Expression, Purification And Pharmacodynamic Studies Of Recombinant Human Endostatin Expressed In Pichia Pastoris

Endostatin, a specific angiogensis inhibitor, was isolated as a 20 kD peptide from mouse endothelioma by O' Reilly in 1997. Amino acid sequence analysis of N-terminal revealed that Endostatin is the c-terminal fragment of collagen XIII generated by hydrolyzation. Endostain retards neovascularization, tumor growth and/or metastasis by inhibition of endothelial cell proliferation and migration.In the study, we aim to optimize the expression of recombinant human Endostatin (rhEndostatin) and to examine the pharmacodynamics of rhEndostain in tumor treatment. We initially compared the expression efficiencies of rhEndostatin in yeast by adding methanol or methanol : glycine mixture (2:1) into standard fermentation condition. The results showed that OD₆₀₀was 795.8 for methanol vs. 799.1 for methanol: glycine, and the expression of rhEndostatin was 71.0 mg/L for methanol vs. 80.1mg/L for methanol:glycine. The purification of rhEndostain was carried out by concentration, dialysis and step-wise HPLC separation using SP Sepharose FF ion exchange column, resulting in highly purified final product, as determined by HPLC (97%), with 87% recovery.The biological activity of rhEndostatin was evaluated in vitro and in vivo. rhEndostatin inhibited proliferation of primary endothelial cells, especially primary HUVEC (EC50 = 160.8 ng/ml), in a dose-dependent manner, but not tumor cell lines, such as B16, SGC-7901 and HL60. The EC₅₀ of the migration of HMEC and HUVEC were 10.5 and 1.7 μg/ml, respectively. The anti-tumor efficiency was determined by treating tumor-bearing mice with intravenous injection of rhEndostatin at the dose of 8, 2, 0.5mg/kg. The inhibitory efficiency of tumor by rhEndostatin was 49.49%, 34.69%, 22.96 % and 44.81%, 30.05%, 17.49% for mouse sarcoma and mouse hepatoma, respectively. The rhEndostatin inhibited tumor growth of all the cell lines tested with appreciable efficiency, including human hepatoma Bel-7402 (52.03%, 33.78%, 18.24%), cervical cancer Hela (68.00%, 46.50%, 24.50%) and gastric cancer SGC-7901 (51.89%, 40.57%, 16.98%). rhEndostatin treatment significantly prolonged the life-span of the tumor-loaded mice. The combination of chemotherapy of CTX with rhEndostatin further inhibited tumor growth up to 99% concomitant with a significant reduction of side-effect.A competitive ELISA assay was developed to examine the pharmacodynamic of rhEndostatin in mice. The concentrations of rhEndostatin in mice receiving intravenous injection of 1.5 or 4.5 mg/kg rhEndostatin were eliminated by a three-compartment model. The terminal t_1/2 was 9.1 and 10.2 hr , and AUC was 2733 ng.h/ml and 7425ng.h/ml, respectively. The CLs were the same, indicating a linear pharmacodynamics of rhEndostatin within the experimental dose range. The pharmacodynamics of subcutaneously injected rhEndostatin at the dose of 1.5% 4.5 mg/kg fitted the first-order kinetics absorption and two-compartment elimination model. And Cmax were 162, 428 ng/ml. AUC was 190, 571 ng.h/ml .Bioavailability was 65.6%, 78.7%, respectively. Once daily intravenous injection of rhEndostatin at 1.5 mg/kg for 7 days revealed that the drug concentration one hour post injection at day 7 was higher than that at day 1. As well, the AUC_0-24 after first and seventh injections was 2936 ng.h/ml and 4719 ng.h/ml, respectively. The accumulative factor (AUC_7th/AUC_1st during the whole treatment period is 1.6, demonstrating that the continuous administration of rhEndostain caused an accumulation of the durg in body.The toxicity effects of rhEndostain were determined by tail vein and intraperitoneal injections. We observed that the LD₅₀ in mice treated by tail vein and intraperitoneal injection was > 500 mg/kg. The antigenicity test of rhEndostain was tested to be negative in guinea pig.In summary, the present study found out the optimized condition for rhEndostatin production and purification and studied the pharmacodynamic, pharmacokinetic and toxicity of rhEndostatin. Thus our study will provide a solid theoretic basis for further clinical study. The endothelial cells are quiescent and do not produce drug-resistance normally. rhEndostain specifically targets active endothelial cells, showing an unique potential to be developed as a novel anti-angiogenesis drug.