Hydrophobicity of HIV Protease Inhibitors by Immobilized Artificial Membrane Chromatography: Application and Significance to Drug Transport

Abstract

Purpose. The feasibility of using hydrophobicity measurements as screens for intracellular availability in T-cells or intestinal permeability in Caco-2 cells was examined.

Methods. T-cell experiments: Cells were counted, collected, then incubated with drug solution at 37°C. At selected time intervals, uptake was quenched by transferring a sample into oil, followed by rinsing, lysis of cells, protein precipitation and analysis by HPLC. Caco-2 cell experiments: Cells were grown on plastic dishes for 7−10 d, then rinsed and incubated with drug solution at 37°C. Uptake was quenched, cells were lysed, protein precipitated and drug was analyzed by HPLC. IAM chromatography: Stock solutions were injected onto an IAM column for HPLC. Mobile phase consisted of varying amounts of acetonitrile in buffer (pH 7.4). The capacity factor, k′_IAM, was calculated using citric acid to measure the void volume and was obtained by extrapolation to pure buffer.

Results. Nine HIV protease inhibitors were studied for uptake by CEM T-cell suspensions or Caco-2 cell monolayers. Capacity factors (log) between IAM and C-18 columns were positively correlated for this series. Caco-2 uptake rates correlated well with T-cell uptake rates when normalized by protein mass. Single-variable regression using IAM or C-18 columns was acceptable for analysis of T-cell data. Correlation coefficients between T-cell uptake and log k′_IAM or log k′_C-18 were not improved with multivariable regression. Correlation between Caco-2 uptake and log k′_IAM was enhanced when molecular weight and hydrogen-bonding potential were included in multivariable regression analysis (from r² of 0.39 to 0.91). Correlations obtained using log k′_IAM, log k′_C-18.or log distribution coefficient (log D) were comparable when regressed against Caco-2 uptake using this approach. Calculated log partition coefficient (ClogP) provided the poorest correlation in the multivariable analysis (r² = 0.57 for T-cell uptake and r² = 0.71 for Caco-2 cell uptake).

Conclusions. Uptake of HIV protease inhibitors by T-cell suspensions or Caco-2 cell monolayers was positively correlated. Uptake by T-cell suspensions was adequately described by hydrophobicity alone. Description of uptake by Caco-2 cell monolayers required multivariable regression analysis in which molecular weight and hydrogen bonding were included. Experimental measures of hydrophobicity (log k′_IAM, log k′_C18 and log D) were superior to ClogP in the correlation analysis.