583-T. Effects of Human Serum on Protease Inhibitor Activity in a Phenotypic Drug Susceptibility Assay (PhenoSense HIV)

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Session 77 Poster Session
Resistance Testing in Drug Selection
Session Time: 4:30-6:30 pm
Room 4E-F

583-T.

Effects of Human Serum on Protease Inhibitor Activity in a Phenotypic Drug Susceptibility Assay (PhenoSense HIV)
K. Limoli*, L. H. Trinh, G. M. Heilek-Snyder, N. S. Hellmann, and C. J. Petropoulos
ViroLogic Inc., South San Francisco, CA

Background: The ability of ViroLogic’s PhenoSense HIV Assay to measure susceptibility of HIV-1 (patient) samples to antiretroviral drugs provides physicians with a powerful tool to individually tailor treatment regimens. To improve the pharmacological relevance of data obtained with this in vitro phenotypic resistance assay, the effects of added human serum (HS) on protease inhibitor (PI) potency were investigated.
Methods: To assess the impact of HS on PI susceptibility, PhenoSense HIV assays were performed in the presence and absence of varying concentrations of HS for 6 FDA approved PIs: nelfinavir (NFV), saquinavir (SQV), ritonavir (RTV), lopinavir (LPV), amprenavir (AMP), and indinavir (IDV). HS and PIs were added to the assay at the time of virus production. Various sources and concentrations of HS and HIV negative plasma (Intergen, Purchase) were evaluated, as well as purified alpha-1 acid glycoprotein (AAG). IC50 values were determined in the presence of HS or purified AAG and were compared to the IC50 values derived using the standard assay conditions (10% FBS).
Results: The data demonstrate reduced potency of PI in the presence of HS. In the presence of 50% HS, NFV exhibited the most substantial drop in potency (average = 22-fold) whereas IDV showed the smallest effect (average = 2-fold). The relative effect of 50% HS in reducing PI potency showed the following order: NFV(22-fold)>SQV(16-fold)>RTV(9-fold)>LPV~AMP(4-fold)>IDV(2-fold). The data also demonstrated a linear relationship between reduced potency (higher IC50) and HS concentrations over a wide range (25-75%), which allowed the extrapolation of PI potency in 100% HS. Purified AAG alone significantly decreased the potency of most PI drugs tested (NFV>AMP>LPV>RTV).
Conclusions: HS and purified AAG significantly reduce PI potency in the PhenoSense HIV Assay. The inverse linear relationship between HS concentration and drug potency provides the means for determining adjustment factors (based on IC50) that permit accurate estimations of drug activity in patient plasma (100% HS). This approach should facilitate more precise calculations of the PI drug concentrations required to inhibit HIV in vivo.