Population pharmacokinetic analysis of raltegravir pediatric formulations in HIV-infected children 4 weeks to 18 years of age.

P1066 is an open-label study of raltegravir in HIV positive youth, ages 4 weeks-18 years. Here we summarize P1066 pharmacokinetic (PK) data and a population PK model for the pediatric chewable tablet and oral granules. Raltegravir PK parameters were calculated using noncompartmental analysis. A 2-compartment model was developed using data from P1066 and an adult study of the pediatric formulations. Interindividual variability was described by an exponential error model, and residual variability was captured by an additive/proportional error model. Twelve-hour concentrations (C12h ) were calculated from the model-derived elimination rate constant and 8-hour observed concentration. Simulated steady-state concentrations were analyzed by noncompartmental analysis. Target area under the curve (AUC0-12h ) and C12h were achieved in each cohort. For the pediatric formulations, geometric mean AUC0-12h values were 18.0-22.6 µM-hr across cohorts, and C12h values were 71-130 nM, with lower coefficients of variation versus the film-coated tablet. A 2-compartment model with first-order absorption adequately described raltegravir plasma PK in pediatric and adult patients. Weight was a covariate on clearance and central volume and was incorporated using allometric scaling. Raltegravir chewable tablets and oral granules exhibited PK parameters consistent with those from prior adult studies and older children in P1066, as well as lower variability than the film-coated tablet.

A sensitive HPLC-MS/MS method for the determination of dolutegravir in human plasma.

A sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) assay was developed and validated to facilitate the assessment of clinical pharmacokinetics of dolutegravir (DTG) in plasma samples. This work describes an assay system requiring only a 20µL aliquot of human plasma that is subjected to a simple acetonitrile protein precipitation containing a stably labeled isotope of DTG used as an internal standard. Chromatography was performed on an XBridge C18, 2.1mm×50mm, reversed phase analytical column, using a 60:40 acetonitrile/water mobile phase containing 0.1% formic acid. Detection of the analyte and internal standard was achieved by ESI positive ionization tandem mass spectrometry. The precursor/product transitions (m/z) monitored were 420.1/136.0 and 428.1/283.1 for DTG and DTG-IS, respectively. The dynamic range of this assay extends from 5 to 10,000ng/mL, with a mean coefficient of determination (r, mean±SD) of 0.9996±0.0003. The mean precision values for calibration standards ranged from 0.7 to 4.1%, while accuracy values were 98.3 to 102.0%. Validation results demonstrated high accuracy (≤6.5% deviation) and high precision (≤9.1% CV) for the quality control samples. This assay system provides an accurate, precise, and sensitive method for DTG quantitation and was successfully applied to clinical research samples as part of a phase I/II pediatric clinical trial.

Novel methodology for antiretroviral quantitation in the female genital tract.

PURPOSE: Challenges exist regarding antiretroviral quantitation in the female genital tract. Endocervical wicking using sterile tear flow test strips is an alternative to conventional methods due to the consistent sample volume obtained. METHODS: A novel method for measuring antiretrovirals in cervicovaginal secretions using Sno-strip wicking was developed and tested by spiking Sno-strips with known concentrations of tenofovir, nevirapine, atazanavir, lopinavir, and ritonavir in blank cervicovaginal lavage fluid. Drug concentrations were determined by high-performance liquid chromatography with ultraviolet or mass spectrometry detection. RESULTS: Mean extraction recoveries were 91% for tenofovir, 89% for nevirapine, 63% for atazanavir, 60% for lopinavir, and 61% for ritonavir relative to controls. Freezing spiked samples for 24 hours at -80 degrees C had no effect on recovery. CONCLUSIONS: Results suggest that the antiretrovirals tested can be efficiently extracted from Sno-strips, although a greater percentage of tenofovir and nevirapine was recovered. Storage of Sno-strip samples up to 24 hours before analysis showed no difference in the percentage of drug recovered compared with immediate analysis. Quantitating antiretroviral penetration into the female genital tract may assist in determining optimal therapeutic antiretroviral regimens to both decrease the risk of HIV transmission and prevent development of HIV drug resistance.

Zidovudine, lamivudine, and nelfinavir concentrations in amniotic fluid and maternal serum.

