Research And Development Of NobilisideA Freeze-drying Liposomes

Nobiliside A, first discovered and isolated from the Holothuria nobilis at Research Center for Marine Drugs, School of Pharmacy, Second Military Medical University, Shanghai, is a new triterpene glycoside. In vitro and in vivo anti-tumor and anti-angiogenesis activities of nobilisde A were studied. The results showed that nobilisde A has both cytotoxic and angiogenesis inhibiting effects. But nobilisde A was observed low bioavailability in oral administration because of gastrointestinal hydrolysis and enzymolysis and it has severe side effects to heart and kidney as well as severe hemolytic activity, all of which limit its development and clinical use. Therefore, to choose a good carrier for reducing the hemolysis and toxicity of nobilisde A is of great importance for its development and clinical use.Considering its properties and requests for therapy, liposome dispersion was prepared using film-dispersion and reverse phase evaporation (REV) with freezing-thawing. Single factor test had been done and the orthogonal design was adopted to obtain the optimized prescription. Film-dispersion was the last choice, the better prescription was that concentration of nobiliside A was 0.5mg/ml, the ratio of phospholipid and cholesterol was 2:1(W/W), the ratio of lipid and drug was 40:1 (W/W) and the EE% could exceed 90%after three freezing thaw.Microcolumn centrifugation method was adopted to separate free drug from nobiliside A liposomes. The concentration of nobiliside A in liposomes was determined.To resolve the instability problem of liposome dispersion, freeze-drying (lyophilization) technique was utilized to prepare freeze-dried liposome. Its main advantage is high stability because of solid form during preservation. It can readily and quickly be reconstituted by adding water and simplely shaking before use. Inspect physico-chemical properties of liposomes before and after freeze-drying, including distribution of particle diameter, content, EE% and so on, it was seen that freeze-drying products can keep original characters and raise the stability of liposomes.However, freeze-drying process can induce serious and irreversible damage to liposomes, including size increasing, membrane rupture, liposome infusion, membrane infolding, and the drug leakage etc. So it's of great importance to investigate each freeze-drying process and cryoprotectant formulation which can preserve the vesicles stable in freeze-drying. Screening from various cryoprotectants, sucrose was ensured to protect nobiliside A liposomes during freeze-drying, the ratio of phospholipid and sucrose was 1:5.Influence tests showed that nobiliside A freeze-drying liposomes were sensitive to temperature and humidity. Accelerating stability test show that nobiliside A liposome freeze-drying products were stable at the temperature of 25℃±2℃and relative humidity of 60%±10% for three months. Long-term stability test show that nobiliside A liposome freeze-drying products were stable at the temperature of 6℃±2℃for three months.The LC/MS/MS methods were established for the determination of nobilside A in rat plasma. The pharmacokinetics in rats of nobilside A liposomes and nobilside A solution were explored. Significant statistical differences were found between the AUC and other parameters of two preparations.