Preparation And Quality Evaluation Of AMHDL

Arginine deiminase（ADI）has shown cytotoxic activities against different types of tumor cells,which has attracted significant attention from researchers for ADI potential use in cancer therapy.ADI can kill cancer cells by depleting arginine.ADI may cure leukemia,hepatocellular carcinoma,melanoma,small cell lung cancer and breast cancer.Usually,arginine depletion aggravates weight loss in cancer patients,but one research article reported that ADI-PEG2000 can treat different types of cancers without influencing whole body protein metabolism or fractional protein synthesis in most tissues.However,as protein drug,ADI has shortcomings such as instability,short half-life and low bioavailability.In this experiment,AMHDL was prepared.We hoped that AMHDL could increase the avtivity of ADI,enhance in vitro and in vivo stability and half life and improve the pharmacokinetic behavior of ADI.The first part,preparation and physicochemical properties of AMHDL.Objective:To prepare AMHDL and determine AMHDL physicochemical parameters.Methods:AMHDL was prepared and its physicochemical parameters were investigated.Results:HA-g-mPEG was synthesized.AMHDL was prepared successfully.The average particle size of AMHDL was 197.33 nm,the average Zeta potential was-8.79 mV,the average conductivity was 8.54 ms·cm^-1,the optimum temperature was 37℃,the optimum pH was 6.5.The K_m of AMHDL was 0.75 mmol·L^-1 and V_max was13.51μmol·min·L^-1,respectively.Conclusion:AMHDL was prepared and had suitable physicochemical parameters.The second part,the stability of AMHDL in vitro.Objective:To investigate the temperature stability（-20℃,55℃and 4℃）,pH stability（pH 5.0⁹.0）,antitryptase stability and plasma stability of AMHDL.Methods:The activity of AMHDL and ADI were measured in different conditions.Results:The activity of AMHDL was higher than that of ADI in all conditions.Conclusion:AMHDL can improve the stability of ADI in different conditions.The third part,the fluorescence spectrum experiment of AMHDL.Objective:To investigate mechanism of AMHDL activity improvement and stability enhancement.Methods:Fluorescence spectroscopy was used to investigate the structure and activity relationship of ADI,the interaction between ADI and B-AMHDL membrane,and the fluorescence intensity changes of AMHDL and ADI after heating.Results:The fluorescence intensity of AMHDL was higher than that of ADI.The fluorescence intensity of ADI was decreased after incubation with B-AMHDL.The fluorescence intensity of both AMHDL and ADI decreased after heating.Compared with ADI,AMHDL fluorescence intensity decreased less.Conclusion:AMHDL can enhance the activity and stability of ADI.The reason might be due to the interaction between ADI and B-AMHDL membranes and AMHDL can protect the three-dimensional structure of ADI.The forth part,study on the pharmacokinetics of AMHDL.Objective:To investigate the pharmacokinetic behavior of AMHDL in rats.Methods:Twelve male SD rates were intravenous administration with same dose of AMHDL.Blood samples were collected and the plasma activities of ADI were determined.Subsequently,main pharmacokinetic parameters were calculated by DAS software.Results:Non-model analysis showed that AUC_{（0-96 h）},C_max,T_max and MRT_{（0-96 h）}of AMHDL were 3.74,1.53,1.10 and3.14 times higher than ADI,respectively.The Cl of ADI was 7.00 times higher than AMHDL.The relative bioavailability of AMHDL was 374.06%of higher than ADI.The results of compartment model analysis were consistent with those of non-compartment model analysis.Both AMHDL and ADI conformed to the three compartment model.The AUC and C_max90%confidence interval of logarithmic ratio of ADI and AMHDL was73.2%⁷5.8%and 79.3%⁹3.5%,repectively.T_maxax was tested by Wilcoxon nonparametric test（P>0.05）.Conclusion:AMHDL can improve the bioavailability of ADI.AMHDL and ADI are not bioequivalent.