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Henri Benech

Publications and source records attributed to Henri Benech.

11 recordsLinked to original sources
PubMed2005

Development and validation of a liquid chromatographic/tandem mass spectrometric assay for the quantitation of nucleoside HIV reverse transcriptase inhibitors in biological matrices.

Besides liquid chromatographic (LC)/UV methods adapted to therapeutic drug monitoring, there is still a need for more powerful techniques that can be used for pharmacological research and clinical purposes. We developed an LC method coupled with tandem mass spectrometry (MS/MS) to separate, detect and quantify with high sensitivity the nucleoside analogues used in multitherapies (zidovudine, stavudine, zalcitabine, didanosine, lamivudine and abacavir) in plasma and in the intracellular medium. We worked on two essential issues: (i) the need to use two ionization modes in order to achieve the best sensitivity, which leads to the optimization of the chromatographic separation of drugs detected in the positive ionization mode and drugs detected in the negative ionization mode, and (ii) the need to optimize the extraction step in order to enhance sample recovery. The peripheral blood mononuclear cells were lysed in Tris buffer-MeOH. A clean-up procedure was performed by solid-phase extraction only for plasma samples. The LC separation was carried out on a Zorbax Stable Bond C(18) column followed by MS/MS analysis after electrospray ionization in either the negative or positive mode. The positive ionization mode was applied at the beginning of the run to detect zalcitabine and lamivudine, then the ionization mode was changed to negative for the detection of didanosine, stavudine, internal standard and zidovudine. The calibration range for all the analytes was 0.5-200 ng ml(-1). The recoveries were between 64 and 90%, with coefficients of variation (CVs) lower than 15%. The inaccuracy (bias) was +/-15% with CVs always lower than 12%. The analytes were stable at room temperature and in the extraction solvent for at least 24 h, after storage at -80 degrees C for 3 months, after three freeze-thaw cycles and in the injection solvent after 48 h at 4 degrees C. Together with the measurement of intracellular triphosphorylated metabolites thanks to the powerful plasma and intracellular assay method for intact drugs, it is possible to describe the behaviour of nucleoside analogues against HIV through plasma pharmacokinetics, cell membrane diffusion including drug transport involvement, and also the intracellular metabolism.

Chromatography, High Pressure Liquid↗
PubMed2005

Measurement of intracellular didanosine and tenofovir phosphorylated metabolites and possible interaction of the two drugs in human immunodeficiency virus-infected patients.

Recent work has demonstrated the existence of a systemic interaction between didanosine (ddI) and tenofovir disoproxyl fumarate (TDF) that leads to a significant increase in plasma ddI levels when coadministered with TDF (40 to 50% increase). These two drugs are, respectively, nucleoside and nucleotide analogues of adenosine and efficiently inhibit the human immunodeficiency virus (HIV) reverse transcriptase when transformed to their triphosphate moieties in the intracellular (IC) medium (ddA-TP and TFV-DP, respectively). Since ddI and TDF partly share the same IC metabolic pathway leading to the active triphosphates, we investigated a putative IC interaction. We used high-performance liquid chromatography-tandem mass spectrometry techniques to determine ddA-TP and TFV-DP IC levels in HIV-infected patients cotreated with both drugs, in comparison with patients treated with just one of the two drugs. These measurements revealed no significant differences in IC levels of the corresponding triphosphates when ddI (250 mg, once a day [QD]) was coadministered with TDF (300 mg, QD) compared to ddI 400 mg (QD) administered without TDF, thus supporting the dose adaptation proposed for this combination. However, we observed that both ddA-TP and TFV-DP have very long IC half-lives, resulting in unusual IC pharmacokinetic profiles with no significant changes in triphosphate concentrations between two dosings. In the case of TFV-DP, this t(1/2) of elimination was roughly estimated to be 180 h (7.5 days). This characteristic is certainly interesting in terms of efficacy but could have some drawbacks in terms of virus resistance for patients discontinuing these drugs.

Adenine↗
PubMed2005

Quantification of the effects on viral DNA synthesis of reverse transcriptase mutations conferring human immunodeficiency virus type 1 resistance to nucleoside analogues.

