Intraindividual Variability in Absolute Bioavailability and Clearance of Midazolam in Healthy Individuals.

BACKGROUND AND OBJECTIVE: Midazolam is the preferred clinical probe drug for assessing CYP3A activity. We have previously shown substantial intraindividual variability in midazolam absolute bioavailability and clearance in patients with obesity before and after weight loss induced by gastric bypass or a strict diet. The objective was to describe intraindividual variability in absolute bioavailability and clearance of midazolam in healthy individuals without obesity. METHODS: This study included 33 healthy volunteers [28 ± 8 years, 21% males, body mass index (BMI) 23 ± 2.5 kg/m2] subjected to four pharmacokinetic investigations over a 2-month period (weeks 0, 2, 4, and 8). Semi-simultaneous oral (0 h) and intravenous (2 h later) midazolam dosing was used to assess absolute bioavailability and clearance of midazolam. RESULTS: At baseline, mean absolute bioavailability and clearance were 46 ± 18% and 31 ± 10 L/h, respectively. The mean coefficient of variation (CV, %) for absolute bioavailability and clearance of midazolam was 26 ± 15% and 20 ± 10%, respectively. Approximately one-third had a CV > 30% for absolute bioavailability, while 13% had a CV > 30% for clearance. CONCLUSIONS: On average, intraindividual variability in absolute bioavailability and clearance of midazolam was low to moderate; however, especially absolute bioavailability showed considerable variability in a relatively large proportion of the individuals.

Determination of the alcohol biomarker phosphatidylethanol 16:0/18:1 and 33 compounds from eight different drug classes in whole blood by LC-MS/MS.

INTRODUCTION: Most bioanalytical LC-MS/MS methods are developed for determination of single drugs or classes of drugs, but a multi-compound LC-MS/MS method that can replace several methods could reduce both analysis time and costs. The aim of this study was to develop a high-throughput LC-MS/MS method for determination of the alcohol biomarker phosphatidylethanol 16:0/18:1 (PEth 16:0/18:1) and 33 other compounds from eight different drug classes in whole blood. METHODS: Whole-blood samples were prepared by 96-well supported liquid extraction (SLE). Chromatographic separations were performed on a biphenyl core shell column with a mobile phase consisting of 10 mM ammonium formate, pH 3.1 and methanol. Each extract was analyzed twice by LC-MS/MS, injecting 0.4 µL and 2 µL, in order to obtain narrow and symmetrical peaks and good sensitivity for all compounds. Stable isotope-labeled internal standards were used for 31 of the 34 compounds. RESULTS: A 96-well SLE reversed phase LC-MS/MS method for determination of PEth 16:0/18:1 and 33 other compounds from eight different drug classes was developed and validated. By using an organic solvent mixture of isopropanol/ methyl tert-butyl ether (1:5, v:v), all compounds, including the polar and ampholytic compounds pregabalin, gabapentin and benzoylecgonine, was extracted by 96-well SLE. DISCUSSION/CONCLUSION: For the first time an LC-MS/MS method for the determination of alcohol biomarker PEth 16:0/18:1 and drugs and metabolites from several different drug classes was developed and validated. The developed LC-MS/MS method can be used for high-throughput analyses and sensitive determinations of the 34 compounds in whole blood.

Dual Transcriptomics of Host-Pathogen Interaction of Cystic Fibrosis Isolate Pseudomonas aeruginosa PASS1 With Zebrafish.

Pseudomonas aeruginosa is a significant cause of mortality in patients with cystic fibrosis (CF). To explore the interaction of the CF isolate P. aeruginosa PASS1 with the innate immune response, we have used Danio rerio (zebrafish) as an infection model. Confocal laser scanning microscopy (CLSM) enabled visualization of direct interactions between zebrafish macrophages and P. aeruginosa PASS1. Dual RNA-sequencing of host-pathogen was undertaken to profile RNA expression simultaneously in the pathogen and the host during P. aeruginosa infection. Following establishment of infection in zebrafish embryos with PASS1, 3 days post infection (dpi), there were 6739 genes found to be significantly differentially expressed in zebrafish and 176 genes in PASS1. A range of virulence genes were upregulated in PASS1, including genes encoding pyoverdine biosynthesis, flagellin, non-hemolytic phospholipase C, proteases, superoxide dismutase and fimbrial subunits. Additionally, iron and phosphate acquisition genes were upregulated in PASS1 cells in the zebrafish. Transcriptional changes in the host immune response genes highlighted phagocytosis as a key response mechanism to PASS1 infection. Transcriptional regulators of neutrophil and macrophage phagocytosis were upregulated alongside transcriptional regulators governing response to tissue injury, infection, and inflammation. The zebrafish host showed significant downregulation of the ribosomal RNAs and other genes involved in translation, suggesting that protein translation in the host is affected by PASS1 infection.