Gemcitabine-containing liposomes

Abstract

Gemcitabine, an anticancer agent, is currently in clinical use for the treatment of several types of cancer. Unfortunately, gemcitabine is rapidly metabolised with a short plasma half-life and its cytostatic action is strongly exposure-time dependent. In order to achieve the required concentration over sufficient periods of time, repeated application of relatively high doses is required. This, in turn, leads to dose-limiting systemic toxicity. In order to improve both the efficiency and the toxicity profile of gemcitabine the use of liposomes appears promising. In literature, only a few attempts to entrap gemcitabine within liposomes are found, however none of these liposomal formulations has reached clinical practice. In this study, an ammonium sulphate gradient was tried for active loading of gemcitabine into liposomes.

Firstly, unsaturated egg phosphatidyl choline liposome dispersion was prepared with ammonium sulphate as hydration medium by the hand shaken method followed by filter extrusions with decreasing filter sizes down to 0.1 µm. Then, a transmembrane ammonium sulphate gradient was generated by removing extra-liposomal ammonium sulphate by size exclusion chromatography. Quantitative determination of the ammonium sulphate concentration, both outside and inside the liposomes, via electric conductivity measurement revealed that a gradient of external to internal ammonium sulphate of about 1:58 was achieved.

Secondly, the liposomes were loaded with gemcitabine by incubation at different conditions. Among the loading conditions tested, a total loading time of 24 hours including heating for 2 hours at 60 ˚C seemed advantageous in achieving efficient loading. A higher starting concentration of gemcitabine resulted in enhanced loading efficiency, calculated on a molar basis. Comparing these results to a VPG passive loading technique, the active loading technique resulted in a gemcitabine:lipid ratio of about 1:20 versus 1:140 for the vesicular phospholipid gel loaded liposomes. Unfortunately, the actively loaded liposomes revealed poor storage stability with 80 % leakage after 24 hours. Further studies are needed in order to optimise loading and stability of the liposomes.