Human immunodeficiency virus type I (HIV-1) reverse transcriptase (RT) resistance mutations reduce the susceptibility of the virus to nucleoside analogues but may also impair viral DNA synthesis. To further characterize the effect of nucleoside analogue resistance mutations on the efficiency and kinetics of HIV-1 DNA synthesis and to evaluate the impact of the depletion of deoxynucleoside triphosphates (dNTP) on this process, DNA synthesis was evaluated by allowing DNA synthesis to proceed with natural HIV-1 templates and primers, either within permeabilized viral particles or in newly infected cells, and quantifying the products by real-time PCR. Three recombinant viruses derived from three pNL4-3 molecular clones expressing mutations associated with resistance to zidovudine: a clone expressing RT mutation M184V, a clone expressing mutations M41L plus T215Y (M41L+T215Y), and clinical isolate BV34 (carrying seven resistance mutations). Following infection of P4 cells, the BV34 mutant, but not viruses expressing the M184V mutation or M41L+T215Y, exhibited a defect in DNA synthesis. Importantly, however, for mutants carrying the M184V mutation or M41L+T215Y mutations, a defect could be detected by using target cells in which dATP pools had been reduced by pretreatment with hydroxyurea. Based on these observations, we developed a recombinant-virus assay to assess the effects of hydroxyurea pretreatment on infectivity of viruses carrying plasma-derived RT sequences from patients with nucleoside resistance. Using this assay, we found that many, but not all, viruses carrying RT resistance mutations display an increased sensitivity to hydroxyurea, suggesting that the impact of RT resistance mutations on viral replication may be more profound in cell populations characterized by smaller dNTP pools.

Anti-HIV Agents↗
PubMed2004

Monitoring of didanosine and stavudine intracellular trisphosphorylated anabolite concentrations in HIV-infected patients.

OBJECTIVE: To determine the concentrations of intracellular active anabolites of stavudine (d4T) and didanosine (DDI) and their interpatient variability in HIV-infected patients and to explore relationships between plasma and intracellular forms. METHODS: This pilot study included 28 antiretroviral-naive HIV-infected patients who received d4T (40/30 mg twice daily), ddI (400/250 mg daily) and efavirenz (600 mg daily). After 6 months of therapy, 7 ml of blood was collected between 0.5 and 16.2 h and 2.5 and 28.5 h after the last dose of d4T and ddI, respectively. Plasma samples were obtained for the determination of d4T and ddI concentrations. Peripheral blood mononuclear cells were prepared for measuring intracellular d4T and ddI triphosphates (d4T-TP and ddA-TP, respectively). RESULTS: d4T-TP and ddA-TP concentrations were above the limit of quantification in 25 of 26 compliant patients: median d4T-TP was 31 fmol/10(6) cells (range, 0-99) and median ddA-TP was 8 fmol/10(6) cells (range, 0-23). The half-life of d4T-TP was calculated as 7 h. Interpatient variability in d4T-TP and ddA-TP concentrations was 48% and 58%, respectively. A significant relationship was observed between plasma d4T and intracellular d4T-TP. No relation was found between ddI and ddA-TP. A linear relation was observed between the intracellular concentrations of d4T-TP and ddA-TP. CONCLUSION: This is the first time that data have been obtained on intracellular concentrations of d4T-TP and ddA-TP, their intracellular pharmacokinetics and interpatient variability. Other similar studies with more patients are needed to enhance knowledge of the intracellular pharmacology of the nucleoside reverse transcriptase inhibitors.

Anti-HIV Agents↗
PubMed2004

ATP binding cassette multidrug transporters limit the anti-HIV activity of zidovudine and indinavir in infected human macrophages.

