Studies of poly(ethylene glycol) modification of HM-3 polypeptides

An RGD modified endostatin-derived synthetic peptide, named HM-3, is a polypeptide angiogenesis inhibitor previously synthesized in our laboratory. Its robust inhibitory effects on endothelial cell migration and tumor growth have been demonstrated by in vivo and in vitro activity assays. The RGD integrin recognition sequence enables the selective binding of HM-3 and its specific targeting to tumor cells that express high levels of integrin. However, the drug has relatively short half-life in vivo, thus requiring administration twice a day to achieve its optimal in vivo antitumor efficacy. In the current study designed to prolong HM-3 half-life, we used methoxy-poly(ethylene glycol)-aldehyde (mPEG-ALD) to specifically modify its N terminus and optimized the reaction condition via monitoring the modification by reverse-phase high-performance liquid chromatograph (RP-HPLC) under varying stoichiometric ratios (n(mPEG10k-ALD):n(HM-3)), reaction times, and pH values. The maximal modification rate was achieved in a reaction when substrates mPEG10k-ALD and HM-3 were mixed at the molar ratio of 2:1 in a pH 6 phosphate buffer after 4 h incubation at room temperature. The reaction product of this optimal reaction was purified to 96% purity by RP-HPLC. Compared with HM-3, the newly modified PEG(10k)-HM-3 was shown to be more active in the inhibition of angiogenesis in the chorioallantoic membrane of chick embryos (CAM), its rate of in vitro degradation in serum was markedly reduced, and its in vivo half-life was prolonged by 5.86-fold relative to unmodified HM-3 after intravenous injection into male SD rats.