Studies On Docetaxel Cationic Liposome

Docetaxel,a semisynthetic taxane derivation,is a wide spectrum antitumor agent specifically effects on cell cycle regulation.Despite having the potent antitumor efficacy,poor water solubility of Docetaxel largely limits its clinical development and application.The commercial drug Taxol Emperor which has great amount of Tween-80 often induces serious adverse reactions.In order to avoid these adverse reactions and fluid retention,repeated doses of glucocorticoid drugs are needed to administrate,which causes much inconvenience to clinical use.Thus,it is necessary to develop a new preparation drug delivery system to improve the solubility of docetaxel.As a gene vector,cationic liposome is widely used in the study of gene transfection and therapy.Recently,it is popular to use cationic liposome as vector of anti-tumor drugs.Cationic liposome is a closed vesicle composed of amphiphilic phospholipids bilayer.The hydrophobic tails of the phospholipids are clustered together to avoid the aqueous phase,but leaving the hydrophilic heads to expose to it.It is supposed that the direct contact with aqueous phase can be avoided if the docetaxel molecule is embedded into the phospholipids bilayer,which significantly improves the solubility of docetaxel.The new microvessels of tumor responsible for the nutrition supplication are growing very fast.The amount of negative charge on the surface of tumor angiogenic endothelial cells is much more than normal microvessels.The cationic liposome can target the angiogenic endothelial cells by electrostatic interaction.They are immediately endocytosed by the cells.Then the cationic liposomes quickly impair the membrane of endosomes resulting in release of the drug into the cytoplasm,which accelerate the transportation of the drug from cytoplasm to nuclear.This mechanism significantly enhances the level of drug accumulation and release in the tumor area.Objective:In this study,docetaxel is loaded into the cationic liposome to improve its solubility.Moreover,the antitumor efficacy of docetaxel is improved by enhanced targeting effect of the cationic liposome to the negative charged tumor angiogenic endothelial cells.In addition,the in vivo/vitro pharmacokinetic(PK)and pharmacological(PD)property of the new preparation was investigated.Methods:First,we established the method to determine the encapsulation efficiency(EE%)and the content of the main materials including docetaxel,DOTAP-C1 and DOPC.Ethanol injection method was employed in preparing the docetaxel cationic liposome and passive loading method was used in the drug loading procedure,according to the preliminary studies.1,2-dioleoyl-3-trimethylammonium-propane(chloride salt)(DOTAP-C1)and 1,2-dioleoylphosphatidylcholine(DOPC)were selected as the structural phospholipids.Trehalose dihydrate solution was used as the aqueous phase;meanwhile the oil phase was formed by dissolving docetaxel and the phospholipids in ethanol.The liposome was obtained by injecting the lipid phase into the liquid phase under stirring.The liposome was then extruded through polycarbonate membranes to adjust to a certain particle size.The extruded liposome was then lyophilized to improve stability.Single factor studies have been designed against the factors which potentially affect the drug stability.Using the size distribution and EE%as the index,the influence of the factors,such as different phospholipids composition,oil phase concentration,water phase concentration,pH value of water phase,phase ratio and injection speed,on the physical property of the liposome was investigated to determine the optimal formulation and process condition of docetaxel cationic liposome.The quality studies including the appearance,pH value,osomlality,size distribution,zeta potential,encapsulation efficiency and content,et al were conducted on liposome prepared by the optimal formulation and process.The 24h stability of docetaxel cationic liposome suspension and the 6 month stability of lyophilized product were carried out.The detection method of docetaxel and DOTAP-C1 was established and then verified.PK study was conducted on male Wistar rats to preliminarily investigate the in vivo behavior of docetaxel liposome.Docetaxel cationic liposome or Taxol Emperor was administrated into the rats by intravenous tail vein injection.Both the dosage was 4 mg·kg-1,and injection speed was 12 mg·kg-1·min-1.The in vitro pharmacological study(PD)was carried out and compared among Docetaxel cationic liposome,blank cationic liposome and Taxol Emperor(Taxotere)by MTT assay.Given that docetaxel was mainly used in the clinical therapy of breast cancer,oophoroma and lung cancer,MCF-7 cell(human breast cancer)and A549 cell(human pancreatic cancer cell)were employed.The in vivo pharmacological study of docetaxel was performed in female BDF1 mice by inoculating LLC cell(mice lung cancer)in the axilla left lower limbs.The tumor inhibition rates of docetaxel cationic liposome,blank cationic liposome and Taxotere were compared.Results:The optimal formulation was determined as:the molar ratio of DOTAP-C1,DOPC and docetaxel was 50:43:7;the total phospholipids content was 1500 mM;the water phase was trehalose dehydrate(110 g/L,pH 4.5-5.5);the oil/water phase ratio is 1:74,and the injection speed is 1.0 mL/min.Exclusion and granulation processes are conducted after injection,the product was freezing stored.The liposome prepared by the optimal formulation and process is a uniform lyophilized white power.It is quickly reconstitutes into opalescence suspension after adding of purified water and gently shaking.The analysis results of 3 batches of product are:the particle size was 160 nm and PDI<0.1;the pH(5.0)and the osomlality(310 mOsmol/Kg)meet the requirements of injection.The drug loading content was up to 1 mg/mL,with the EE%is more than 98%.The reconstituted liposomes are stable in 24 hr by 4? or-20?.There are no significant changes of the lyophilized product after 6 months'storage by 4?.Docetaxel cationic liposome displays a much similar PK property with Taxotere,both reaching maximum 1 min after injection beginning,followed by quickly decrease.But the PK property of DOTAP-C1 was different,which reached top point 30 min after injection beginning,with little fluctuation in 5 to 60 min and subsequently decreased smoothly.In vitro PD study results showed all three preparations,docetaxel cationic liposome,blank cationic liposome and Taxotere,can inhibit the growth of MCF-7 cell and A549 cell.When compared with Taxotere in low concentration range,docetaxel cationic liposome displayed stronger inhibition effect to the cells.The in vivo PD study results showed that the LLC tumor inhibition rate of docetaxel cationic liposome,blank cationic liposome and Taxotere was 58.1%,12.9%and 29.6%respectively,indicating that the tumor inhibition effect of docetaxel cationic liposome is much more better than Taxotere(P<0.01).Conclusion:Docetaxel cationic liposome is of simple and controlled process,high encapsulation efficiency,and stable physical properties.The in vivo PK property of the cationic liposomal docetaxel is similar with Taxotere.The therapeutic efficacy is significantly improved compared to the commercial product Taxotere(P<0.01).