Study On Nano - Micelles Containing Anti - Tumor Compound Zeylenone

Zeylenone was a kind of polyoxygenated cyclohexenes, which was firstly isolated from Uvaria grandiflora Roxb by institute of medicinal plant development, Chinese Academy of Medical Sciences & Peking Union Medical College. Previous results showed that zeylenone displayed strong cytotoxicity to cultured cancer celles and showed equal potency against sensitive and multidrug resistant cell lines. Study on structure-actively relationship suggested that the two benzoic ether groups in zeylenone were the activity groups and the cytotoxicity would disappear if the benzoic ether was hydrolyzed. These earlier results indicated that zeylenone had the potential to be developed as a new kind anticancer drug. However, the poor aqueous solubility and stability remain a challedge for the formulation development. As a result, the main objective of this study was to prepare mPEG-PLGA nano-micelles to improve the solubility and stability of zeylenone with also a view to achieving sustained release.Firstly, an HPLC method was developed to determine zeylenone, and the physical and chemical properties of zeylenone were systemically evaluated. The results showed the purity of zeylenone was 99.7%, melting point was 160.37℃; solubility in water was only 11.3μg/ml and log P was 2.97. These indicated zeylenone was insoluble in water but soluble in organic solvents. The half life time of degradation of zeylenone in PBS (pH 7.0) was 17.3 h, and the most stable condition was at pH 4.0.Secondly, mPEG-PLGA loaded zeylenone nano-micelles were prepared by solvent evaporation method. Both DSC and 1H NMR methods were used to confirm the formation of micelles and the properties of micelles were characterized by several methods. The solubility of drug loaded in nano-micelles were over 2 mg/mg and easily redissloved in water with the drug loading content and encapsulate efficiency being 2.55% and 97.12%, respectively. The average size diameter was approximaetly 35 nm with high degree of monodispersability. The drug loaded micelles effectively prevented zeylenone from hydrolysis and released zeylenone with nearly zero-order pattern in vitro.Thirdly, a highly sensitive LC-MS/MS method was developed for the determination of zeylenone in rat plasma and lung tissues using emodin as internal standard and NaF-BNPP as esterase inhibitors. The analytes were extracted with liquid-liquid extraction by ethyl acetate. The absolute recovery of both plasma and lung tissues were over 85%, indicating that NaF-BNPP had good inhibiting effect on the enzymatic hydrolysis of zeylenone. The assays were linear over the concentration ranges of 2.68-1340 ng/ml in plasma and 1.34-670 ng/ml in lung tissues with limits of quantification of 2.68 and 1.34 ng/ml, respectively. The accuracy was between 93.5-111.9% and the precision was 1.56-16.60%, respectively.Moreover, the pharmacokinetics of zeylenone as free drug or loaded in micelles via intravenous and intratracheal administrations were determined by the validated LC-MS/MS methods. The results showed that the micelles prolonged the effective time of zeylenone compared with free drug after intravenous injection, indicating sustained release of zeylenone in micelles. Micelles also decreased toxicity because the concentration of zeylenone in plasma was lower than free drug during the 90 min after injection. The pulmonary bioavailability of free drug and micelles were 15.8% and 11.5%, respectively. The lung content of free drug was higher than that in micelles.Finally, the toxicity to the lung after intratracheal administration of free drug and micelles were evaluated by histopathological section. The micelles group did not show obvious change but some light pathological changes emerged in free drug group, such as slight hurt in mucosa of bronchiolar epithelium and occasional inflammatory cells infiltration. Intratracheal delivered zeylenone either loaded in miclles or as free drug did not lead to serious pulmonary toxicity.