationally carboplatin target AUCs as a function of pretreatment or concomitantly administered chemotherapies. For example, an AUC of 5 mg/mL · min is associated with a risk of grade 3 or 4 thrombocytopenia of 2% in combination with paclitaxel versus 38% with gemcitabine in a non-pretreated patient

Circulating HDL and Non-HDL Associated Apolipoproteins and Breast Cancer Severity

Plasma lipids are carried within lipoproteins with various apolipoprotein content. This study evaluates the interest of measuring the apolipoproteins of circulating lipoproteins in breast cancer. Patients with early-stage breast cancer (n = 140) were included. Tumors differed by the expression of estrogen and progesterone receptor (HR&minus; and HR+ for negative and positive expression) and the proliferation marker Ki-67 (&le;20% or &ge;30%). Apolipoprotein concentrations were determined in plasma, HDL and non-HDL fractions, and results are given in mg/dL, median (25th&ndash;75th). Patients did not differ in their plasma and lipoprotein lipid concentrations. HDL apoC-I and non-HDL apoC-II were reduced (1.34 (1.02&ndash;1.80) vs. 1.61 (1.32&ndash;2.04), p = 0.04; 0.31 (0.18&ndash;0.65) vs. 0.63 (0.39&ndash;1.02), p = 0.01; respectively), in RH-/high Ki-67 patients in comparison to RH-/low Ki-67 patients, while plasma apoD and HDL apoD were higher (3.24 (2.99&ndash;4.16) vs. 3.07 (2.39&ndash;3.51), p = 0.04; 2.74 (2.36&ndash;3.35) vs. 2.45 (2.01&ndash;2.99), p = 0.04; respectively). When RH+/high Ki-67 patients were compared with RH+/low Ki-67 patients, HDL apoC-I and HDL apoC-III were higher (1.56 (1.20&ndash;1.95) vs. 1.35 (1.10&ndash;1.62), p = 0.02; 2.80 (2.42&ndash;3.64) vs. 2.38 (1.69&ndash;2.96), p = 0.02; respectively). The distribution of exchangeable apolipoproteins, such as apoC-I, apoC-II, apoC-III, apoD, between lipoproteins is linked to the severity of breast cancer

Le carboplatine (participation à un protocole de recherche clinique)

Le besoin permanent d'améliorer les pratiques médicales se ressent particulièrement en oncologie puisque les pathologies prises en charge sont lourdes, les traitements toxiques et le nombre de malades en constante augmentation. Les avancées thérapeutiques passent aujourd hui obligatoirement par la recherche clinique. En octobre 2006, au Centre Régional de Lutte Contre le Cancer René Gauducheau, une participation à un protocole de recherche pour optimiser l'utilisation du carboplatine, m'a été proposée. Après avoir rappelé les bases de la chimiothérapie, étudié en détail le carboplatine et le contexte de cet essai, j'ai réalisé les dosages de platine sur le plasma et l'ultrafiltrat des patients inclus, par spectrométrie d'absorption atomique. Les résultats des dosages ne sont que des résultats préliminaires, qui ne permettent pas aujourd'hui de répondre aux questions posées dans le cadre de l'essai. A l'issue de cette année de travail, j'ai pu suivre dans sa globalité ce protocole de recherche clinique : de sa conception jusqu'à sa réalisation pratique. L'implication dans un tel projet m'a permis de mieux comprendre quel peut être le rôle du pharmacien en recherche clinique et d'envisager le médicament sous un aspect différent.NANTES-BU Médecine pharmacie (441092101) / SudocSudocFranceF