Partition chromatography of drugs on immobilized liposomes and biomembranes : A method applicable to screening in drug design

Sammanfattning: One of the main pathways for drug passage into cells is diffusion across the lipid bilayer ofthe cell membrane. A key factor in this process is the drug partitioning into the bilayer, whichcan be translated into a retention in a chromatographic system. Liposomes, proteoliposomesand cytoskeleton-depleted red cell membrane vesicles were therefore entrapped in gel beads tobe used as biomimetic models in drug partition chromatography. Red cells were adsorbed ongel particles for similar use as a stationary phase, whereby ghosts were formed. Each retentionvolume was divided by the amount of immobilized phospholipids, as determined byphosphorus analysis, to define a capacity factor (Ks). I verified that the chromatographicretention volumes were proportional to the amounts of immobilized phospholipids. The loss oflipids with time was small and allowed many series of runs. Short columns containing smallamounts of lipids allowed quick analysis of highly lipophilic drugs. The logarithm of Ksvalues for positively charged drugs on negatively charged liposomes decreased as the ionicstrength was increased, increased with increasing negative charge of the liposomes as the pHwas increased and varied linearly with the temperature. On red cells/ghosts the range of thelog Ks values was more narrow than on liposomes and vesicles. The log Ks values on neutralliposomes decreased with increasing cholesterol fraction and mostly increased as the temperature was increased. Insertion of transmembrane proteins into lipid bilayers changed the log Ksvalues for charged drugs, and in some cases also for neutral drugs. Comparison of log Ksvalues with values from the literature for octanol/water distribution ratios, apparent partitioning in liposome suspensions, retention on immobilized artificial membranes, calculateddynamic polar molecular surface areas, permeability values on cultured monolayers of Caco-2cells and absorption of orally administered drugs in humans showed different recti- or curvilinear correlations. The results obtained have shown that immobilized liposome or biomembrane chromatography is a reproducible, robust and simple method for characterization ofdrug partitioning. The method is applicable for drug screening.