PURPOSE: The objective of this study was to examine lamivudine (3TC), zidovudine (ZDV), nelfinavir (NFV), and its active nelfinavir metabolite (M8) concentrations in paired maternal plasma and amniotic fluid samples to determine antiretroviral penetration or accumulation in the fetal compartment. METHOD: Ten paired amniotic fluid and maternal plasma samples were obtained during caesarian section for pharmacokinetic analysis. Antiretroviral concentrations were measured in both matrices using high-performance liquid chromatography (HPLC) and mass spectrometry (LC/MS) methodologies. RESULTS: Median maternal plasma concentrations for NFV, M8, 3TC, and ZDV were 456, 244, 176, and 794 ng/mL, respectively, while median amniotic fluid concentrations were 118, 21, 2537, and 1483 ng/mL, respectively. The median NFV amniotic fluid to maternal plasma ratio was 0.44; the median M8 ratio was 0.11. Median 3TC and ZDV amniotic fluid to plasma ratios were 11.9 and 1.5, respectively. CONCLUSIONS: NFV and M8 exhibited partial drug transfer and/or accumulation in the amniotic compartment, whereas ZDV and 3TC concentrations mostly exceeded that in maternal plasma. Overall, all drugs achieved exposures in the amniotic fluid in excess of their wild-type viral susceptibilities. Amniotic fluid is an important compartment in the prevention of mother-to-child transmission; a further understanding of protease inhibitor and other antiretroviral drug penetration into amniotic fluid is warranted.

A sensitive HPLC-MS-MS method for the determination of raltegravir in human plasma.

This work describes an assay system that has been developed to quantify raltegravir concentrations in human plasma using a liquid-liquid extraction technique paired with HPLC separation and MS-MS detection. The dynamic range of this assay extends from 1 to 3000 ng/mL, with a coefficient of determination (r(2), mean+/-SD) of 0.9992+/-0.0002. The mean precision values for calibration standards ranged from 0.6% to 3.0%, while accuracy values were 96.5-104.3%. This procedure is an accurate, precise, and sensitive method for raltegravir quantitation and was successfully validated using external proficiency testing.

Persistence of nevirapine in breast milk after discontinuation of treatment.

The objective of this study was to serially quantitate the concentration of nevirapine in breast milk after discontinuation of treatment. Samples were collected from both breasts of a human immunodeficiency virus-infected patient for 3 weeks. Nevirapine was quantifiable for up to 17 days after discontinuation of therapy; total nevirapine concentrations remained above the 90% inhibitory concentration for 6 days, and no differences were observed between breasts.

Development of a sensitive and specific liquid chromatography/mass spectrometry method for the determination of tenofovir in human plasma.

A sensitive and specific method for the quantitation of tenofovir (TFV) in human plasma by liquid chromatography/electrospray ionization mass spectrometry was developed and validated. Plasma samples were prepared by solid-phase extraction performed on Waters Oasis cation-exchange cartridges (30 mg). Chromatographic separation was performed isocratically on a reversed-phase Waters Atlantis dC18 column (2.0x100 mm, 3 microm). The mobile phase consisted of a hydroxylamine/acetic acid buffer (pH 6.75) and methanol (93:7, v/v). The acquisition was performed in selected ion monitoring mode for the protonated molecular ions [M+H]+ of m/z 288.2 for TFV and 212.2 for the internal standard, zalcitibine. The method was fully validated to determine its specificity, recovery, linearity and sensitivity, accuracy and precision. The analytical range was set at 1-750 ng/mL using a 200 microL plasma sample, with a mean coefficient of determination (r2) of 0.9969. The mean accuracies for the calibration standards ranged from -5.0 to 4.3%, while the precisions were within 1.2 and 6.4%. Intra-assay and inter-assay mean accuracies for three quality control concentrations (2, 60, and 600 ng/mL) ranged from -6.1 to 10.7%, while the precisions were within 1.3 and 9.1%. TFV was shown to be stable under normal storage and assay conditions; no degradation was seen when stored at -20 degrees C or -80 degrees C for up to 6 months, and after 16 h at room temperature in the injection matrix. The present method provides an accurate, precise, and sensitive tool for TFV quantitation and was successfully applied to an external proficiency-testing program and pharmacokinetic analysis.