Immunocompromised individuals tend to suffer from influenza longer with more serious complications than otherwise healthy patients. Little is known about the impact of prolonged infection and the efficacy of antiviral therapy in these patients. Among all 189 influenza A virus infected immunocompromised patients admitted to ErasmusMC, 71 were hospitalized, since the start of the 2009 H1N1 pandemic. We identified 11 (15%) cases with prolonged 2009 pandemic virus replication (longer than 14 days), despite antiviral therapy. In 5 out of these 11 (45%) cases oseltamivir resistant H275Y viruses emerged. Given the inherent difficulties in studying antiviral efficacy in immunocompromised patients, we have infected immunocompromised ferrets with either wild-type, or oseltamivir-resistant (H275Y) 2009 pandemic virus. All ferrets showed prolonged virus shedding. In wild-type virus infected animals treated with oseltamivir, H275Y resistant variants emerged within a week after infection. Unexpectedly, oseltamivir therapy still proved to be partially protective in animals infected with resistant virus. Immunocompromised ferrets offer an attractive alternative to study efficacy of novel antiviral therapies.

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After solid organ transplantation, tacrolimus is given to prevent rejection. Therapeutic drug monitoring is used to reach target concentrations of tacrolimus in whole blood. Because the site of action of tacrolimus is the lymphocyte, and tacrolimus binds ~80% to erythrocytes, the intracellular tacrolimus concentration in lymphocytes is possibly more relevant. For this purpose, we aimed to develop, improve and validate a UPLC–MS/MS method to measure tacrolimus concentrations in isolated peripheral blood mononuclear cells (PBMCs). PBMCs were isolated using a Ficoll separation technique, followed by a washing step using red blood cell lysis. A cell suspension of 50 μL containing 1 million PBMCs was used in combination with MagSiMUS‐TDMPREP. To each sample we added 30 μL lysis buffer, 20 μL reconstitution buffer containing 13C2H4‐tacrolimus as internal standard, 40 μL MagSiMUS‐TDMPREP Type I Particle Mix and 175 μL Organic Precipitation Reagent VI for methanol‐based protein precipitation. A 10 μL aliquot of the supernatant was injected into the UPLC–MS/MS system. The method was validated, resulting in high sensitivity and specificity. The method was linear (r2 = 0.997) over the range 5.0–1250 pg/1 × 106 PBMCs. The inaccuracy was <5% and the imprecision was <15%. The washing steps following Ficoll isolation could be performed at either room temperature or on ice, with no effect of the temperature on the results. A method for the analysis of tacrolimus concentrations in PBMCs was developed and successfully validated. Further research will be performed to investigate the correlation between concentrations in PBMCs and clinical outcome.

This method allows quantification of APAP and 6 metabolites, which serves purposes for research, as well as therapeutic drug monitoring. The advantage of this method is the combination of minimal injection volume, a short runtime, an easy sample preparation method, and the ability to quantify APAP and all 6 metabolites.

Background Intravenous salbutamol is used to treat children with refractory status asthmaticus, however insufficient pharmacokinetic data are available to guide initial and subsequent dosing recommendations for its intravenous use. The pharmacologic activity of salbutamol resides predominantly in the (R)-enantiomer, with little or no activity and even concerns of adverse reactions attributed to the (S)-enantiomer. Objective Our aim was to develop a population pharmacokinetic model to characterize the pharmacokinetic profile for intravenous salbutamol in children with status asthmaticus admitted to the pediatric intensive care unit (PICU), and to use this model to study the effect of different dosing schemes with and without a loading dose. Methods From 19 children (median age 4.9 years [range 9 months-15.3 years], median weight 18 kg [range 7.8-70 kg]) treated with continuous intravenous salbutamol at the PICU, plasma samples for R-and S-salbutamol concentrations (111 samples), as well as asthma scores, were collected prospectively at the same time points. Possible adverse reactions and patients' clinical data (age, sex, weight, drug doses, liver and kidney function) were recorded. With these data, a population pharmacokinetic model was developed using NONMEM 7.2. After validation, the model was used for simulations to evaluate the effect of different dosing regimens with or without a loading dose. Results A two-compartment model with separate clearance for R-and S-salbutamol (16.3 L/h and 8.8 L/h, respectively) best described the data. Weight was found to be a significant covariate for clearance and volume of distribution. No other covariates were identified. Simulations showed that a loading dose can result in higher R-salbutamol concentrations in the early phase after the start of infusion therapy, preventing accumulation of S-salbutamol. Conclusions The pharmacokinetic model of intravenous R-and S-salbutamol described the data well and showed that a loading dose should be considered in children. This model can be used to evaluate the pharmacokinetic-pharmacodynamic relationship of intravenous salbutamol in children, and, as a next step, the effectiveness and tolerability of intravenous salbutamol in children with severe asthma. Nienke J. Vet and Brenda C. M. de Winter contributed equally to this work.In this prospective study, we developed a population pharmacokinetic model of intravenous R-and S-salbutamol in children with status asthmaticus admitted to the intensive care unit.The model described the data well and showed that a loading dose seems valid to reach higher initial R-salbutamol concentrations with a possible therapeutic advantage.This model can be used to evaluate the pharmacokinetic-pharmacodynamic relationship of intravenous salbutamol.

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Currently, pharmacokinetic-pharmacodynamic studies of sedatives and analgesics are performed in neonates and children to find suitable dose regimens. As a result, sensitive assays using only small volumes of blood are necessary to determine drug and metabolite concentrations. We developed an ultra-performance liquid chromatographic method with tandem mass spectrometry detection for quantification of midazolam, 1-hydroxymidazolam, hydroxymidazolamglucuronide, morphine, morphine-3-glucuronide and morphine-6-glucuronide in 100 microL of plasma. Cleanup consisted of 96 wells micro-solid phase extraction, before reversed-phase chromatographic separation (ultra-performance liquid chromatography) and selective detection using electrospray ionization tandem mass spectrometry. Separate solid-phase extraction methods were necessary to quantify morphine, midazolam and their metabolites because of each group's physicochemical properties. Standard curves were linear over a large dynamic range with adequate limits of quantitation. Intra- and interrun accuracy and precision were within 85-115% (of nominal concentration using a fresh calibration curve) and 15% (coefficient of variation, CV) respectively. Recoveries were >80% for all analytes, with interbatch CVs (as a measure of matrix effects) of less than 15% over six batches of plasma. Stability in plasma and extracts was sufficient, allowing large autosampler loads. Runtime was 3.00 min per sample for each method. The combination of 96-well micro-SPE and UPLC-MS/MS allows reliable quantification of morphine, midazolam and their major metabolites in 100 microL of plasma.

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