The Study On Paclitaxel-PLGA Microsphere

Paclitaxel, an anti-tumor drug isolated from Pacific Ocean Yew bark, has been demonstrated with significant therapeutic effects on many kind of stereo-tumors, including breast cancer, advanced ovarian cancer, lung cancer, cerebral and cervical tumors, acute leukemia and hormone independent prostatic carcmorma. However, Paclitaxel was proved very hard to dissolve in water and other commonly used drug solvents. As a substitution, Cremorphor EL was used as the solvent of Paclitaxel in medical injections, although many side effects, such as severe allergy, have been discovered in clinincal applications. To solve this problem, researchers have manipulated Paclitaxel into different drug delivery systems, such as liposomes, microemulsions, microspheres and nanoparticles. These modifications have been test effectively to avoid the side effects of Cremophor EL and diminish toxicities of Paclitaxel, as well as extending drug effects and improving the drug bioefficiency.As a new kind of sustained-release drug delivery systems, microspheres have been widely investigated. It exerts sustained-release effects through the specific skeleton material, PLGA, which could biodegrade into harmless water and CO₂. It was used as an adjuvant approved by FDA. So, it is rational to manipulate Paclitaxel into microspheres for cancer chemotherapy. The advantages were obvious, including lower toxicity, elongated and stronger effects, higher efficiency and convenience.In present study, Paclitaxel-PLGA microspheres were prepared with different molecular weights (MW) of PLGA and ratios of LA/GA. Then we carried out a serial studies, including active content determination, evaluation method for in vitro releasing characteristics, optimization of microsphere recipes and preparation techniques, in vitro releasing method and mechanism, stability and in vivo pharmacodynamic evaluation, blood toxicity, biocompatibility and Pharmacokinetics.1. Optimization of Paclitaxel-PLGA microsphere recipes and preparation techniquesPaclitaxel-PLGA microsphere was prepared with modified single emulsion solvent evaporation method. Then the recipes and preparation techniques were optimized based on single factor analysis statistical results and orthogonal design. The evaluation parameters were microsphere morphological form, diameter, distribution, drug loadings, stirring rate and in vitro releasing curve. The results showed that the microspheres produced with the optimized recipes and preparation technique displayed smooth surface, consistent diameters, mean diameter less than 10μm, encapsulation efficiency more than 80%, and in vitro releasing period over 20-30 days.2. Establishment of evaluation method for in vitro releasing effect and stability studyThe in vitro releasing conditions of microspheres, such as different releasing medium, container and antibiotics, were investigated. Then we established a new fast-releasing method to enhance releasing and to shorten the evaluation period. To qualify the new method, we performed point-to-point relevant curve with data from sustained-releasing method and fast-releasing method. The results showed that the new fast-releasing method was similar to sustained-releasing method. So it is reasonable to use data from fast-releasing method to mimic that of sustained-releasing method.The stability of microsphere was evaluated with parameters, such as morphological change, drug contents and in vitro releasing behavior, under different conditions including high temperature, high moisture, light exposure and ⁶⁰Co irradiation sterilization. The results showed that the microspheres were obviously instable under aforementioned conditions except ⁶⁰Co irradiation sterilization.3. The releasing mechanism studyThe releasing mechanism was investigated with microspheres degradation process evaluated with agravic method and GPC determination of PLGA. Different statistical models, including Higuchi, zero order, first order equation, and Ritger-Peppas exponential model, were applied to analyze microsphere solubility and inflation level, which were evaluated with diameter variation at different time points. The results showed that the releasing curves of PLGA14000 and PLGA26000 matched with Higuchi equation, which suggested the diffusion mechanism play a key role in microsphere releasing.4. The in vivo pharmacodynamic effects studyWe determined the inhibition effects of Paclitaxel-PLGA microsphere on S-180 tumor developed in mice and PC-3m tumor developed in naked mice. For comparison, we also determined the in vitro inhibition effects of Paclitaxel-PLGA microsphere on MDA-MB-435 cell line, which was derived from breast cancer. The results from both of in vivo and in vitro experiments suggested that, the inhibition effects of Paclitaxel-PLGA microsphere were dose-dependent and higher than that of Paclitaxel injection.5. Pharmacokinetics studyThe pharmacokineitics of Paclitaxel-PLGA microsphere was accessed with rat model. The results showed that Paclitaxel-PLGA microsphere afeter s.c. matched with 2-compartment model, and the Cmax was 68.63ng/ml, Tmax was 3.28d, MRT was 17.32d. In the studies of in vivo- in vitro correlation, in vivo release rate was calculated by measuring the amount of drug remaining in microspheres excised from the injection site. The results showed that a good correlation was obtained between in vivo releasing percent and in vitro releasing percent.6. The blood toxicity and biocompatibility studyThe blood toxicity of Paclitaxel-PLGA microsphere was evaluated with number of murine white blood cells. The results showed that the white blood cell level decreased slightly after treatment and then kept steady afterwards. As a comparison, Paclitaxel injection resulted significant decrease of white blood cell level in the first 2 weeks after treatment. So it implied that Paclitaxel-PLGA microsphere might have less blood toxicity than that of Paclitaxel injection.The biocompatibility of Paclitaxel-PLGA microsphere was evaluated with pathological examination of mice skin after treatment. The results showed that Paclitaxel-PLGA microsphere treatment induced slight inflammation response in mice skin, but no sign of infiltration liquid accumulation was found, and neither did of obvious angiogenesis and fiber hyperplasia. So it implied that Paclitaxel-PLGA microsphere possessed good biological compatibility.The patent, "PLGA microspheres of Paclitaxel and its derivatives intended for local injection " , was in process of application. The application code was 200420029829.0.