OBJECTIVES: To investigate whether P-glycoprotein (P-gp) and multidrug resistance proteins (MRPs), which limit the bioavailability of HIV protease inhibitors (PIs) and nucleoside reverse transcriptase inhibitors (NRTIs), modulate the anti-HIV activity of NRTIs, non-NRTIs and PIs in vitro. DESIGN: We used primary cultures of major HIV target cells: human monocyte-derived macrophages (MDMs) and lymphocytes. METHODS: P-gp and MRP expression in response to long-term zidovudine (3'-azido-3'-deoxythymidine; AZT) or indinavir treatment was quantified by RT-PCR. MDM and lymphocytes were infected in vitro with HIV-1/Ba-L and HIV-1-LAI, respectively, and treated with antiretroviral drugs. We evaluated the activity of these drugs in combination with PSC833, a P-gp inhibitor, and/or probenecid, an MRP1 inhibitor. Intracellular AZT triphosphate derivative (AZT-TP) was quantified by HPLC-MSMS. P-gp ATPase activity was measured with inside-out native membrane vesicles enriched in P-gp. RESULTS: Levels of MDR1, mrp4 and mrp5 mRNA were high following AZT treatment. In infected MDM, PSC833 and probenecid increased the anti-HIV activity of AZT and indinavir. AZT (5 nM) decreased HIV replication by 34% alone and by 72% in combination with P-gp/MRP inhibitors. Indinavir (10 nM) gave 14% inhibition alone and 81% in combination. The increase in anti-HIV activity of AZT was correlated with an increase in intracellular AZT-TP concentration. However, unlike PIs, neither AZT nor its metabolites interacted with P-gp. CONCLUSION: AZT increases the expression of multidrug transporters, thereby decreasing its pharmacological activity. The cellular efflux of AZT probably involves MRP4 or MRP5. In contrast, increases in indinavir anti-HIV activity require the inhibition of both P-gp and MRP1.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2003

Liquid chromatography-tandem mass spectrometry assays for intracellular deoxyribonucleotide triphosphate competitors of nucleoside antiretrovirals.

This study was aimed to apply an LC-MS-MS method previously developed for intracellular nucleoside reverse transcriptase inhibitors-triphosphate (NRTI-TPs) to the determination of natural deoxyribonucleotides (dNTPs) in human peripheral blood mononuclear cells. The LC-MS-MS method was directly used in assay of dATP and dTTP. Interferences by ribonucleotides (rNTPs) prevented direct application to the two other analytes: dGTP and dCTP. A periodate oxidation procedure was therefore optimized to remove rNTPs from the cell medium in order to quantitate dCTP and dGTP. The determination of the intracellular ratio of NRTI-TP/dNTP in HIV-infected patients now involves use of the same chromatographic system for simultaneous assay of several analytes.

Chromatography, Liquid↗
PubMed2003

Interferon-ribavirin in association with stavudine has no impact on plasma human immunodeficiency virus (HIV) type 1 level in patients coinfected with HIV and hepatitis C virus: a CORIST-ANRS HC1 trial.

A randomized, open-label trial was performed to study virological and intracellular interactions between stavudine and ribavirin in 30 patients coinfected with human immunodeficiency virus (HIV) and hepatitis C virus (HCV). Patients were randomized to receive either interferon and ribavirin or no treatment for HCV infection for 3 months. Intracellular peripheral blood mononuclear cells' stavudine-triphosphate (TP) concentrations were assessed. Plasma HIV RNA levels did not change significantly between baseline and month 3. There was a nonstatistically significant trend for a lower median residual concentration of intracellular stavudine-TP in the treated group, compared with the control group. The same trend was also observed for peak concentrations. Coprescription of ribavirin and stavudine has no short-term impact on plasma HIV RNA level in HIV-HCV-coinfected patients treated with stavudine as a part of their antiretroviral treatment; this coprescription can be safely used, although an in vivo interaction between ribavirin and stavudine is possible.

Adult↗
PubMed2003

A strategy for liquid chromatography/tandem mass spectrometric assays of intracellular drugs: application to the validation of the triphosphorylated anabolite of antiretrovirals in peripheral blood mononuclear cells.

