| Parenteral submicro-emulsion, with high biocompatibility, low toxicity and irritation, has been widely used as an excellent drug delivery carrier. Teniposide (VM-26), one of the most broadly effective anticancer agents, is included in a wide variety of cancer chemotherapy protocols. However, because of its poor water solubility, teniposide is currently administered in the form of a nonaqueous formulation (Vumon(?)), which contains Cremophor(?)EL (polyethoxylated castor oil), dehydrated alcohol and other non-aqueous solvents. Vumon(?) therapy is associated with life-threatening hypersensitivity reactions and a danger that teniposide may crystallize during the process of dilution. Thus, the aim of this study was to prepare and evaluate a stable teniposide sub-microemulsion injection (TSE).An HPLC method was established for the analysis of teniposide in vitro. The method validations were carried out and approved. Several physicochemical parameters of teniposide were investigated, such as solubility, degradation parameter and so on. The solubility of teniposide in LCT and MCT were lower than 0.5mg·mL-1, so solubilizers would be used in the formulation of TSE. The study of degradation of teniposide in the buffers with the pH 5.0,6.0, 6.5,7.0,8.0 at 80℃shows that pH 6.5 was the most stable pH for teniposide with the half-life 8.36h.High pressure homogenization was used to prepare TSE. HPLC, dynamic light scattering and electrophoretic light scattering technology, and ultracentrifugation were also employed. Taking physical appearance, pH, particle size distribution (PSD),ξ-potential, content, entrapment efficiency and thermal sterile stability as index, the final formulation and preparation process for directly loaded TSE were as follows:as quality percentage, oil phase was composed of 0.05% teniposide,10% MCT,10% LCT,2% VE,2%PEG-400,2.4% soybean lecithin,0.06% oleic acid; the water phase was composed of 2.5% glycerol,0.4% F-68,0.4% Tween-80 and 0.05% EDTA-2Na; and the oil phase and water phase were both heated to 70℃; the homogenization pressure and cycles were 70MPa and 8 times; the pH was adjusted to 6.0-6.5 and then TSE was sterilized in a rotating water bath at 100℃for 30min.Through the study of different pH and sterile ways, the sterile stability of directly loaded TSE was investigated systematically. The results indicated that, with pH 6.5, the physicochemical characteristics of TSE were stable after sterilizing in a rotating water bath at 100℃for 30min. After 1 month room temperature storage, directly loaded TSE showed bad physical appearance. So the phospholipid complex TSE was investigated in the further study.The reaction solvents, the ratios and types of phospholipids were investigated, and the teniposide-phospholipid complex was finally prepared with teniposide and soybean lecithin at a ratio of 1:40 in dehydrated alcohol at 80℃for 2h, and then the reaction solvent was evaporated to obtain dry residue. DSC, IR, and X-ray diffraction were also employed to identify the formation of the complex and analyze the possible interaction between teniposide and phospholipids.The final formulation and preparation process for TSE using teniposide- phospholipid complex were as follows:as quality percentage, oil phase was composed of 0.05% teniposide in the form of the complex,20% MCT,2% VE,2%PEG-400,2.4% soybean lecithin,0.06% oleic acid; the water phase was composed of 2.5% glycerol,0.4% F-68,0.4% Tween-80 and 0.05% EDTA-2Na; and the oil phase and water phase were both heated to 70℃; the homogenization pressure and cycles were 70MPa and 8 times; the pH was adjusted to 6.0-6.5, and then TSE was sterilized in a rotating water bath at 100℃for 30min.The characteristics including PSD,ξ-potential, drug content and entrapment efficiency of TSE using teniposide-phospholipid complex were 162.4±51.14nm,-23.14mV,101.1%, and 91.4%. The dilution test of TSE with Sodium chloride injection solution (0.9%) and dextrose (5%) suggested that after diluting TSE was stable. The shelf-life of TSE was calculated to be 22 days by constant temperature accelerated test. However, the long term stability study showed that the physicochemical characteristics of TSE were still stable at 4±2℃after 2 months. The rheology change of the emulsion demonstrated to be the reason for the extension of the shelf-life detected compared with the shelf-life calculated through the viscosity test.An UPLC/MS/MS method was established to determine the concentration of teniposide in rat plasma. Both TSE using teniposide-phospholipid complex and teniposide solution (TS) fitted three-compartment model and their pharmacokinetic behaviors were similar. The AUC0-t and t1/2 of TSE and TS were (24065.096±6320.831) and (10590.352±2054.728)μg/L*h, and (0.936±0.163) and (0.962±0.264) h, respectively. |
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