| Lung cancer is a common malignant tumor, which is a serious threat to human health and life. By2025, Amounts of Chinese lung cancer will be the largest in the world. Lung cancer consisits of two categories, non-small cell lung cancer (NSCLC) and small cell lung cancer. About85%of lung cancer belongs to a non-small cell carcinoma. The treatment of lung cancer and the selection of drugs are becoming increasingly diverse. The drugs which are high efficiency, low toxicity and targeting effect, have been attracted more and more attention.Bexarotene (Targretin(?)) is a synthetic retinoid analog. Its oral soft capsule and gels for external use were approved for the treatment of skin T-cell lymphoma by FDA in2000. Now, the phase Ⅲ clinical trials has been carried out in abroad, mainly used for the treatment of non-small cell lung cancer, breast cancer and psoriasis. Bexarotene showed a significant effect in the treatment of lung cancer, but it belonged to the Biopharmaceutics Classification System (BCS) Class Ⅱ compounds. Its poor solubility and low bioavailability greatly limit the clinical application. In order to solve these problems, Bexarotene was studied to provided the basis for applying Bexarotene to the treatment of lung cancer. Bexarotene, the model drug in this topic research, was made into nanosuspensions by precipitation-combined microfluidization method. The nanodrug delivery systems,method for in vitro samples and in vivo samples analysis, pharmaceutics properties, pharmacokinetics, organs distribution characters of Bexarotene nanosuspensions, and the antitumor activity in vivo and in vitro was studied.These provided theoretical and experimental basis for the development of targeted agents for lung cancer.The main contents of this study included:1. Preformulation study of Bexarotene nanosuspensionsAccording to the preformulation study, Bexarotene samples was scanned by using UV spectrophotometry, then detection wavelength and the determine method by HPLC were established. Methodological study results show that Bexarotene has a good linear relationship in the concentration range0.05～40.0μg·mL-1. And within-day precision, between-day precision, stability, reproducibility and recoveries were corresponded to technology requirement.2. Process and formulation optimization of Bexarotene nanosuspensionsBexarotene nanosuspensions formulation was optimized and screened by orthogonal design method, and zeta-potential was slected as the investigate index. The amount and type of stabilizers were investigated. The experiment revealed that, the impact of various factors on the of zeta-potential was:lecithin＞F68＞PVP K30. Nanosuspension was thermodynamically unstable systems. In order to improve the stability of nanosuspensions, nanosuspensions were prepared into lyophilized powder by using the freeze-drying method. In the lyophilization process, to ensure good physical and chemical stability of the dispersion, the freeze-drying process was investigated. By determining eutectic point and screening lyoprotectants, the freeze-drying process was identified:prepared nanosuspensions by precipitation-combined microfluidization method were added5%mannitol as lyoprotectant, and set into freeze-drying machine for24h. Then freeze-dried powder (NC-Bexarotene) was obtained.4. The study of pharmacokinetic in ratsThis study established a HPLC method for detection of Bexarotene, and the pharmacokinetics in rats was studied after oral administration and intravenous administration, and the effect of Nano-preparation in improving Pharmacokinetics was discussed.The pharmacokinetics in rats was studied for oral and intravenous administration, respectively. According to the pharmacokinetic parameter of oral administration, the control group of Bexarotene and the group of NC-Bexarotene fitted the two compartment model. The T1/2of NC-Bexarotene is significant longer than the control group, and the AUC0-∞almost2times of the control group. Drug release systems of NC-Bexarotene remained relatively durable and stable, mainly because of its good biological adhesion characteristics, that prolong the residence time of pharmaceutical in gastrointestinal tract and greatly improved the drug bioavailability. For intravenous administration, the NC-Bexarotene had a rapid dissolution in blood circulation. NC-Bexarotene using injection water as solvent, avoided the stimulation and the damage to the body from organic solvent, had a better compliance than the control group.5. The study of the tissue