Studies of factors influencing drug efflux rates from liposomes and their impact on antitumor efficacy

The anti-tumor efficacy of liposomal formulations of cell cycle dependent anticancer drugs is dependent on the rates at which the drugs are released from the liposomes. Previous work on liposomal formulations of vincristine has shown increasing efficacy for formulations with progressively slower release rates. This thesis examined methods for increasing drug retention in vitro and in vivo for various antineoplastic agents as well as enhancing the circulation lifetime of the drug delivery system.; In Chapter 2 the effects of very high drug-to-lipid (D/L) ratios on vincristine release rates were investigated, and the antitumor efficacy of these formulations characterized in human xenograft tumor models. It is shown that the half-times (T1/2) for vincristine release from egg sphingomyelin/cholesterol liposomes in vivo can be adjusted from T1/2 = 6.1 h for a formulation with a D/L of 0.025 (wt/wt) to T1/2 = 117 h (extrapolated) for a formulation with a D/L ratio of 0.6 (wt/wt). The increase in drug retention as the D/L ratio is increased is attributed to the presence of drug precipitates in the liposomes. Variations in the D/L ratio did not affect the circulation lifetimes of the liposomal vincristine formulations. The relationship between drug release rates and antitumor efficacy was evaluated using a MX-1 human mammary tumor model. It was found that the antitumor activity of the liposomal vincristine formulations increased (tumor growth delay 21 d to greater than 60 d respectively) as the D/L ratio was increased from 0.025 to 0.1 (wt/wt) (T1/2 = 6.1--15.6 h respectively) but decreased (tumor growth delay 29 d) at higher D/L ratios (D/L=0.6, wt/wt, T1/2 =117 h). Free vincristine exhibited the lowest activity of all formulations examined. These results demonstrate that varying the D/L ratio provides a powerful method for regulating drug release and allows the generation of liposomal formulations of vincristine with therapeutically optimized drug release rates.; In Chapter 3, it has been demonstrated that the release properties of ciprofloxacin, a drug that does not precipitate following accumulation into large unilamellar vesicles, are not affected by the D/L ratio, whereas the release properties of liposomal doxorubicin are strongly dependent on the D/L ratio. It has also been shown that the crystalline intravesicular doxorubicin precipitates observed as the D/L ratio is raised from 0.05 to 0.46 (wt/wt) adopt increasingly unusual morphologies. Linear crystals are observed at the lower D/L values (0.05), however triangular and rectangular variations are observed as the D/L ratio is increased that induce considerable distortion in the vesicle morphology. It is noted that the trapping efficiency following uptake of external doxorubicin into LUV is reduced from nearly 100% at a D/L ratio of 0.05 (wt/wt) to less than 50% at an (initial) D/L ratio of 0.8 (wt/wt). It is suggested that this arises, at least in part, from membrane-disrupting effects of internal drug crystals as they increase in size.; In Chapter 4, it was shown that replacement of egg sphingomyelin (ESM) by dihydrosphingomyelin (DHSM) in sphingomyelin/cholesterol (55/45; mol/mol) large unilamellar vesicles (LUV) results in substantially improved drug retention properties both in vitro and in vivo. In the case of liposomal formulations of vincristine, for example, the half-times for drug release (T1/2) were approximately 2-fold longer for DHSM/Chol LUVs as compared to ESM/Chol LUVs, both in vitro and in vivo. Further increases in T1/2 could be achieved by increasing the D/L ratio of the liposomal vincristine formulations. In addition, DHSM/Chol LUVs also exhibit improved circulation lifetimes in vivo as compared to ESM/Chol LUV. For example, the half-time for LUV clearance (Tc1/2) at a low lipid dose (15 mumol lipid/kg, corresponding to 8.4 mg lipid/kg body weight) in mice was 4.3 h for ESM/Col LUVs compared to 6.7 h for DHSM/Chol LUVs. It is shown that the clearance pro...