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Characterization of cholesterol-free liposomes for use in delivery of anti-cancer drugs Dos Santos, Nancy


Improving existing therapies with lipid based carriers has been successfully applied to drugs that have narrow therapeutic indices, such as anti-cancer agents. It is known that the addition of cholesterol to a lipid matrix of gel phase lipids (> CI 8), increases the permeability of lipid membranes below the phase transition temperature (Tc) of the bulk phospholipid species used, and thus it is predicted that these formulations may retain drugs that are not compatible with conventional (cholesterol-containing) liposome formulations. Liposomes composed of 1,2- distearoyl-sn-phosphatidylcholine (DSPC), without added cholesterol, were effectively stabilized by incorporation of PEG-lipids, where stability was defined by parameters including prevention of surface-surface interactions and extending blood residence times. Cholesterol-free liposomes as carriers for anti-cancer drugs are hampered, in part, because standard pH gradient-based loading methods rely on high temperatures (> Tc of the phospholipids used), which can collapse the ion gradient and / or result in unstable loading. Doxorubicin, for example, could not be loaded efficiently into cholesterol-free DSPC liposomes, a problem that was circumvented by the addition of 10% (v/v) ethanol, as a permeability enhancer. Another more hydrophobic anthracycline, idarubicin, could be encapsulated in cholesterol-free liposomes without the aid of ethanol as a permeability enhancer. Cryo-transmission electron microscopic studies indicated that idarubicin formed a precipitate within the liposomes. Pharmacokinetic studies demonstrated that liposome encapsulation manifested a 66-fold increase (1.97 μmole h ml⁻¹) in the mean plasma area-underthe- curve (AUC) as compared to free idarubicin (0.03 μmole h ml⁻¹). Further alterations in lipid composition, including decreasing PEG-lipid and internal citrate (osmolarity) concentrations, resulted in stepwise improvements in drug retention and blood residence times. The optimized lipid formulation, DSPC / DSPE-PEG[sub 2000] (98:2 mole ratio, 150 mM citrate), mediated a 175- fold (7.0 μmole h ml⁻¹) increase in mean plasma AUC and 5.5-fold (6.74 h) increase in the plasma half-life (T[sub ½]) when compared to free idarubicin. Antitumor activity of liposomal idarubicin was assessed in a P388 lymphocytic leukemia model, the median survival at the maximum tolerable dose (3 mg/kg) was 22 days (175 % ILS) for liposomal idarubicin and 19.5 days (144 % ILS) for free idarubicin. These results warranted further investigation to improve the therapeutic activity of liposomal idarubicin through use of combination drug treatments. In order to assess this, a liposomal formulation of gemcitabine was prepared and the antitumor activity of a combination treatment consisting of liposomal gemcitabine and liposomal idarubicin was evaluated. Gemcitabine was passively encapsulated in DSPC / CH / DSPE-PEG[sub 2000] (50:45:5 mole ratio) at a 0.1 drug-to-lipid mole ratio. Pharmacokinetic studies indicated that encapsulation of gemcitabine in liposomes mediated a 135-fold (15.4 μmole h ml⁻¹) and 8-fold (14.3 h) increase in the mean plasma AUC and plasma half-life, respectively. An increase in median survival was observed in liposomal treatment groups of gemcitabine (3.4 mg/kg) / idarubicin (2 mg/kg) administered at 0.66 maximum tolerable doses for individual drugs. The median survival time was 30 days (281 % ILS) for liposomal gemcitabine / liposomal idarubicin, as compared to 16 days (100 % ILS) for 5 mg/kg liposomal gemcitabine, and 20.5 days (156 % ILS) for 2 mg/kg liposomal idarubicin. Assessment of the combination of these liposomal drugs yielded supra-additive effects.

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