| Docetaxel is a semi-synthetic taxane compound that shows anti-tumor effect by disrupting the microtubule network in cells,which is essential for mitotic and interphase cellular functions.Because of its poor solubility in water,currently docetaxel is dissolved in Tween 80 by manufacturer for clinical injections.However,Allergic reaction,fluid retention and other serious side effects have been noted in the clinical treatment,which partially come from Tween 80.Therefore,the development of docetaxel liposome will effectively remove Tween 80 and greatly benefit the clinical use.Objective:Effectively remove Tween 80 existing in docetaxel injecting solution by docetaxel liposome,reduce the toxicity of docetaxel,enhance the efficiency of docetaxel by modifying docetaxel liposome with FA and other accessories to extend the circulation time of drug in the body,and increase the distribution of drugs in tumor tissue.Method:(1)We have developed detection methods,including encapsulation efficiency,particle size,Zeta potential,to test the quality of docetaxel liposome.(2)We have carried out preformulation studies.The stability,solubility and LogP in different PH values of docetaxel were examined.(3)Thin film vesicles(TFV)method was used to prepare docetaxel liposome,and key conditions for preparing docetaxel liposome were optimized.Encapsulation efficiency being used as parameter,the optimized formulation of docetaxel liposome was screened out by a single factor and orthogonal test.The effect of the amount of FA and cholesterol in liposomal formulations on the in vitro release profiles of docetaxel liposome was studied.(4)The appearance and distribution of rehydrated particle sizes of freeze-dried liposome were used as indicators to investigate the freeze-dried process of docetaxel liposome,and the protective effect of various cryoprotectants at different gradient concentrations on the freeze-dried liposome was studied.(5)Studied the quality standards for freeze-dried docetaxel liposome,and tested the stability of freeze-dried docetaxel liposome based on this standard.(6)Research on efficacy of docetaxel liposome was carried out in the model of ICR mice transplanted with S180.(7)Using tumor-bearing ICR mice,pharmacokinetics,tumor tissue and marrow distribution of docetaxel liposome were preliminary studied and compared with commercial docetaxel injecting solution.Result:(1)A method for determining of docetaxel concentration in docetaxel liposome by HPLC,a method for determining of encapsulation efficiency by Sephadex G-50 column,and methods for determining the size and Zeta potential of docetaxel liposome were established.(2)Docetaxel is unsteady at high temperature and by illumination,but stable at relatively high humidity.Docetaxel is soluble in methanol and acetone,slightly soluble in ethanol and chloroform,and barely soluble in water.The LogP of docetaxel is 3.26.(3)Key conditions for preparing docetaxel liposome are listed below.The film-forming solvent is ethanol,the preparation temperature of the film is 50?,rotational speed for preparing membrane is 90?100r/min,materials passed through 0.2?m and 0.1 ?m membrane three times to control the size of docetaxel liposome after homogenization.The best formulation is composed of docetaxel(DOC),Egg phosphatidylcholine(EPC),Cholesterol(CHOL),Dimyristoyl phosphatidic acid(DMPA)and FA.Among them,the drug to lipid molar ratio is 1:26,CHOL to EPC molar ratio is 1:12,and the FA content of total lipid is 1.5 mol%.The amount of CHOL in prescription has a significant impact on the in vitro releasing rate of docetaxel liposome,but FA does not.(4)The best freeze-drying protocol includs following steps:pre-freezing at-42 centigrade,flash freezing,19 hours of sublimation-drying and 9 hours of desorption-drying.The optimal cryoprotectant is 20%trehalose.(5)The quality standard of freeze-dried docetaxel liposome was established.The stability test showed that docetaxel liposome was unstable under the condition of high temperature or light exposure,while it was stable for 24 months if kept in darkness at 4?8?.(6)Pharmacodynamic studies showed that the docetaxel liposome in which the FA content of total lipid was 1.5 mol%had the highest tumor inhibitory effect and was similar to that of commercial docetaxel injecting solution.(7)The half-life of docetaxel liposome in tumor-bearing ICR mice was 330min,while that of docetaxel injecting solution was 210 min.Area under curve(AUC)of docetaxel liposome was 4367?g*min/ml,which was 3.4times bigger than that of docetaxel injecting solution(1297.5?g*min/ml).These results suggest that liposome can be eliminated from the organism more slowly than commercial injecting solution.In tumor tissue of tumor-bearing mice,the contents of docetaxel liposome were significantly higher than that of docetaxel injection,which shows good tumor targeting of docetaxel liposome.There was no significant difference in the contents of docetaxel in the marrow of tumor-bearing mice between the group of docetaxel liposome and the group of docetaxel injecting solution,which implied that docetaxel liposome did not increase marrow toxicity.Conclusion:In this study,the prepared docetaxel liposome had a good performance,stable technology and good reproducibility.It was confirmed by preliminary experiments in vivo showing that docetaxel liposome had a longer circulation time in vivo,good distribution of tumor tissue and good anti-tumor activity.Docetaxel liposome is expected to be developed into a new drug. |
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