Investigation Of Preparation And Pharmacokinetics For Freeze-dried Docetaxel Liposome

Background:Docetaxel is a semi-synthesized anti-cancer drug of the taxoid group which represents a new class of cytotoxin with a novel mechanism of action. Cytotoxins exert their lethal effects on dividing cancer cells by various mechanisms all of which result either in failure of the cell to divide or subsequently to grow and develop. It showed high anti-tumor activity in vitro and in vivo. Docetaxel, with poor solubility in water, is formulated using Tween 80 (polysorbate 80) and 13% ethanol. However, acute hypersensitivity reactions and other toxic effects have been noted in the majority of patients, which partially come from using of Tween80. In view of these observations there is a strong rationale for reformulating docetaxel using a safer better-tolerated vehicle than Tween 80. Among drug carrier systems, liposome is a relatively non-toxic technology with considerable potential for encapsulation of both lipophilic and hydrophilic drugs, and is currently use to treat a number of cancers. Thus, in this study we intend to prepare the docetaxel liposome (L-DOC), to improve its solubility, pharmacokinetics characters,to avoid the toxic reaction coming from Tween80 and to enhance anti-tumor activity,which can make foundation for preclinical study of L-DOC.Objective:1. To develop a novel liposome dosage form of docetaxel with high encapsulation efficiency and high drug content.2. To establish the quality standard for determining docetaxel in L-DOC.3. To study the influential factors so as to optimize the formulation of L-DOC.4. To evaluate the stability of L-DOC under different conditions, and to offer information for the next research and storage.5. To compare the pharmacokinetics characters of L-DOC and M-DOC after single intravenous injection in rats. 6. From above research, to make a foundation for further investigation.Method:1. To prepare L-DOC with high encapsulation efficiency (EE %) and uniform size distribution by rotary evaporation followed by lyophillization.2. To determine the docetaxel content and the encapsulation efficiency of L-DOC by high performance liquid chromatography (HPLC) combined with ultracentrifugation.3. To evaluate the particle size and polydispersity index (PI) of L-DOC by photon correlation spectroscopy (PCS).4. To investigate the effect of PC/CHOL, pH of buffer and temperature of hydration on recovery of docetaxel and EE%.5. To optimize the formulation of L-DOC with the uniform experimental design by determining EE%.6. To observe the stability of L-DOC under 4℃and in accelerated test, respectively.7. To determine the docetaxel concentration in blood after single intravenous injection of L-DOC or M-DOC in rats. The pharmacokinetics parameters were studied with 3P97 software.Results:1. L-DOC was prepared with stable concentration and high encapsulation efficiency, and its mean particle size is about 214.9 nm.2. The HPLC method for determining of docetaxel concentration in L-DOC was established. The retained time of L-DOC is about 11.062 min, and the standard curve of L-DOC was linear in the range of 3.64 ~ 7.80μg/ml, r=0.9996. The average recovery was 99.18%. The within-day and between-day RSD did not exceed 2.0% and 2.5%, respectively. The stability, repetition, and line relation of this HPLC method is very well.3. The analysis showed that (PC/CHOL) 4:1, (DOC/ (PC+CHOL)) 1:50 and (pH of buffer) 5 were the optimal formulation. Under the optimal formulation, the drug recovery, EE%, particle size and PI was (99.96±1.87)%, (91.61±1.69)% ,(231.6±100.3) nm and (0.35±0.06), respectively.4. The preliminary results indicated that the liposome are stable under 4℃, it does not deteriorate after 30 days accelerated test. 5. From the view of pharmacokinetics study, L-DOC showed higher plasma concentration levels, longer elimination half life, and larger area under the curve values comparing with M-DOC.Conclusion:1. Establish a well stable and high repetitive HPLC method with well linear relation to determine docetaxel concentration in L-DOC successfully.2. Establish ultracentrifugation method to determine the encapsulation efficiency of L-DOC.3. Establish PCS method to determine the size distribution of liposomes successfully.4. Under the optimal formulation, the drug recovery, the EE%, the particle size and PI of L-DOC are satisfactory5. L-DOC is stable at low temperature.6. In the comparative pharmacokinetics studies with M-DOC, L-DOC shows longer T1/2 and smaller CL in rats.