Stability of association of 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine with liposomes is composition dependent.

The ether lipid, 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-OCH3), has anticancer activity, but it has serious side-effects, including hemolysis, which prevent its optimal use. We surmised if ET-18-OCH3 could be stably associated with liposomes, less free ET-18-OCH3 would be available for lytic interaction with red cells. Liposome composition variables investigated included acyl chain saturation, phospholipid head group and mole ratio of Chol and ET-18-OCH3. It was found that attenuation of hemolysis was strongly liposome composition dependent. Some ET-18-OCH3 liposome compositions were minimally hemolytic. For example, whereas the HI5 (drug concentration required to cause 5% human red cell lysis) was 5-6 microM for free ET-18-OCH3, it was approximately 250 microM for DOPC (dioleoylphosphatidylcholine):Chol (cholesterol):DOPE-GA (glutaric acid derivatized DOPE):ET-18-OCH3, (4:3:1:2) and 640 microM for DOPE (dioleyolphosphatidylethanolamine):Chol:DOPE-GA:ET-18-OCH3 (4:3:1:2) liposomes. Efflux of carboxyfluorescein (CF) from liposomes and Langmuir trough determinations of mean molecular area of lipids in monolayers (MMAM) were used as indicators of membrane packing and stability. Incorporation of ET-18-OCH3 in liposomes reduced the MMAM. Reduction in CF permeation was correlated with reduction in hemolysis. The most stable liposomes included components, such as cholesterol, DOPC and DOPE, which have complementary shapes to ET-18-OCH3.

Combination of antitumor ether lipid with lipids of complementary molecular shape reduces its hemolytic activity.

Because the therapeutic use of the antitumor ether lipid 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphorylcholine (ET-18-OCH3) is restricted by its hemolytic activity we explored the use of lipid packing parameters to reduce this toxicity by creating structurally optimized ET-18-OCH3 liposomes. We postulated that combination of ET-18-OCH3, which is similar in structure to lysophosphatidylcholine, with lipid molecules of complementary molecular shape (opposite headgroup/chain volume) would likely yield a stable lamellar phase from which ET-18-OCH3 exchange to red blood cell membranes would be curtailed. To quantitate the degree of shape complementarity, we used a Langmuir trough and measured the mean molecular area per molecule (MMAM) for monolayers comprised of ET-18-OCH3, the host lipids, and binary mixtures of varying mole percentage ET-18-OCH3. The degree of complementarity was taken as the reduction in MMAM from the value expected based on simple additivity of the individual components. The greatest degree of shape complementarity was observed with cholesterol: the order of complementarity for the ET-18-OCH3-lipid mixtures examined was cholesterol >> DOPE > POPC approximately DOPC. Phosphorus NMR and TLC analysis of aqueous suspensions of ET-18-OCH3 (40 mol%) with the host lipids revealed them to all be lamellar phase. For ET-18-OCH3 at 40 mol% in liposomes, the hemolytic activity followed the trend of the reduction in MMAM and was least for the ET-18-OCH3/cholesterol system (H50 = 661 microM ET-18-OCH3) followed by ET-18-OCH3/DOPE (H50 = 91 microM) and mixtures with POPC and DOPC which were comparable at H50 = 26 microM and 38 microM, respectively: the H50 concentration for free ET-18-OCH3 was 16 microM. This experimental strategy for designing optimized liposomes with a reduction in exchange, and hence toxicity, may be useful for other amphipathic/lipophilic drugs that are dimensionally compatible with lipid bilayers.