| Curcumin (Cur) is a natural poly-phenolic compound isolated from turmeric (Curcuma longa L) which has demonstrated anti-cancer, anti- inflammatory, anti-virus, antioxidant properties. However, due to its water insolubility, instability and low bioavailability in vivo, the application of curcumin as a potential anti-tumor drug has been strongly impeded. To design Cur dosage forms which have high bioavailability and low dose has been highlighted as a major problem.self-microemulsifying drug delivery system (SMEDDS) is an liquid dosage form which consists of a mixture of drug, oil, surfactant and cosurfactant, and the gentle mixing of these ingredients in aqueous media can generate oil-in-water microemulsion droplets of solubilized drugs with a mean droplet size≤100 nm. It is considered that such SMEDDS may increase drug solubility, improve the absorption of drugs and bioavailability, thereby it has been proven possible to formulate preparations suitable for hydrophobic drugs.Sub-microemulsion is a novel drug delivery system which has been employed as carriers for lipid soluble drugs especially anti-cancer drugs, anti- inflammatory drugs and anesthetic drugs. This preparation possesses a number of advantages such as increasing stability, improving drug distribution in vivo, target function and administrating of hydrophobic drugs in the way of intravenous injection.In this article,the physic-chemical properties, stability, solubility of Cur were investigated, and Cur SMEDDS and sub-microemulsion were prepared. The formulation and preparation technology were optimized by orthogonal design and single- factor study, the quality was evaluated, and the pharmacokinetics and pharmacodynamics in vitro and in vivo were investigated. Part one Pre-formulation studyThe stability, solubility and oil/water partition were determined in order to provide some evidence for the design of Cur SMEDDS and sub-microemulsion.METHODS:(1)The method of high performance liquid chromatography (HPLC) was set up to determine the concentration of Cur.(2)The stability of Cur at different pH phosphate buffered saline (PBS), light and high temperature were determined;(3)The stability of Cur-MCT solution at high temperature was measured;(4)The oil/water partition coefficient of Cur was determined by using octanol-water system;(5)The solubility of Cur was detemined in different ratio soybean oil and medium-chain triacylglycerol (MCT) mixtures, surfactant and cosurfactant were investigated.Results:(1)HPLC was accurate and suitable for the determination of Cur in SMEDDS and sub-microemulsion;(2)Cur was relatively stable in ethanol-water, ethanol-pH 4 PBS and ethanol-pH 5.8 PBS at room temperature, but it degradated about 17% in pH6.8 PBS within 48h,white precipitation produced immediately when Cur ethanol solution mixed with pH3,pH7.8 PBS solution;(3) Cur ethanol-water solution was relatively stable at in door light and 4500lux light condition; it degraded about 35% at 80℃water bath within 24h,t1/2 was 92.40 h-1, while Cur MCT solution was relatively stable at 80℃;(4) The oil/water partition coefficient of Cur was 19204±192(Log P为4.210±0.005) which demonstrated strongly hydrophobic property;(5)The composition of oil phase had influence on the solubility of Cur. The solubility of Cur in LCT was lowest which was 1067μg·g-1,while it was highest in MCT which was 3332μg·g-1;Among the oil phase of SMEDDS, the solubility of Cur in isopropyl myristate was more than ethyl oleate; Among the cosurfactant, the solubility of Cur in ethanol was more than 1.2-propylene glycol.Part two Formulation optimization, preparation technology and quality evaluation of Cur SMEDDSBased on orthogonal experiment, pseudo-ternary phase diagram conduction, self-emulsifying efficacy and particle size determination, the formulation of Cur SMEDDS was studied, then the quality was evaluated.METHODS:(1) Pseudoternary phase diagrams were constructed and orthogonal design was used to compare the o/w microemulsion forming capacity of different oil/surfactant/co-surfactant;(2) The solubility of curcumin in various oils and cosurfactants was investigated to find suitable ingredients with a good solubilizing capacity;(3) Droplet size and self-emulsifying efficacy were determined to obtain the concentration range of oil, surfactant and cosurfactant for forming stable microemulsion;(4) The drug loading was determined by solubility test and dilution test;(5) To evaluated the quality of the optimized formulation of Cur SMEDDS, pH, self-emulsifying time, particle size, morphologic characteristics, surface charge, stability and release behavior in vitro were determined.RESULTS:(1)Among the oil phase of SMEDDS, the self-emulsifying capacity of IPM and ethyl oleate were superior to soybean oil; Among the cosurfactant, ethanol and 1.2-propylene glycol were superior to PEG 400; Among the surfactant, Cremophor RH40 was superior to Tween 40 and Cremophor EL; (2) According solubility test, IPM, Cremophor and ethanol were selected as the excipients of Cur SMEDDS; (3) Droplets analysis showed that SMEDDS composed of 20% ethanol, 60% RH 40, 20% IPM would be a stable dosage form for curcumin; (4) The solubility of Cur in SMEDDS was 107.60 mg·g-1,which was 249 thousands times of that of water. But in consideration to the status of micro-emulsion, drug loading was defined to 5%; (5) Cur self-microemulsifying delivery system can emulsified completely within 4min. Mean particle size of the resultant emulsion was 31.98nm, Zeta potential was -19.69mV , and pH approximated to neutral. The dissolution of curcumin self-microemulsifying formulation at 10min was 100% and the content of drug