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 mmx50 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.
References
1.
ShahV.P., MidhaK.K., FindlayJ.W.Bioanalytical method validation–a revisit with a decade of progress. Pharm Res2000; 17: 1551–1557.
2.
Food and Drug Administration.Guidance for industry on bioanalytical method validation; availability. Federal register, Vol. 66, no. 100, p. 28526. (Updated 23 May 2001. Accessed 4 July 2010.) Available from http://www.gpo.gov/fdsys/pkg/FR-2001-05-23/pdf/01-12908.pdf.
3.
HollandD.T., DiFrancescoR., ConnorJ.D., MorseG.D.Quality assurance program for pharmacokinetic assay of antiretrovirals: ACTG proficiency testing for pediatric and adult pharmacology support laboratories, 2003 to 2004: a requirement for therapeutic drug monitoring. Ther Drug Monit2006; 28: 367–374.
4.
HollandD.T., DiFrancescoR., StoneJ., HamzehF., ConnorJ.D., MorseG.D.Quality assurance program for clinical measurement of antiretrovirals: AIDS clinical trials group proficiency testing program for pediatric and adult pharmacology laboratories. Antimicrob Agents Chemother2004; 48: 824–831.
5.
MatuszewskiB.K., ConstanzerM.L., Chavez-EngC.M.Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Anal Chem2003; 75: 3019–3030.
6.
ViswanathanC.T., BansalS., BoothB.Quantitative bioanalytical methods validation and implementation: best practices for chromatographic and ligand binding assays. Pharm Res2007; 24: 1962–1973.
7.
RigottiA., MiettinenH.E., KriegerM.The role of the high-density lipoprotein receptor SR-BI in the lipid metabolism of endocrine and other tissues. Endocr Rev2003; 24: 357–387.
8.
CataneseM.T., AnsuiniH., GrazianiR.Role of scavenger receptor class B type I in hepatitis C virus entry: kinetics and molecular determinants. J Virol2010; 84: 34–43.
9.
DreuxM., Dao ThiV.L., FresquetJ.Receptor complementation and mutagenesis reveal SR-BI as an essential HCV entry factor and functionally imply its intra- and extra-cellular domains. PLoS Pathog2009; 5: e1000310.
10.
SyderA.J., LeeH., ZeiselM.B.Small molecule scavenger receptor BI antagonists are potent HCV entry inhibitors. J Hepatol2011; 54: 48–55.
11.
Wong-StaalF., SyderA.J., McKelvyJ F.Targeting HCV entry for development of therapeutics. Viruses2010; 2: 1718–1733.