The pharmacokinetics of intracellular drugs have recently aroused new interest because monitoring a drug's behaviour near the site of action can enhance knowledge of its efficacy and toxicity. Liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) is particularly attractive for intracellular analytes. Very few papers deal precisely with special features encountered in intracellular drug assay or with how closely the assay matches the actual recommendations. Particular problems are encountered mainly because the analytes are located intracellularly. This mainly concerns the handling of biological media, including provision of blank samples using Ficoll gradient separation, cell counts, optimisation of cell lysis, sample extraction, plotting standard curves using either fmol/10(6) cells or fmol/ml of extract or fmol/sample, the matrix effect as a function of the number of cells, stability before and during cell separation, as well as in storage conditions using clinical samples, biological matrix replacement and interference by endogenous compounds. This paper describes a strategy for the full validation and routine use of an LC/MS/MS assay applied to the simultaneous intracellular determination of the triphosphorylated anabolites of didanosine (2',3'-dideoxyadenosine triphosphate or ddA-TP) and stavudine (2',3'-didehydro-3'-deoxythymidine triphosphate or d4T-TP), two nucleoside reverse transcriptase inhibitors of HIV, in human peripheral blood mononuclear cells (PBMCs), as a guide for further LC/MS/MS assay of intracellular drugs.

Anti-HIV Agents↗
PubMed2002

Development of a direct assay for measuring intracellular AZT triphosphate in humans peripheral blood mononuclear cells.

Direct LC/MS/MS methods have recently been developed for measuring triphosphate anabolites of several nucleosidic reverse transcriptase inhibitor (NRTI) in peripheral blood mononuclear cells (PBMCs) from HIV-positive patients. Whereas AZT is one of the most-used NRTIs, no such method has been developed for AZT-TP, its active anabolite, mainly because of the presence of endogenous nucleotides that interfere with such an assay. In this paper, we first describe the development of two enzyme immunoassays (EIA) of AZT-TP in PBMCs: one directly measuring AZT-TP content; the other, measuring the nucleoside AZT after selective extraction of AZT-TP and dephosphorylation. The precision of these two assays was too low to achieve precise determination of AZT-TP in PBMC samples. Direct LC/MS/MS is not specific enough for AZT-TP, since at least two interfering endogenous nucleotides (same m/z ratio and fragment as well as retention time close to that of AZT-TP) are found in the intracellular medium of PBMCs. The off-line combination of immunoaffinity extraction (IAE) and LC/MS/MS proved to be a successful strategy allowing without dephosphorylation appropriate specificity and sensitivity (limit of quantification established as 9.3 fmol/10(6) cells) to determine AZT-TP in PBMCs from 7 mL of blood of HIV-infected patients. Validation of this IAE-LC/MS/MS method demonstrated CV percent for repeatability and intermediate precision lower than 15%. More than 150 samples/week can be analyzed by one analyst, making this method suitable for routine analysis during clinical studies.

Anti-HIV Agents↗
PubMed2002

Improved method for the simultaneous determination of d4T, 3TC and ddl intracellular phosphorylated anabolites in human peripheral-blood mononuclear cells using high-performance liquid chromatography/tandem mass spectrometry.

There is still a need for direct determination (i.e. without dephosphorylation) of nucleoside reverse transcriptase inhibitor (NRTI) triphosphorylated nucleotides in the peripheral-blood mononuclear cells (PBMCs) of HIV-positive patients. The objective of this paper was first to improve our previously described direct liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay for stavudine triphosphate (d4T-TP). Preparation of PBMCs was modified to reduce degradation of d4T-TP during cell preparation and to simplify this step for routine use in clinical units. The performance of several HPLC columns was compared in order to improve the stability of peak shape over time. The SMT C(18) column was replaced by a Supelcogel ODP-50, thereby reducing two-fold the concentration of the first standard. Various internal standards were compared to optimize peak shape and remove an interfering peak in LC. 2-Chloroadenosine 5prime prime or minute-triphosphate was chosen as the most appropriate internal standard. Substitution of the narrowbore column by a microbore column (150 x 0.32 mm) is also presented and discussed. Secondly, this improved method was successfully applied to the simultaneous determination of d4T-TP, dideoxyadenosine triphosphate (ddA-TP) and lamivudine triphosphate (3TC-TP) in PBMCs, which is useful in view of the common use of NRTI combinations. The method was subsequently applied to clinical samples from HIV-positive patients receiving antiretroviral therapy containing d4T, ddl and/or 3TC. This method can be used simply and routinely on approximately 200 samples per week, using commercially available instruments and with a simple cell lysis as sample treatment.

Anti-HIV Agents↗