Investigation On The Tumor Cell Active Targeting Characteristics Of Mitoxantrone-Insulin Conjugate

Coupling of the ligand with its receptor has the characteristics of high specificity, selectivity and affinity. Since recent researches showed higher expression and better affinity of insulin receptors on the cytomembrane of many kinds of tumor cells compared with normal cells, using insulin as the vector of the active targeting drug delivery system was expected to lead the anticancer drug into tumor cells to enhance anticancer effect and reduce serious side effects of the drug. The fact that insulin maintained receptor-coupling competence after it was conjugated with some compounds through covalent bond makes the expectation more feasible.Mitoxantrone (Mit), a broad spectrum anti cancer drug, was used as the model drug, and hepatocellular carcinoma was used as targeting objective in this investigation. The tumor cell active targeting characteristics of mitoxantrone-insulin conjugate (Mit-Ins) were investigated.The direct conjugation of insulin with mitoxantrone (IM) and the conjugation through a spacer of insulin with mitoxantrone (ISM) were prepared to obtain two kinds of mitoxantrone-insulin conjugates. The intermediate and final products were characterized by melting point, UV, IR, ~1H NMR, MS and HPLC. The molar ratio of mitoxantrone to insulin was 0.78 in IM, and that in ISM was 1.53. The ratio of free Mit content to total Mit content in IM was 1.1%, and that in ISM was 4.7%.Because of its higher drug conjugation rate and good solubility, ISM was select to carry out further investigation. The lyophilized powder of solution type was prepared by using 3% mannitose as the supporting agent with ISM content of 1 mg per bottle, whose comprehensive properties were evaluated. The preparation, having hygroscopic property, should keep airtight storage. Stored for 3 months, the preparation had a good stability at 4℃, while its insulin activity diminished at 25 ℃, which means it should be stored in the lower temperature.In vitro stability trials showed Mit-Ins were stable in buffers with different pH value (2-8) at 37°C within 120 h (less than 3% of free Mit released), and were also stable in mouse plasma within 48 h (less than 2% of free Mit released).In vivo study on tumor-bearing mice (H22 hepatocellular carcinoma) showed that, compared with Mit, the conjugates had better tumor-targeting efficiency. Pharmacokinetic analysis in plasma showed that AUCMit-ins was 7.70 times of AUCMit, ti/2B of Mit-Ins was 3.74 times of that of Mit, which means slower drug clearance in blood of Mit-Ins compared with Mit. In tumor, AUCMit-ins was 1.67 times of AUCMit; t^Mit-ins was 1.75 times of ti/2Mit; CLsMit-ins was 0.6 times of CLsMit; CeMit-ins was 2.00 times of CeMit- These results mean the higher initial drug concentration and lower clearance in tumor of Mit-Ins compared with Mit, which lead to durative higher drug concentration of Mit-Ins compared with Mit. Good tumor targeting effect of the conjugate was showed by the parameters as follows: Re=1.67, Rre=5.65-81.00, R%t =12.64. In organs of heart, liver, spleen, lung and kidney, the ratios of AUCMit-ins to AUCMit were 0.08, 0.30, 0.12, 0.02 and 0.23 respectively; the ratios of CLsMit-ins to CLsMit were 12.42, 3.38, 8.00, 43.00 and 4.43 respectively; Re were from 0.02 to 0.30; Ce were 0.26, 1.26, 0.46, 0.34 and 1.09 respectively; R%twere 0.37, 1.50, 0.55, 0.06 and 1.05 respectively. On the whole, these parameters reflected lessening of drug distributions of the conjugate compared with free drug in the organs. Although the initial drug concentrations of Mit-Ins and Mit were similar in the two organs, the clearance of Mit-Ins were faster than Mit in the liver and kidney, which lessened the poisonous side effect of the conjugate to the organs compared with the free drug. In conclusion, the conjugate, compared with the free drug, had longer Ua in plasma, good targeting effect to the tumor and lessened drug distribution in normal organs.MTT method was used to inspect tumor cell suppression and normal cell poisonous effect of the conjugate and free drug. The results showed that the higher drug concentration and the longer drug action period the better SMMC-7721 hepatocarcinoma cells suppression effect; the tumor cell suppression effect of Mit-Ins was doubled compared with Mit before drug action period of 12 h; from 24 h to 72 h, the suppression effect of the conjugate and free drug had no distinctdifference. It meant the conjugate entered into tumor cells easily, and kept the activity to kill the tumor cells. The results also showed that the higher drug concentration and the longer drug action period the severer poisonous effect to Chang normal liver cells; the conjugate had milder normal cell poisonous effect than free drug, especially in higher drug concentration.Summarily, the conjugate prepared, with good stability in vitro, had better targeting effect to the tumor, lessened poisonous side effect to the organs, enhanced tumor cell suppression and lessened poisonous effect to normal cells compared with the free drug. The results suggest the feasibility to promote the curative effect and to lessen side effect in cancer chemotherapy by using insulin as the vector of anti-cancer drugs, which means the objective of this investigation was obtained.