title: Validation and Application of a Liquid Chromatography-Tandem Mass Spectrometry Method To Determine the Concentrations of Sofosbuvir Anabolites in Cells.
abstract: Sofosbuvir (SOF) is a highly efficacious and well-tolerated uridine nucleotide analog that inhibits the hepatitis C virus (HCV) NS5B polymerase enzyme. SOF is administered as a prodrug, which undergoes intracellular phosphorylation by host enzymes to a monophosphate, diphosphate, and finally a pharmacologically active triphosphate. In order to fully understand the clinical pharmacology of SOF, there is a great need to determine the intracellular phosphate concentrations of the drug. We describe the validation and utilization of a method to characterize SOF's disposition into various in vivo cell types, including hepatocytes, peripheral blood mononuclear cells (PBMC), and red blood cells (RBC). Standard bioanalytical validation criteria were applied to lysed cellular matrices, with a validated linear range of 50 to 50,000 fmol/sample for each phosphate moiety. The assay was utilized to collect the first data demonstrating concentrations of phosphorylated anabolites formed in PBMC, hepatocytes, and RBC in vivo during SOF therapy. Median concentrations in PBMC were 220 (range, 51.5 to 846), 70.2 (range, 25.8 to 275), and 859 (range, 54.5 to 6,756) fmol/10(6) cells in the monophosphate, diphosphate, and triphosphate fractions, respectively. In contrast, RBC triphosphate concentrations were much lower than those of PBMC, as the median concentration was 2.91 (range, 1.14 to 10.4) fmol/10(6) cells. The PBMC triphosphate half-life was estimated at 26 h using noncompartmental approaches, while nonlinear mixed-effect modeling was used to estimate a 69 h half-life for this moiety in RBC. The validated method and the data it generates provide novel insight into the cellular disposition of SOF and its phosphorylated anabolites in vivo.
authors.affiliation: University of Colorado, Skaggs School of Pharmacy and Pharmaceutical Sciences, Aurora, Colorado, USA.
authors.forename: Joseph E
authors.initials: JE
authors.lastname: Rower
authors.affiliation: University of Colorado, Skaggs School of Pharmacy and Pharmaceutical Sciences, Aurora, Colorado, USA.
authors.forename: Leah C
authors.initials: LC
authors.lastname: Jimmerson
authors.affiliation: University of Colorado, Skaggs School of Pharmacy and Pharmaceutical Sciences, Aurora, Colorado, USA.
authors.forename: Xinhui
authors.initials: X
authors.lastname: Chen
authors.affiliation: University of Colorado, Skaggs School of Pharmacy and Pharmaceutical Sciences, Aurora, Colorado, USA.
authors.forename: Jia-Hua
authors.initials: JH
authors.lastname: Zheng
authors.affiliation: University of Colorado, Skaggs School of Pharmacy and Pharmaceutical Sciences, Aurora, Colorado, USA.
authors.forename: Ariel
authors.initials: A
authors.lastname: Hodara
authors.affiliation: University of Colorado, Skaggs School of Pharmacy and Pharmaceutical Sciences, Aurora, Colorado, USA.
authors.forename: Lane R
authors.initials: LR
authors.lastname: Bushman
authors.affiliation: University of Colorado, Skaggs School of Pharmacy and Pharmaceutical Sciences, Aurora, Colorado, USA.
authors.forename: Peter L
authors.initials: PL
authors.lastname: Anderson
authors.affiliation: University of Colorado, Skaggs School of Pharmacy and Pharmaceutical Sciences, Aurora, Colorado, USA jennifer.kiser@ucdenver.edu.
authors.forename: Jennifer J
authors.initials: JJ
authors.lastname: Kiser
mesh_terms: D000998:Antiviral Agents / Q000032:analysis* / Q000493:pharmacokinetics; D002851:Chromatography, High Pressure Liquid; D004912:Erythrocytes / Q000737:chemistry; D006207:Half-Life; D006526:Hepatitis C / Q000097:blood / Q000378:metabolism; D022781:Hepatocytes / Q000737:chemistry; D006801:Humans; D009000:Monocytes / Q000737:chemistry; D017711:Nonlinear Dynamics; D010710:Phosphates / Q000032:analysis; D010766:Phosphorylation; D011786:Quality Control; D015203:Reproducibility of Results; D000069474:Sofosbuvir / Q000032:analysis* / Q000493:pharmacokinetics; D053719:Tandem Mass Spectrometry
chemical_list: D000998:Antiviral Agents; D010710:Phosphates; D000069474:Sofosbuvir