Acta Pharmacologica Sinica 2008 November; 29 (11): 1357-1369; doi: 10.1111/j.1745-7254.2008.00865.x

Acta Pharmacologica Sinica 2008 November; 29 (11): 1357-1369; doi: 10.1111/j.1745-7254.2008.00865.x

Original Article

Bioactivity, pharmacokinetics, and immunogenicity assays in preclinical and clinical trials for recombinant human endostatin¹

Bi HU², Hao-wen ZHU⁴, Li-ping ZHU², Chen LI², Zhi-gang RONG⁴, Jia-ming XU⁴, Zhi-wei WU³, Jian-jun WANG^2,5, Gen-xing XU^3,4,5

²Department of Biological Science and Technology and State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210093, China; ³Center for Public Health Research, Nanjing University, Nanjing 210093, China; ⁴Jiangsu Research Center for Gene Pharmaceutical Engineering and Technology, Suzhou 215128, China

Aim: To determine the in vitro and in vivo bioactivity of recombinant human endostatin (rhEndostatin) and to analyze its pharmacokinetics and immunogenicity in rhesus monkeys and patients.

Methods: The physical chemical characteristics of rhEndostatin were detected according to Pharmacopoeia of the People's Republic of China (2005 edition, part III). Its in vitro and in vivo bioactivities were assayed via proliferation_inhibition on human umbilical vein endothelial cells and their inhibitory effect on tumor-bearing mice models. Serum concentrations of rhEndostatin in monkeys and patients were determined by an enzyme immunoassay method.

Results: The corresponding specific in vitro activities of rhEndostatin obtained from the cell counting method, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and lactate dehydrogenase assay, respectively, were 6.4×10⁷, 6.7×10⁷, and 3.8×10⁸U/mg, and the in vivo antitumoral potency was 4.04×10⁷U/mg. In rhesus monkeys, there were no gender differences in all pharmacokinetic parameters. Serum anti-rhEndostatin immunoglobulin (Ig)G antibodies were generated quickly after intravenous (iv) administration and decreased rapidly when therapy was stopped. In phase I clinical trials, linearity in the pharmacokinetics of rhEndostatin was indicated by dose-proportionate increases in the area under the curve and the maximum serum concentration. Serum rhEndostatin reached a steady-state level after 7 d of successive administration with the average concentration at a steady state of 272.44±91.98 ng/mL. Neither IgG nor IgM antibodies against rhEndostatin were observed in patients.

Conclusion: RhEndostatin exhibited a definite proliferation_inhibition effect on HUVEC, and significant antitumoral activity in mice. The immunoreactivity of rhesus monkeys to rhEndostatin is common, and rhEndostatin showed no immunogenicity in patients in this trial. The results provide a basis for further clinical trials.

Keywords: recombinant human endostatin; bioactivity; pharmacokinetics; immunogenicity

¹This work was supported by the 863 Project from the State Ministry of Science and Technology of China (No 2006AA02Z19E), the 985-II Project from Nanjing University to Gen-xing XU, the National Natural Science Foundation of China (No 30670671), the Natural Science Foundation of Jiangsu Province, China (No BK2006713), and a Rising Fountain Development Program (RFDP) grant from the State Educational Ministry of China to Jian-jun WANG (No 20050284025).

⁵Correspondence to Prof Gen-xing XU and Prof Jian-jun WANG.
Phn/Fax 86-25-8359-7570.
E-mail genxingxu@nju.edu.cn (Gen-xing XU)
Phn/Fax 86-25-8359-2714.
E-mail jjwang@nju.edu.cn (Jian-jun WANG)
Received 2008-06-02 Accepted 2008-07-13

[ Full text]