| Doxorubicin(DOX) has the advantages of broad-spectrum antitumor activity, higher therapeutic index, cheaper and easily available. It is widely used in the treatment of breast cancer, bladder cancer and other solid tumors. But the serious cardiac toxicity, bone marrow suppression and high drug resistance rate limit its clinical application. Due to the high selectivity on tumor tissue, less distribution in normal tissue of the large molecular targeted anti-tumor drugs, it become a hot spot for new drug research and development. Objective:In this study, a novel tumor targeted macromolecule drug FA-PEG-hyd-DOX was synthesized. In order to further evaluate the drug like properties of FA-PEG-hyd-DOX, we investigated the preparation, pharmacokinetics and pharmacodynamics of FA-PEG-hyd-DOX. This may lay a foundation for the research and development of FA-PEG-hyd-DOX. Methods:By using folic acid as the tumor targeting ligand, PEG(polyethylene glycol) as macromolecular drug carrier and DOX as active drug, FA-PEG-hyd-DOX was prepared. According to the second appendix XIXC in Chinese Pharmacopoeia(2010 edition, the guidance of active pharmaceutical ingredients and pharmaceutical preparation stability test) and(ICHQ1-Stability testing of new APIs and formulations), the stability of FA-PEG-hyd-DOX raw materials and FA-PEG-hyd-DOX freeze dried powder were investigated. HPLC-DAD method was used to detect the changes of DOX mass percentage of each drug in the experiment of influencing factors, accelerated experiment and long-term experiment. The change of the melting point of the related drugs was measured by the X-4 micro melting point measuring instrument. HPLC-DAD method was used to study the pharmacokinetics of FA-PEG-hyd-DOX in rats. Tumor model was constructed by subcutaneous implantation of Hep G2 cells in nude mice. After the treatment with 0.9% sodium chloride, free DOX(5 mg/kg) and FA-PEG-hyd-DOX(2.5 mg/kg, 5 mg/kg, 10 mg/kg) by tail vein injection, the body weight, tumor size and survival rate of nude mice were monitored. At the end of the administration, the morphological changes in normal tissue and tumor tissues were observed by H&E staining. The distribution of DOX in brain, heart, liver, spleen, kidney, lung and tumor tissues in nude mice was observed by in vivo imaging apparatus and fluorescence microscopy. Results:1. The results of spectroscopy indicated the synthesis product was in accordance with the target product.2. Influence factors experimental results showed that the stability of DOX and FA-PEG-hyd-DOX raw material were closely related to temperature, humidity and light. In the high temperature, high humidity and light environment for 10 days, the quality percentage content of free DOX was 31.95%, 66.36% and 61.27%, respectively. And the melting point was 195 ℃, 194 ℃ and 194 ℃. In the high temperature, high humidity and strong light environment, the content of DOX in FA-PEG-hyd-DOX connection was 2.71%, 3.85% and 3.14%, respectively. The melting point was 169~175 ℃, 170~175 ℃ and 170~175 ℃. In high temperature, high humidity and strong light environment for 10 days, the contents of DOX in the freeze-dried powder of FA-PEG-hyd-DOX was 3.80%, 5.76% and 4.80%. Under the same conditions, the stability of FA-PEG-hyd-DOX raw material was higher than that of DOX. The stability of freeze dried powder of FA-PEG-hyd-DOX in 10% mannitol was significantly higher than that of FA-PEG-hyd-DOX raw material. In accelerating conditions for 6 months, and in the long term experimental for 12 months, the stability of the freeze dried powder of FA-PEG-hyd-DOX in 10% mannitol also showed good stability. The above results indicated that the freeze-dried powder of FA-PEG-hyd-DOX in mannitol could increase the stability of FA-PEG-hyd-DOX raw materials, and it was suitable for saving in sealing, avoiding light, and storing at room temperature.3. After SD rats were administered with 1.75 mg/kg free DOX by tail vein, the half-life and AUC of DOX were 7.0 h, 278 μg·min·L-1, repectively. After SD rats were administered with 1.75 mg/kg FA-PEG-hyd-DOX(1.75 mg DOX /kg, 3.5 mg DOX /kg, 7 mg DOX/kg) by tail vein, the half-life of DOX was 36.4 h, 38.5 hand 36.7 h, respectively. The AUC0-72 was 542 μg·min·L-1, 1093 μg·min·L-1 and 1782 μg·min·L-1, respectively. The experimental results showed that FA-PEG-hyd-DOX increased the circulation time of DOX in SD rats.4. In nude mice of free DOX treated group, the average body weight of nude mice decreased from 20.3 g to 17.0 g, and the weight of the nude mice decreased by 16%, along with poor mental state, dull eyes, emaciated and sluggish. In the end, all of tumor bearing nude mice in the free DOX treated group died, while the other groups were in good condition. This reflects the systemic toxicity of DOX. By giving saline, 5 mg/kg free DOX, 2.5 mg DOX/kg FA-PEG-hyd-DOX, 5 mg DOX/kg FA-PEG-hyd-DOX and 10 mg DOX/kg FA-PEG-hyd-DOX, the tumor volume of each group increased by 1322%, 278%, 510%, 156%, 13%, respectively. Compared with normal saline and free DOX treated group, FA-PEG-hyd-DOX showed good antitumor activity. Compared with the normal saline and DOX, FA-PEG-hyd-DOX prolonged the survival time of tumor bearing nude mice. After tumor bearing nude mice treated with free DOX, the tissue of heart showed a greater change in morphology, and there was a small amount of neutrophil infiltration and necrosis in the tumor. However, in FA-PEG-hyd-DOX treated tumor bearing nude mice, there was no obvious morphological changes in normal tissues, while the tumor tissue showed a large number of inflammatory cells and neutrophil invasion. After nude mice were given free DOX, DOX were distributed in all organs including the brain, heart, liver, spleen, lung, kidney and tumor. After treated with FA-PEG-hyd-DOX, the accumulation of DOX in tumor tissues was significantly increased, and the accumulation of DOX in normal tissues was significantly decreased. The accumulation of DOX in the tumor site was significantly higher in 24 h than that in 12 h after administration of FA-PEG-hyd-DOX. After giving free DOX, the red fluorescence was distributed in the slide of brain, heart, liver, spleen, lung, kidney and other tissues of tumor bearing nude mice. After giving FA-PEG-hyd-DOX, DOX red fluorescence was mostly distributed in the slide of tumor tissue. Conclusion:Compared with free DOX and FA-PEG-hyd-DOX, the freeze dried powder of FA-PEG-hyd-DOX in 10% mannitol showed higher stability. FA-PEG-hyd-DOX increased the circulation time of DOX in SD rats, prolonged the survival time of the tumor bearing nude mice, reduced systemic toxicity of DOX in tumor bearing nude mice, and improved the antitumor activity of DOX. |
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