Estimation of serum-free 50-percent inhibitory concentrations for human immunodeficiency virus protease inhibitors lopinavir and ritonavir

Abstract

Using measured free fraction and 50% inhibitory concentration (IC50) values for the human immunodeficiency virus protease inhibitors lopinavir (LPV) and ritonavir (RTV) in tissue culture media with various protein concentrations ranging from 5 to 50%, we estimated serum-free IC50 values for each drug. The range of serum-free IC50 values (0.64 to 0.77 ng/ml for LPV and 3.0 to 5.0 ng/ml for RTV) did not exhibit a trend with increasing protein concentrations, despite a 10-fold difference in the free fraction value (0.006 to 0.063) for LPV and a 5-fold difference in the free fraction value (0.013 to 0.057) for RTV. The mean serum-free IC50 by the MTT-MT4 assay (0.69 ng/ml for LPV and 4.0 ng/ml for RTV) may be the most accurate parameter for the estimation of the inhibitory quotient (IQ), a relative measure of in vivo potency defined as the ratio of the minimal free drug concentration in plasma (C(trough,free)) for a specific patient population and the serum-free IC50. Using this approach, we calculated the average IQs for protease inhibitor-naïve patients for LPV and RTV to be 67 and 5.6, respectively.

FIG. 1.

Effect of FCS on free fractions of [¹⁴C]LPV and [¹⁴C]RTV in RPMI 1640-PS. Binding was determined at 0.1 (•)-, 1 (▪)-, and 10 (▴)-μg/ml drug concentrations. The means of triplicate experiments are shown with error bars indicating the standard deviations. When no error bars are apparent, the errors fall within the area of the symbol. Percent free is the free fraction × 100. (a) LPV; (b) RTV.

FIG. 2.

Effect of FCS on drug IC₅₀ (♦) and mean % free drug (at 0.1 μg/ml; •) in RPMI 1640-PS. For IC₅₀ values, the symbols represent the means of duplicate determinations. For protein binding, the means of triplicate experiments are shown with error bars indicating the standard deviations. When no error bars are apparent for protein binding, the errors fall within the area of the symbol. (a) LPV; (b) RTV.

FIG. 3.

Estimation of serum-free IC₅₀ of LPV and RTV. Each symbol represents the product of the mean IC_50,total for virus inhibition (n = 2) and the fraction of unbound drug in the medium determined in triplicate at 0.1 μg/ml by equilibrium dialysis. The horizontal lines represent the mean IC_50,free values. (a) LPV; (b) RTV.