In vitro studies of intestinal drug absorption : Determination of partition and distribution coefficients with brush border membrane vesicles

Abstract

Brush border membrane vesicles (BBMV) were isolated from rat small intestine and characterized in terms of relative enrichment of specific organelle marker enzymes (20-fold enrichment; 20% yield), contamination by other subcellular organelles (<1%) and functional integrity (Na +-dependent glucose uptake). Using these vesicles, techniques were developed for the determination of partition and distribution coefficients for the model solutes, nitrobenzene, toluene and benzoic acid. No gender, age or regional variation along the small intestine in partition coefficient (log P) values was detected. There was no temperature (10–40°) or pH (4.5–8.0) dependence in partition coefficients of nitrobenzene and toluene. Fair agreement was obtained for log P and log D values for these two solutes determined with BBMV and those reported with octanol and propylene glycol dipelargonate. Selective removal of proteins, both ecto-brush border and micro-villus core proteins, did not alter the partition coefficients of the three model solutes. In contrast, depletion of the BBMV of non-esterified fatty acids significantly decreased the partition coefficients. Liposomes prepared from BBMV lipid extracts were also used for partition coefficient determinations and gave similar values to intact BBMV; addition of increasing amounts of cholesterol to the lipid extract caused small increases in the partition coefficients of the model solutes in the liposomes. It was concluded that the partition coefficients of the BBMV were related to the lipid and not to the protein composition of the vesicles. The method offers a rapid and reliable means of measuring the partition coefficient of non-protein bound drugs and nutrients in isolated intestinal BBMV and should assist in the subsequent modelling and prediction of intestinal absorption in vivo.