Fabien Xuereb

Virological, intracellular and plasma pharmacological parameters predicting response to lopinavir/ritonavir (KALEPHAR study).

Objectives: To assess the impact of HIV-1 protease mutations and intracellular and plasma lopinavir minimum concentrations (Cmin) on virological success or failure on lopinavir/ritonavir-containing highly active antiretroviral therapy (HAART). Design: HIV-1-infected HAART-experienced patients included in an observational study, received lopinavir/ritonavir (400/100 mg twice a day) plus two to three nucleoside reverse transcriptase inhibitors (NRTI) or one NRTI plus one non-NRTI. A viral load less than 50 copies/ml at month 6 defined virological success. Methods: Intracellular and plasma lopinavir concentrations were determined by high-pressure liquid chromatography with mass-spectrometry detection. Reverse transcriptase and protease genes were sequenced at baseline and the time of virological failure. Results: When the 38 patients started the lopinavir/ritonavir-based regimen, baseline median (25–75th percentile) values were: CD4 cell count 218 cells/μl (133–477); plasma HIV-1-RNA load 5.3 log10 copies/ml (3.8–5.1); number of lopinavir mutations four per protease gene (two to six). Univariate analysis associated virological success or failure at month 6 (21/38 patients) with the number of baseline lopinavir mutations, intracellular and plasma lopinavir Cmin, and the genotype inhibitory quotient (GIQ) at months 1 and 6. Multivariate analysis showed that the number of baseline lopinavir mutations and intracellular and plasma lopinavir Cmin were independently associated with virological success or failure. We defined the most discriminating intracellular and plasma lopinavir Cmin efficacy thresholds (8 and 4 μg/ml, respectively) and GIQ thresholds (1 and 3, respectively). Conclusion: The monitoring of lopinavir/rironavir-based HAART efficacy should include the number of baseline lopinavir/ritonavir mutations, intracellular and plasma lopinavir Cmin and GIQ calculation.

Alveolar concentrations of piperacillin/tazobactam administered in continuous infusion to patients with ventilator-associated pneumonia.

Objectives:To determine the steady-state serum and alveolar concentrations of piperacillin/tazobactam administered in continuous infusion to critically ill patients with ventilator-associated pneumonia and various degrees of renal failure. Design:Prospective comparative study. Setting:An intensive care unit and research ward in a university hospital. Patients:Forty patients with microbiologically documented ventilator-associated pneumonia. Interventions:Patients were randomized to receive piperacillin/tazobactam daily continuous infusions of 12/1.5 g or 16/2 g after a loading dose of 4/0.5 g. The serum and alveolar piperacillin/tazobactam concentrations were determined at steady-state with high performance liquid chromatography. Measurements and Main Results:The median (interquartile) serum and alveolar piperacillin concentrations were respectively 25.3 mg/L (23.1–32.6) and 12.7 mg/L (6.7–18.0) for 12/1.5 g/day, and 38.9 mg/L (32.9–59.6) and 19.1 mg/L (14.0–21.5), respectively, for 16/2 g/day in patients with no/mild renal failure. In patients with moderate/advance renal failure, the median (interquartile) serum and alveolar piperacillin concentrations were 102.4 mg/L (97.4–112.6) and 44.1 mg/L (33.4–48.3), respectively, for 12/1.5 g/day, and 135.3 mg/L (119.5–146.2) and 54.9 mg/L (45.2–110.3), respectively, for 16/2 g/day. Our results show great variability in piperacillin/tazobactam concentrations, with an alveolar percentage penetration of 40–50% for piperacillin and 65–85% for tazobactam and a negative association between serum or alveolar concentrations and creatinine clearance. Conclusions:A target piperacillin serum concentration of at least 35–40 mg/L is probably required to provide alveolar concentrations exceeding the susceptibility breakpoint for Gram-negative bacteria (16 mg/L) during ventilator-associated pneumonia. In patients with no/mild renal failure, a continuous daily dose of piperacillin/tazobactam 16/2 g allows reaching this target concentration, which might be not observed with 12/1.5 g/day. In patients with moderate/advanced renal failure, both dosages achieve serum concentrations far above the 35–40 mg/L threshold, suggesting that in that case, therapeutic drug monitoring should be performed in order to adjust the daily dose.

A sensitive liquid chromatography coupled with mass spectrometry method for the intracellular and plasma quantification of raltegravir after solid-phase extraction

Introduction  Liquid chromatography coupled with mass spectrometry for the quantification of raltegravir in human plasma and peripheral blood mononuclear cells has been developed.