distribution in mice.In order to investigate the effect of nano preparation on drug distribution in the body, the tissue distribution in mice of NC-Bexarotene was studied. Compared with Bexarotene-Sol, NC-Bexarotene has significantly reduced the initial concentration of drug, and made AUC more stable and durable. MRT increased from3.82h to16.58h, AUC boosted to18.65h·μg·g-1from12.68h·μg·g-1. Compared with Bexarotene-Sol, either through oral administration or intravenous administration, NC-Bexarotene could reduce the highest drug concentration in heart and kidney, and avoid the high concentration of drug adverse effects on the heart and kidney function. Because NC-Bexarotene obviously prolonged the drugs in vivo. The MRT in heart increased from1.96h to3.15h(oral administration), from1.73h to2.90h(intravenous administration), but the percentage of distribution greatly decreased than the control group. According to the results of liver tissue distribution, in the oral administration MRT of NC-Bexarotene in liver had little increase, but the AUC and the percentage of distribution were not changed obviously. In the intravenous administration, MRT, AUC and percentage of distribution all had a little bit increased. After oral and injected with NC-Bexarotene, drug distribution in the spleen was obvious increase, and AUC and MRT was obvious increase. r e is1.50(oral administration) and1.25(intravenous administration), which indicated that the liver targeting property was obvious. In the lung tissue, the distribution of drugs in the lungs was almost similar to the distribution of spleen. AUC, AUQ and MRT all increased. In the oral administration AUC increased from48.25h·μg·g-1to79.93h·μg·g-1, MRT prolong from1.96h to3.80h. In the intravenous administration, AUC increased from32.85h·μg·g-1to54.94h·μg·g-1, MRT prolong from2.69hto6.34h. rCe=1.7(lung> spleen＞liver). And according to the relative percentage distribution figure, preparation group was significantly improved compared to the solution group. Compared with the solution group, the drug concentration of NC-Bexarotene group was obviously decreased at any time in renal. Therefore NC-Bexarotene could decrease the renal toxicity from Bexarotene.6. Pharmacodynamics study of Bexarotene nanosuspensions on the cell of Lung adenocarcinoma A549and nude mice TumorFrom the result of experiment in cell and nude mice Tumor, NC-Bexarotene and Bexarotene can inhibit the proliferation of cell of Lung adenocarcinoma A549. NC-Bexarotene can retardant G1in cell cycle and induce the cell of Lung adenocarcinoma A549death. From result of content determination of Lactate dehydrogenase (LDH) and DNA fragmentation analysis proved NS-Bexarotene has no toxic effect. During the period of drug delivery, nude Tumor mice group has a significant body weight decrease, and NC-Bexarotene group has lowest death rate. NC-Bexarotene and Bexarotene both can suppress the grow of transplantation tumor, but not much significant different from the control group; NC-Bexarotene can suppress the transplantation tumor volume after administration at13th and17th day. The investigation of the volume of transplantation tumor after autopsy showed that NC-Bexarotene has the lowest volume, But there was no significant difference between groups. From the result of study, the extension of drug administration time is needed in order to get better therapeutic effect. However, because of the high invasiveness of the cell of Lung adenocarcinoma A549, the dead rate of nude mice has increase after drug administration, especially the tumor-burdened group of nude mice model. Compared with Bexarotene, NC-Bexarotene can better inhibit Cell proliferation, induce apoptosis, and case cell cycle arrest, and tumor-burdened rat mortality is low and without obvious side effects.It was the first time to build Nano-suspension agent drug release system of Bexarotene, and it was also the first report on the pharmacokinetics and tissue distribution of Bexarotene nanosuspensions after oral and intravenous administration, and the first Pharmacodynamics study of Bexarotene nanosuspensions on the cell of Lung adenocarcinoma A549and nude mice Tumor. This paper enriched the research of passive targeting of nano-drug release system, and was helpful for the development and application of oral and intravenous formulations of nanosuspensions of Bexarotene. It has important significance on the clinical development of anti-cancer drugs of Bexarotene. |
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