Preparation and stability of N-terminal mono-PEGylated recombinant human endostatin.

Endostatin can specifically inhibit endothelial proliferation and potently inhibit angiogenesis and tumor growth. N-Terminal site-specific mono-PEGylation of recombinant human endostatin (mPEG-rhES) was accomplished by using methoxy poly-ethylene glycol (mPEG) propionaldehyde with an average molecular weight of 5000 Da through a reactive terminal aldehyde group. The site-specific mPEG conjugation was conducted under optimal conditions, which were identified through a statistical L(9)(3(4)) orthogonal test. In this study, we have investigated the stability and antitumor activity of mPEG-rhES. SDS-PAGE, RP-HPLC, and UV spectrophotometric analysis were used to identify the purity and stability of mPEG-rhES. When incubated with protease or placed in an extreme environment, mPEG-rhES was more stable than rhES. The unmodified and PEGylated rhES were tested for their ability to inhibit the tumor growth of mouse H22 liver cancer in male mice. In a multiple versus single doses comparison study, daily administration of 0.25, 0.50, and 1.00 micromol/kg of unmodified rhES for 7 days resulted in 26.9%, 43.0%, and 64.9% reductions in tumor weight, respectively, while single doses of 0.13, 0.25, and 0.50 micromol/kg of the PEGylated protein per day resulted in 24.8%, 38.0%, and 64.5% reductions, respectively. Both treatments resulted in statistically significant reductions in mean tumor weight as compared to the physiological saline solution (control)-treated mice, with the dose of mPEG-rhES being a half of rhES, respectively, while the tumor inhibition rates were similar. Therefore, it is suggested that PEGylation enhances the stability of rhES and improves its antitumor activity.