HYDROPHOBICITY OF HIV PROTEASE INHIBITORS BY IMMOBILIZED ARTIFICIAL MEMBRANE CHROMATOGRAPHY - APPLICATION AND SIGNIFICANCE TO DRUG TRANSPORT

Purpose. The feasibility of using hydrophobicity measurements as scree ns for intracellular availability in T-cells or intestinal permeabilit y in Caco-2 cells was examined. Methods. T-cell experiments: Cells wer e counted, collected, then incubated with drug solution at 37 degrees C. At selected time intervals, uptake was quenched by transferring a s ample into oil, followed by rinsing, lysis of cells, protein precipita tion and analysis by HPLC. Caco-2 cell experiments: Cells were grown o n plastic dishes for 7-10 d, then rinsed and incubated with drug solut ion at 37 degrees C. Uptake was quenched. cells were lysed, protein pr ecipitated and drug was analyzed by HPLC. IAM chromatography: Stock so lutions were injected onto an IAM column for HPLC. Mobile phase consis ted of varying amounts of acetonitrile in buffer (pH 7.4). The capacit y factor, k '(IAM), was calculated using citric acid to measure the vo id volume and was obtained by extrapolation to pure buffer. Results. N ine HIV protease inhibitors were studied for uptake by CEM T-cell susp ensions or Caco-2 cell monolayers. Capacity factors (log) between IAM and C-18 columns were positively correlated for this series. Caco-2 up take rates correlated well with T-cell uptake rates when normalized by protein mass. Single-variable regression using IAM or C-18 columns wa s acceptable for analysis of T-cell data. Correlation coefficients bet ween 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 p otential were included in multivariable regression analysis (from r(2) 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 regre ssed against Caco-2 uptake using this approach. Calculated log partiti on coefficient (ClogP) provided the poorest correlation in the multiva riable analysis (r(2) = 0.57 For T-cell uptake and r(2) = 0.71 for Cac o-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 hydrophobicit y alone. Description of uptake by Caco-2 cell monolayers required mult ivariable regression analysis in which molecular weight and hydrogen b onding were included. Experimental measures of hydrophobicity (log kj, ,, log k '(C18) and log D) were superior to ClogP in the correlation a nalysis.