A new reliable, transposable and cost-effective assay for absolute quantification of total plasmatic bevacizumab by LC–MS/MS in human plasma comparing two internal standard calibration approaches

The quantification of monoclonal antibodies (mAbs) such as bevacizumab, a recombinant humanized immunoglobulin G1 (hIgG1), in biological fluids, is an essential prerequisite to any pharmacokinetic preclinical and clinical study. To date, reference techniques used to quantify mAbs rely on enzyme-linked immunosorbent assay (ELISA) lacking specificity. Furthermore, the commercially available ELISA kit to quantify bevacizumab in human plasma only assesses the free fraction of the drug. However, the conditions of storage and analysis of plasma samples could alter the physiological equilibrium between the free, bound and partially bound forms of bevacizumab and this could result in over- or underestimation of drug concentration. We developed a new assay for absolute quantification of total fraction of bevacizumab by liquid chromatography tandem mass spectrometry (LC-MS/MS) basing identification and quantification of bevacizumab on two specific peptides. In this report we compare our assay with two internal standard (IS) calibration approaches: one using a different human mAb (Trastuzumab) and the other using a stable isotope labeled specific peptide. After enrichment by affinity chromatography on protein A and concentration by ultrafiltration, human plasma samples were proteolyzed by trypsin. Linearity was established from 12.5 to 500μg/mL with an interday accuracy ranging from 101.7 to 110.6% and precision from 7.0% to 9.9%. This study demonstrates the importance of the choice of the IS in quantifying bevacizumab in human plasma and highlights the difficulty of reaching a reliable proteolysis with a sufficient recovery. We developed a reliable and cost-effective LC-MS/MS method to quantify total plasmatic fraction of bevacizumab in human plasma. Through our development we proposed a generic methodology easily transposable to quantify all IgG1 subclass very useful for clinical pharmacokinetics studies.

An in vitro study of epoetin β intravenous injection site at the end of hemodialysis

The aim of this study (SITEPO) is to evaluate the influence of the intravenous injection site (drip chamber injection site, venous injection site or venous fistula needle) on plasma concentration of epoetin beta (Neorecormon, Roche), a recombinant Human Erythropoietin (rHuEPO), at the end of in vitro hemodialysis sessions. No practical administration guidelines are available. Twenty 1-h dialysis sessions are performed. Before each dialysis, the circuit is filled with 270ml, of heparinized total human blood whose hematocrit is adjusted to 35%. A common dosage of epoetin beta in clinical practice (3000IU) is studied for the three injection sites and for reference experiments in which rHuEPO is not injected into the dialysis circuit. Plasma concentrations of erythropoietin are measured by ELISA. The physiologically endogenous erythropoietin concentration is systematically determined and removed from the total epoetin beta concentration. Average epoetin beta plasma levels returned are not significantly different between the three injection sites and no significant rHuEPO loss is observed after injection into the drip chamber, the venous injection site and the venous fistula needle compared with reference experiments. The three intravenous injection sites of rHuEPO can be used at the end of dialysis without significant epoetin beta loss.

Chemical stability of azacitidine suspensions for injection after cold-chain reconstitution of powder and storage

PURPOSE The 72-hour chemical stability of refrigerated syringes of azacitidine suspension prepared via a cold-chain method is investigated. METHODS Three 25-mg/mL azacitidine suspensions were prepared from different lots of powdered drug. The suspensions were stored in 2-mL polypropylene syringes at 2-8 °C and protected from light. The concentrations of azacitidine and the mean area under the concentration-time curve (AUC) values for its degradation products were determined after 0, 1, 2, 3, 4, 6, 8, 12, 24, 48, and 72 hours using high-performance liquid chromatography with ultraviolet detection. RESULTS The degradation process was slow during the first 48 hours and then accelerated. During the first 48 hours of storage, 4.23% of the azacitidine was lost relative to the mean concentration measured at time zero, which complied with International Conference on Harmonisation (ICH) guidance specifying a maximum change of 5% from the initial measured value. Two degradation products were present immediately after syringe preparation; N-formylribosylguanylurea formed rapidly (as indicated by a 35.62% increase from the baseline AUC in the first 12 hours), whereas ribosylguanylurea formation occurred more slowly (a 7.69% mean increase from the baseline AUC at 12 hours) but then rapidly accelerated. The study results indicate that properly prepared azacitidine syringes for injection can be administered up to two days later while maintaining conformance with ICH stability standards. CONCLUSION Azacitidine 25-mg/mL suspensions reconstituted with refrigerated water (2-8 °C) and stored in propylene syringes were chemically stable during the first 48 hours when stored protected from light at 2-8 °C.