Partition of chlorpromazine into lipid bilayer membranes: the effect of membrane structure and composition.

Partition coefficients, kp, of chlorpromazine between the aqueous phase and lipid bilayer vesicles were determined as function of drug concentration, lipid chain length, cholesterol content and temperature encompassing the range of the lipid phase transition. Radioactivity and absorption measurements were performed to determine the kp values. Up to a concentration of 3 . 10(-5) M, the partition coefficient is independent of chlorpromazine concentration, whereas it decreases drastically at higher chlorpromazine concentrations, at which membrane lysis is observed. Membrane structure is not disturbed at less than 3 . 10(-5) M chlorpromazine, as was concluded from electron paramagnetic resonance studies measuring TEMPO partitioning and order degree. However, the lipid phase-transition temperature decreases and is broadened at higher chlorpromazine concentrations. From fluorescence measurements, we conclude the formation of chlorpromazine micelles at concentrations higher than 5 . 10(-5) M in chlorpromazine in the absence of lipids and the formation of mixed micelles in the presence of lipids. The effect of lipid chain length on kp values was investigated. The partition coefficient decreases from 8100 in dilauroyl- to 3400 in dipalmitoylphosphatidylcholine vesicles, both at 50 degrees C, that is, above their corresponding phase-transition temperature tt. At t less than tt the kp values are strongly reduced, by at least a factor of 10, depending on lipid chain length and membrane composition. It is possible to establish a lipid phase-transition curve from the temperature-dependent measurements of the kp values. Cholesterol within the lipid membrane strongly decreases kp. At 20 mol% cholesterol in dipalmitoylphosphatidylcholine membranes, the partition coefficient is reduced from 3400 to 2300. This value is well comparable to the kp value obtained in erythrocyte ghosts. In contradiction to earlier experiments by Conrad and Singer (Biochemistry 20 (1981) 808-818), this value in a biological membrane could be obtained by the hygroscopic desorption as well as the centrifugation method. From our experiments we are justified in further considering artificial bilayer membranes as models for biological membranes.

Lateral diffusion of small solutes and partition of amphipaths in defect structures of lipid bilayers.

The excimer formation technique has been applied to investigate the mechanism of the lateral diffusion in the crystalline P beta' phase of dipalmitoylphosphatidylcholine vesicles. This became possible at low pyrene concentrations. From the shape of phase-transition curves, from the effect of pressure up to 150 bar and from the perturbation induced by cholesterol, we conclude that the free volume model could also be applied to the P beta' phase. However, the diffusion is thought to occur in defect structures that are considered to form fluid pathways between domains of crystalline lipid. Partition coefficients of amphipaths provide a basis for testing for the role of defects. The amphipath chlorpromazine partitions into fluid membranes with a partition coefficient, kp, of 3200, into the crystalline phase with kp = 200 but into the P beta' phase with a value of kp = 800. This again gives rise to the assumption that the P beta' phase contains fluid domains that behave like the fluid L alpha phase and make up about 10-20% of the total amount of lipid in the bilayer. Cholesterol is known to interfere especially with defect structures in the P beta' phase. It fills up the gaps, and therefore reduces the partition coefficient to almost zero in the P beta' phase.

Membrane solubility of chlorpromazine. Hygroscopic desorption and centrifugation methods yield comparable results.

Binding of the positively charged drug chlorpromazine to artificial and erythrocyte bilayer membranes was investigated by the filtration method called hygroscopic desorption [Conrad & Singer (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 5202-5206] and by the conventional centrifugation method. Only minor differences in the partition coefficients were observed using the two methods. Our finding is not consistent with the observation of Conrad & Singer that amphipaths are completely excluded from biological membranes. However, the partition coefficient is dependent on membrane composition, which means dependent on the physical properties of a membrane.