in microemulsion maintained above 94% with 8h. Cur SMEDDS was stable at low temperature (4-8℃) and room temperature.Part Three Formulation optimization, prepare technology and quality evaluation of Cur sub-microemulsionThe formulation and preparation technology of Cur sub-microemulsion were optimized by single-factor experiment, then the quality was evaluated.METHODS:(1)Using stability constant, particle size and distribution as indexes, the formulation of Cur sub-microemulsion was optimized by single-factor experiment ;( 2 ) Cur sub-microemulsion was prepared by high pressure homogenization. The temperature, pressure and cycle times during homogenization were investigated to optimized the technology;(3)Sterilization was determined by evaluating the pH,appearance,particle size and content before and after treatment;(4)To evaluated the quality Cur sub-microemulsion, pH, viscosity, particle size, morphologic characteristics,surface charge,yield were evaluated. (5) Haemolysis test was used to evaluated the safety; (6) Stability was investigated by influential factor and long term stability experiment.RESULTS:(1) The optimized formulation was as follows: MCT15%,egg yolk lecithin 1.8%,pluronic F-68 2%,oleic acid 0.5%,glycerol 2.25%,added water to 100%,drug loading ratio was 3mg/mL. (2) Cur sub- microemulsion was prepared as follows: oil phase (lecithin, MCT, Cur and oleic acid) and water phase (glycerol, pluronic F68 and water) were warmed up to 80℃, the oil phase was dropped into water phase under stirring, the mixture was pre-emulsified using the ultrasonic at 800W for 10min. Final emulsification was carried out by passing the coarse emulsion through a high pressure homogenizer 11 times at a pressure of 900bar, then sealed after adding nitrogen, flowing steam sterilized at 100℃for 45min.(3)Mean particle size of Cur sub-microemulsion was 247nm, Zeta potential was -40.78mV, and pH was 5.83, viscosity was 5m Pa·s, yield was 98.24%,haemolysis test indicated that Cur sub-microemulsion had no hard effect on blood. (4) Cur-microemulsion should be stored in a dark, low temperature area. Stability test indicated that there was no change with appearance, particle size and content after it was stored at 6±2℃for 3 months.Part Four Pharmacokinetics of Cur SMEDDS and sub-microemulsionBy analysing the concentration of drug in plasma, the pharmacokinetics of Cur SMEDDS after oral administration and Cur sub-microemulsion after intravenous administration were investigated, which would provide some evidents for anti-cancer efficacy.METHODS:Cur in plasma were extracted by liquid-liquid extraction, drug concentration was determined by HPLC, pharmacokinetics parameters were obtained by using DAS software, pharmacokinetics behaviors of Cur SMEDDS after oral administration and Cur sub-microemulsion after intravenous injection were compared with Cur suspension and Cur solution.RESULTS: (1) Pharmacokinetic behaviors of Cur micro-emulsion and suspension after gavage in mouse were accorded with one-compartment model. Compared with suspension, absorption of micro-emulsion speeded up 4 times, Cmax increased 3 times, T1/2 prolonged 8 times. The developed SMEDDS formulation improved the oral bioavailability of curcumin significantly, and the relative oral bioavailability of SMEDDS compared with curcumin suspension was 1213%. (2) Pharmacokinetic behaviors of Cur sub-microemulsion and solution after intravenous injection were accorded with two-compartment model. K10 of sub-microemulsion decreased which was one tenth of that of solution, t1/2 prolonged 6 times. MRT prolonged 6 times which was 16.73min and 2.576min,respectively. AUC of sub-microemulsion was 1.3 times of that of solution. All the results demonstrated that Cur sub-microemulsion had some sustained released effect.Part Five The anti-cancer effect of Cur SMEDDS and sub-microemulsion in vivo and in vitroAnti-tumoral action and relevant mechanism induced by Cur microemulsion and sub-microemulsion in K562 cells were investigated, then the anti-tumor effects of two formulations were evaluated in vivo by mouse H22 transplanted tumor model.METHODS:(1) Trypan blue exclusive staining and Colony assay were used to observe the inhibitory effects of two formulations on the growth of K562; (2) Morphologic change was observed by light microscope; (3) AO/EB fluorescent staining was to observe the apoptosis; (4) FITC-Annexin-V/PI flow cytometry was used to detect apoptotic rate; (5) Western blotting was used to analyze the impact of Cur formulations on cell proliferation apoptosis-related signaling molecules and partner protein; (6) anti-tumor effects of two formulations were evaluated in vitro by mouse H22 transplanted tumor model.RESULTS: (1) Cur microemulsion and sub-microemulsion inhibited K562 cells growth with time- and dose-dependent manners and the inhibitory rates were equilibrant or higher than Cur DMSO solution; (2) Two formulations inhibited K562 CFUs; (3) AO/EB fluorescent staining showed that K562 cell apoptosis was induced by Cur formulations; (4) Cur formulations up-regulated Hsp 70 level, down-regulated P210bcr/abl, Erk1/2, p-Erk, p-AKT, NF-КB, PKC, Bcl-2, Hsp 90 level in K562 cells; (5) Significant anti-tumor effect on H22 transplantation tumor was observed after Cur microemulsion oral administration. After treatment at dose of 50,100,200 mg·kg-1, the inhibitory rates were 33.70%,47.95%,57.79% , respectively; (6) After intraperitoneal injection at dose of 60,120,180mg·kg-1/d, the inhibitory rate of Cur sub-microemulsion was 42.50%,35.57%,45.55%,respectively, which showed significant differences compared with control(P<0.05 or 0.01).
|
PDF Full Text Request