Studies On PH Sensitive Docetaxel-Pluronic P123 Conjugate Micelles

With the increased incidence and mortality, tumors especially malignant tumors are threatening human health. Hence, developing the clinical application of the anticancer drugs to improve their efficiency has become a hot topic in pharmaceuticals industry.The taxanes ((paclitaxel, docetaxel, and cabazitaxel) represent a novel class of antineoplastic agents that used widely in treating a broad range of malignancies including lung cancer, breast cancer, gastric cancer, ovarian cancer, and other carcinomas. However, when used clinically, these conventional taxane agents are often associated with serious side effects, which hindered their application to a great extent. With poor solubility, the conventional taxane formulations often contains high concentration of Cremophor(?) EL and Tween-80 as the solubilizers, which often caused the acute hypersensitivity infusion reactions and peripheral neuropathy. In addition, as small cytotoxicity agents, taxanes have poor distribution and selectivity for tumors that there are lots of drugs detained in other tissues, which restricts the efficiency of the taxanes and induces side effects. Therefore, it is critical to develop new formulations in improving the solubility and biodistribution of the taxanes.The year of 2007 has seen the prosperity of polymer micelles in tanxne agents delivery that two paclitaxel-loaded micelles were stepped into market for metastatic or recurrent breast cancer and non-small cell lung cancer:Genexol(?)-PM micelles by Samyang Pharma in South Korea and Nanoxel micelles by Dabur Pharma in India. Polymer micelles could be used as the delivery system of taxane agents with follow advantages:for one thing, the tanxnes could be encapsulated into hydrophobic core of the micelles and sheltered by the hydrophilic corona to improving their solubility and safety; on the other hand, designing nano-sized micelles could target and accumulate to the tumors by EPR effect, which is benefited to improving antitumor efficiency and reduce their side effect. Herein, Genexol(?)-PM and Nanoxel showed better solubility and improving safety and tumor-target issue.Drug-polymer conjugate miclle is an special drug delivery system that based on polymer materials. Compared with micelles formed by physical encapsulation, the drug is bond to the polymer via covalent bonds in conjugate miclles, which plays an important role in controlling drug release:in the process of transport, the drug hold tightly with the carriers because of the strong chemical bond that prevent the premature release of drug and make the conjugates stable; while in the tumor tissues, the linker can be designed to be sensitively broken to release drug and acheive a better curative effect. Recently, paclitaxel-bonded conjugates OpaxioTM had been designated by the FDA for the treatment of glioblastoma.The specific breakage of the linkers at tumor tissues will have an vital impact on conjugate micelles. In this regard, the lower pH environment at tumor tissues can be a strategy to resolve the situation and the hydrazone bond can be employed as the linkers. In 2013, the epirubicin-loaded conjugates NC-6300 had been stimulated into clinical stage.Based on advantages of the polymer micelles in taxane agent delivery and the drug controlled release ability of the pH sensitive drug-polymer conjugate micelles, we want to design and prepare a novel drug-polymer conjugate micelles:the more active taxane agents-docetaxel (DTX,2- to 4-fold more potent than paclitaxel) as the model drug and the Pluronic P123 with better biocompatibility as the carrier. Considering the lower pH value in tumor tissues and intracellular organelles, we employed the hydrazone bond as the linker to prepare the DTX-Pluronic P123 conjugate micelles with the controllable drug-release ability. More than that, in order to evaluate the influence of the hydrazone bond number, the single or double-hydrazone bond were employed to prepared the DTX-Pluronic P123 conjugate micelles.The details of the preparation:First, design the single-hydrazone bond contained conjugate P123-S-DTX and the the double-hydrazone bond contained conjugate P123-L-DTX. Second, the conjugate micelles were obtained with the dialysis method: the P123-S-DTX-CM micelles were obtained from the P123-S-DTX and the P123-L-DTX-CM micelles from the P123-L-DTX.The research contained two main parts:(1) the preparation and the physicochemical properties of the P123-S-DTX-CM and the P123-L-DTX-CM; (2) the evaluation of the P123-S-DTX-CM and the P123-L-DTX-CM in vitro and in vivo. The main methods and results were as follows:1. The preparation and the physicochemical properties of the P123-S-DTX-CM and the P123-L-DTX-CMThe research prepared two pH sensitive conjugate micelles with chemical methods and their preliminary physicochemical properties such as size and morphology were evaluated. Firstly, the LEV and ADH were employed to synthesize the derivative of DTX contained hydrazone bond (DTX-L-A). The Pluronic P123 were also treated to achieve two derivative of P123 that contained carboxyl group or carbonyl group respectively. The two pH sensitive DTX-Pluronic P123 conjugate micelles contained single or double hydrazone bonds as the linkers respectively were prepared by the reaction between DTX-L-A and the two derivative of P123:the P123-S-DTX conjugates and the P123-L-DTX conjugates. After the synthesis of the conjugates, the research prepared the two pH sensitive DTX-Pluronic P123 conjugate micelles by the dialysis method:the P123-S-DTX-CM and the P123-L-DTX-CM. As evaluated, both of the P123-S-DTX conjugate micelles and P123-L-DTX conjugate micelles (with the high drug loading efficiency as 11.20±0.32% and 10.37±0.36% respectively) showed good morphology with uniform size of 131.6±19.2 nm and 109.1±9.8 nm respectively.2. The in vitro and in vivo evaluation of the P123-S-DTX-CM and the P123-L-DTX-CMThe research evaluated t the P123-S-DTX-CM and the P123-L-DTX-CM in vitro and in vivo. As determined, the critical aggregation concentration value (CAC) of P123-S-DTX and P13-L-DTX were 28.357 μg/mL and 28.377 μg/mL respectively, lower thab the CAC of Pluronic P123 and the P123-S-DTX-CM and the P123-L-DTX-CM were proved to be more stable against dilution and plasma by the corresponding trials.As evaluated the total release extents of the drug in mediums with different pH values (7.4,6.5,5.0), both of the two conjugate micelles showed well release extents above 80% at pH 5.0, higher than their release extents at pH 7.4 (13.39±2.18% and 38.10±2.06% respectively). In addition, at pH 6.5, the release extent of P123-L-DTX-CM was 75.08±3.16%, higher than the P123-S-DTX-CM (21.68±0.73%), proved that the employ of the linker of double hydrazone bonds could elevate the drug responding release pH value to some extent.In the in vitro study of the conjugate micelles, the P123-S-DTX-CM and the P123-L-DTX-CM showed an concentration dependent cytotoxicity. The IC50 value of the conjugate micelles were 21.74±3.27 μg/mL and 12.65±3.18 μg/mL. In addition, the proliferation inhibition activity of P123-L-DTX-CM were little superior to that of the P123-S-DTX-CM. In vivo antitumor efficacy of the P123-S-DTX-CM and the P123-L-DTX-CM were studied on B16 melanoma-bearing Kunming mice. Among the treatment in 3 weeks, both of t the P123-S-DTX-CM and the P123-L-DTX-CM could control tumor volume of the mice at the range around 1000 mm3 (898.31±128.67 mm3 and 1099.31±86.03 mm3 respectively), better than the free DTX (2063.45±333.26mm3). More than that, Among the process of treatment, the relative body weight of the mice treated by free DTX showed a significant loss, while treated by both of t the P123-S-DTX-CM and the P123-L-DTX-CM exhibited little weight change, similar with the mice treated by saline. These results showed that the two prepared pH sensitive DTX-Pluronic P123 conjugate micelles had better tumor suppression ability and improving safety.