Enhancement of the specific activities of PEGylated proteins by a simple procedure using a reversible amino-protective reagent

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We developed a novel method for the chemical modification of proteins with synthetic polymers to increase the therapeutic efficacy of the former in vivo. A pH-reversible amino-protective reagent, dimethylmaleic anhydride (DMMAn), was used for modification of interleukin-6 (IL-6) with polyethylene glycol (PEG). The novel PEGylated IL-6 (DmPEG-IL-6), which had been pretreated with DMMAn before PEGylation, showed up to a 140% increase in in vitro specific activity compared with PEG-IL-6 that had been synthesized by the previous method. Moreover, DmPEG-IL-6 caused thrombopoiesis more potently in mice than PEG-IL-6. The DmPEG-IL-6, having 3-4 PEG chains attached to the cytokine, showed the strongest thrombopoietic effect among the DmPEG-IL-6s with different molecular sizes that were tested. PEG-IL-6 had a 500-fold higher potency in stimulating thrombopoiesis than native IL-6, and DmPEG-IL-6 Fr.1 achieved a threefold higher thrombopoietic effect than PEG-IL-6. In addition, side-effects, such as an increase in the plasma fibrinogen level, were not observed after injection of either PEG-IL-6s or DmPEG-IL-6s. Additionally, we also found that PEGylation of tumor necrosis factor-alpha and G-CSF using DMMAn increased their specific activities. These results suggest that PEGylation with DMMAn pretreatment may become a useful means for clinical cytokine delivery.