Extended activity in cynomolgus monkeys of a granulocyte colony-stimulating factor mutein conjugated with high molecular weight polyethylene glycol.

The activity of a granulocyte colony-stimulating factor (G-CSF) mutein (nartograstim; [NTG]) conjugated with an average of two polyethylene glycol (PEG) chains per protein molecule was examined in cynomolgus monkeys following a single s.c. injection. Groups of monkeys were given 10 microg/kg, 30 microg/kg, or 100 microg/kg. For comparison, one group of monkeys was given 5 microg/kg of recombinant human G-CSF (rHuG-CSF) daily for six days. In monkeys given 100 microg/kg of PEG-NTG, neutrophil levels reached a peak one day after injection approximately 20-fold higher than baseline levels. Neutrophil numbers in these animals were still significantly elevated six days after injection. In contrast, peak neutrophil levels in monkeys given six injections of rHuG-CSF reached a peak only on day 6 and were approximately the same as that in monkeys given a single dose of PEG-NTG six days before. Pharmacokinetics of PEG-NTG in these monkeys indicated that the area under the plasma concentration time curve (AUC) increased with increasing the dose from 497 ng x h/ml at 10 microg/kg, 6,140 ng x h/ml at 30 microg/kg to 27,900 ng x h/ml at 100 microg/kg. In a separate study, the effects of single doses of 100 microg/kg of PEG-NTG, rHuG-CSF, and unmodified NTG were compared. In this experiment, peak numbers of neutrophils were reached two days after injection in animals receiving PEG-NTG and one day after in animals given unmodified proteins. The pharmacokinetic parameters demonstrated increased exposure for PEG-NTG relative to the unmodified proteins with an AUC0. of 21,012 ng x h/ml compared with 5,492 ng x h/ml for rHuG-CSF and 5,153 ng x h/ml for NTG. These results demonstrate that conjugation of a G-CSF mutein with high molecular weight PEG results in a preparation that can induce prolonged elevation of neutrophils in normal nonhuman primates following a single injection.