Relationship between molecular mass and duration of activity of polyethylene glycol conjugated granulocyte colony-stimulating factor mutein

Proteins conjugated with polyethylene glycol (PEG) have increased in vivo a ctivity compared to native proteins. We examined the activity of a variety of PEG conjugates prepared with a recombinant mutein of granulocyte colony- stimulating factor (nartograstim [NTG], KW-2228), The total PEG mass was va ried by the number and size of the PEG molecules conjugated, In vitro activ ity, determined using a proliferation assay with G-NFS-60 cells, demonstrat ed an inverse relationship between PEG mass and concentration required for half-maximal proliferation. In vivo activity was examined by injecting comp ounds subcutaneously into normal mice and determining neutrophil counts at various times. Initial experiments in C57BL/6J mice indicated that neutroph il levels were significantly elevated 5 days after a single injection of 25 mu g/mouse of each PEG-NTG preparation. More detailed experiments were per formed with several of the preparations in C3H/HeJ mice lacking endotoxin r eceptors, The results demonstrated that the time after injection at which n eutrophil numbers reached a maximum increased with increasing size of PEG. Similar results were obtained with purified preparations containing 1, 2, o r 3 units of 20-kDa PEG per molecule of NTG, showing that increasing the ex tent of PEGylation also increases in vivo activity, Dose-response studies w ith the 20-kDa PEG-NTG demonstrated a plateau at doses >2.7 mu g/mouse at d ay 3, The plateau dose increased to 8.4 mu g/mouse at day 5, and no plateau was evident at the highest dose tested (50 mu g/mL) at days 7 and 10, Thes e results demonstrate that elevated neutrophil levels can be maintained for extended periods following single administration of high-molecular-weight PEG-NTG, (C) 1999 International Society for Experimental Hematology. Publis hed by Elsevier Science Inc.