Carboxyl-directed pegylation of brain-derived neurotrophic factor markedly reduces systemic clearance with minimal loss of biologic activity

Purpose. Brain-derived neurotrophic factor (BDNF) was modified by carboxyl-directed protein pegylation in order to both retain biologic activity of the neurotrophin and reduce the rate of systemic clearance of this cationic protein in vivo. Since the modification of surface lysine residues of neurotrophins results in loss of biologic activity, the present studies examine the feasibility of placing polyethyleneglycol (PEG) polymers on carboxyl residues of surface glutamate or aspartate residues of BDNF. Methods. PEG molecules with terminal hydrazide (Hz) moieties of molecular weight 2,000 (PEG(2000)-Hz) or 5,000 (PEG(5000)-Hz) Daltons were coupled to BDNF carboxyls using carbodiimide. Results. The systemic clearances of the BDNF-PEG(2000) and BDNF-PEG(5000) were reduced 67% and 91%, respectively, compared to unconjugated BDNF. The brain volume of distribution (V-D) of BDNF-PEG(5000) was not significantly different from the cerebral plasma volume. Cell survival studies and TrkB auto-phosphorylation assays showed that the biologic activity of BDNF was not changed following pegylation with PEG(2000), and was minimally impaired following pegylation with PEG(5000). Conclusions. These experiments describe the first carboxyl-directed pegylation of a neuropeptide, and show this formulation substantially reduces the systemic distribution and elimination of the neurotrophic factor. The biologic activity of the neurotrophin is retained with carboxyl-directed pegylation.