Preliminary Research Of Gemcitabine Liposomes

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 improved thin-film hydration procedure was tried for active loading of gemcitabine into liposomes.Establishing the HPLC analytical method and making quality evaluation in vitro and in vivo,the main work were as follows:Part one:ReviewThe application,development and new kinds of liposmes are introduced in this review;We outlined the physico-chemical property,mechanism of action,pharmacokinetics property and the application of anti-tumor drug gemcitabine.These may be regarded as a basis for our succeeding research.Part two:The establishment of analytical method in vitro about gemcitabineAfter comparing several methods such as:centrifugation,cation exchange resin,Sephdex G50 and dialysis to separate the free drug and liposomes,then combining with the HPLC to determine the entrapment efficiency of gemcitabine liposomes.Finally,we choose the cation exchange resin-HPLC method.It shows that this method is easy,accurate and with good reproducibility.Part three:Preparation of gemcitabine liposomeWe try to find an effective preparation method and properate material to improve the entrapment efficiency of water-soluble drug in liposomes.Many kinds of preparation methods are tested:thin-film hydration procedure,improved thin-film hydration procedure,reverse-phase evaporation,solvent injection,ammonium sulfate gradient method and so on.After comparing those methods,we apply the improved thin-film hydration procedure.Many single factors which may influence the appearance,entrapment efficiency and stability of liposome preparation were observed,such as solvent,drug and phospholipids ration,phospholipids and cholesterol ration,freeze-thawing temperature, type and quantity of phospholipids,hypersound time and waterbath temperature.The optimization formula was got after orthogonal experiment about principal influential factors.The detail was below:drug: lipid=1:15,phospholipids 360mg,waterbath temperature 50℃,freeze-thawing temperature 30℃.Part four:Quality evaluation of gemcitabine liposome in vitroWe observed many indexes to evaluate the quality of gemcitabine liposome in vitro,such as the morphology,particle size,encapsulation efficiency,release profile,stability and so on.The results indicated that the preparation of liposome has small particle diameter,high encapsulation efficiency,and no break release phenomenon.The mean particle diameter is 195nm,and the entrapment efficiency is 63%.The preparation was stable in the storage.leaking only a few drugs. Comparing with before accelerated testing,there was no obvious change. It proved that the preparation has a fine stability.Part five:Pharmacokinetics in rats of gemcitabine liposomeThe concentration of free and gemcitabine liposome in plasma was determined after i.v.administration with test and reference preparations separately,taken by the five rats in a randomized cross-over test.The pharmacokinetics date was calculated with compartment and non-compartment model.After statistical analysis,we get the result that with the compartment calculation,the gemcitabine liposome and the controlled preparation are both two-compartment model,t_1/2α、t_1/2β、AUC_0-τ of the gemcitabine liposome is:4.2h、12.6h and 30.36hμg/ml,the gemcitabine liposome compared with the controlled preparation t_1/2α、t_1/2β、AUC_0-τhave been improved 5.92、5.92 and 9.99 times;with the non-compartment calculation the result is:t_1/2、AUC and MRT of the gemcitabine liposome:3.55h、30.36hμg/ml�'�10.92h,the gemcitabine liposome compared with the controlled preparation t_1/2、AUC and MRT have been improved 3.32、9.99 and 7.74 times.Part six:Initial study of tissue distribution about gemcitabine liposome in ratsThe concentration of free and gemcitabine liposome in organism was determined after i.v.administration with test and reference preparations separately,taken by the three rats in a randomized cross-over test.After statistical analysis,we get the result at 1h the gemcitabine mainly dispersed in blood,spleen and lung.The distribution of gemcitabine liposome compared with the controlled preparation,in blood、spleen、lung is improved;in heart is nearly the same,in liver and kidney is diminished..