title: ITX 5061 quantitation in human plasma with reverse phase liquid chromatography and mass spectrometry detection.
abstract: BACKGROUND
  ITX 5061 is a highly potent small molecule inhibitor of scavenger receptor-B1, an integral transmembrane protein that is found in liver cells and is actively involved in the transport of HCV into hepatocytes. Currently, ITX 5061 is being investigated in monoinfected hepatitis C patients in a proof-of-concept clinical trial carried out by the AIDS Clinical Trial Group (ACTG).
  
  
  METHODS
  To provide quantitative results in human plasma for pharmacokinetic analysis, an assay for ITX 5061 was validated. ITX 5061 and the internal standard, a deuterated analogue, were separated by isocratic reverse phase chromatography using a Polar RP column (Phenomenex Synergi(™); 2.0 mm × 50 mm, 4 µm) and detected via electrospray coupled to a triple quadrupole mass spectrometer with a run time of 5 min. Multiple reaction monitoring in positive mode was used with ITX 5061 at 585/114 m/z and the internal standard at 592/122 m/z with a linear range of 2.50-5,000 ng/ml. Human plasma was extracted using a protein precipitation combing 400 µl of acetonitrile with 100 µl of EDTA plasma.
  
  
  RESULTS
  The interassay variation ranged from 1.19 to 13.2%, while the intraassay variation ranged from 0.394 to 12.9% over 6 days of testing. The method was successfully applied to the samples collected for the ACTG Protocol A5277. Plasma concentrations at 1 h and 24 h following 150 mg ITX 5061 daily in HCV monoinfected patients (n=3) ranged from 138 to 518 ng/ml and 33 to 111 ng/ml, respectively.
  
  
  CONCLUSIONS
  The ITX 5061 assay is accurate and reproducible with a wide linear range and will be used for pharmacokinetic analysis and dose-finding studies in HCV-monoinfected patients.
authors.affiliation: AIDS Clinical Trials Group Pharmacology Specialty Laboratory, Translational Pharmacology Research Core, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, Buffalo, NY, USA.
authors.forename: Jill
authors.initials: J
authors.lastname: Hochreiter
authors.forename: Jill
authors.initials: J
authors.lastname: Lapham
authors.forename: Flossie
authors.initials: F
authors.lastname: Wong-Staal
authors.forename: Jeffrey
authors.initials: J
authors.lastname: McKelvy
authors.forename: Mark
authors.initials: M
authors.lastname: Sulkowski
authors.forename: Marshall J
authors.initials: MJ
authors.lastname: Glesby
authors.forename: Victoria A
authors.initials: VA
authors.lastname: Johnson
authors.forename: Gene D
authors.initials: GD
authors.lastname: Morse
mesh_terms: D056148:Chromatography, Reverse-Phase; D006526:Hepatitis C / Q000097:blood / Q000188:drug therapy; D006801:Humans; D013058:Mass Spectrometry; D010655:Phenylenediamines / Q000737:chemistry / Q000493:pharmacokinetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013449:Sulfonamides / Q000737:chemistry / Q000493:pharmacokinetics*
chemical_list: C583499:ITX-5061; D010655:Phenylenediamines; D013449:Sulfonamides