| Paclitaxel is one of the most important anti-tumor agent from a natural source.Because of the extremely hydrophobic, it is difficult to formulate. Present-day cancerchemotherapy with paclitaxel frequently causes hypersensitivity reactions due to theuse of Cremophor EL?. To alleviate the adverse effect and enhance the efficacy ofanti-cancer growth and anti-metastasis of the injection preparation, the new drugdelivery must be developed for paclitaxel. In this paper, the drug delivery systemcomposed of cytotoxic agent and non-cytotoxic agent were designed and prepared.The new formulation of preparation for injection contains paclitaxel, hyaluronan,surfactant etc. Its stability, drug release property and anti-tumor activities in vitro andin vivo were investigated, and were associated with the composition of preparation forinjection to establish the composition-activity relationship. In the dissertation, thework is mainly focused on:1. The formulation of preparation for injection is developed. It is mainly composed ofactive agent-paclitaxel, carrier-hyaluronan and surfactant, and manufactured byfreezing-dried method. To meet the requirment of fast dissolution rate for clinicalapplication, the propylene glycerin, mannitol or dextrose were added. Thephysicochemical properties of preparation for injection were determined, includingdissolution time, compatibility of components and stability of preparations. Thestudies showed that the formulation is feasibility and stable at the ambienttemperature. It can be dissolved in less than 3mins and the solution is clearancewithout paclitaxel precipitation from 48hrs to 72hrs, which demonstrated thepreparation is stable in aqueous solution,too.2. To investigate the effect of components on the stability of formulation, theinteraction among components was studied by fluorescence method, surface tension,viscosity determination and circular dichroism etc. The results showed that theinteraction between HA and non-ionic surfactant is very weak. The solubility ofsurfactant on the hydrophobic drug was not affected by the addition of HA. But theeffect of saccharide or ployols on the solubility correlated with the concentration ofsurfactant. If NaCl was added to the solution of HA/surfactant, the solubility behaviorof surfactant will be enhanced. However, the behavior of SDS (with the anion charge) hasbeen influenced by the addition of HA, through the hydrogen bond or hydrophilicforce. The HA/SDS system has the lower cmc than the pure SDS solution from 0.2%to 0.16%. The results above mentioned showed that the interaction amongcomponents has no effect or very weak on the stability of preparation.3. The in vitro inhibition activity of paclitaxel-HA against Hela cell and non small celllung cancer cell lines was investigated. The IC50 data indicated that the paclitaxel andHA showed better effects on the tumor cell lines than that of injection of paclitaxel.Although HA can protect cell from being destroyed, the number of cells entering thecell cycle were significantly decreased in certain concentration of HA. The synergiceffect of paclitaxel and HA would inhibit the tumor cell growth in vitro. The releaserate of paclitaxel from preparation for injection associated with the ratio HA andpaclitaxel and displayed better anti-tumor activities. Morphological studiesdemonstrated that the new preparation killed the tumor cells by inducing apoptosis ofcell.4. The inhibition activities in vivo of paclitaxel-HA against murine U14 cervix cancer,S180 melanoma and Lewis lung cancer were investigated using paclitaxel-HA as themodel drug. At a dose level of 5 mg·kg-1, the increased life span of U14 tumor-bearingmice reached 135.5%, the tumor inhibition values for S180 was 42.7% andanti-metastasis rate of Lewis lung cancer was 36.3% , which stated the high activity ofthis new preparation on the three tumor models. Compared with taxol, the efficacy ofthe new formulation was higher, which might result from the syneric effect of HAwith paclitaxel. Judging from the body weight changes of experiment mice, the newpreparation seemed to be less toxic than formulated paclitaxel. The reduced toxicitywas ascribed to the controlled release of taxol from HA or the targeted delivery ofpaclitaxel by HA ,through the specific binding between HA and CD44 and/orRHAMM receptors. The data of pharmacokinetics showed that the retention time ofnew preparation in vivo was prolonged and the plasma level of drug was increased.This results explained the reason of efficacy of paclitaxel was enhanced.5. The variation of proteins expression in mice serum was analyzed by usingtwo-dimensional polyacrylamide after the mice were injected for HA, paclitaxel andHA/paclitaxel respectively. Some difference proteins expression was found, and someof construction and function were determined with the help of mice proteins database.The results showed that the expression of vitamin D binding proteins(DBP) wasobviously up-regulated by injecting the HA-paclitaxel composition preparationcompared with the other samples. DBP is the activator of macrophage. Becausemacrophage activation is the first step in the inflammatory primed immune cascade,this would lead to activiation of the immune system in cancer patients. Hence, theanti-tumor efficacy of HA-paclitaxel was significantly increased.6. The comprehesive analysis of HA-paclitaxel freeze-drying preparation on its anti-tumor activity in vitro and in vivo, drug release property and pharmacokineticscharacteristics suggest that the lyophilized preparation has great potential applicationto cancer and immune defect disease and deserved further attention. |
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