PUTRESCINE-MODIFIED NERVE GROWTH-FACTOR - BIOACTIVITY, PLASMA PHARMACOKINETICS, BLOOD-BRAIN NERVE BARRIER PERMEABILITY, AND NERVOUS-SYSTEM BIODISTRIBUTION/

Previous investigations from our laboratory have demonstrated that the covalent modification of a variety of proteins, including antioxidant enzymes, with the naturally occurring polyamines-putrescine (PUT), sp ermidine, and spermine-dramatically increases their permeability coeff icient-surface area product (PS) at the blood-brain and blood-nerve ba rriers after parenteral administration. In the present study, we have covalently modified nerve growth factor(NGF) with PUT by targeting car boxylic groups for their graded modification by controlling the ioniza tion of these groups with pH, Sodium dodecyl sulfate-polyacrylamide ge l electrophoresis, western, and isoelectric focusing analyses demonstr ated conversion of NGF to its polyamine-modified derivatives at differ ent pH values. Although the immunoreactivity of PUT-NGF determined by ELISA and western analysis decreased with decreasing pH, the biologica l activity of PUT-NGF was not affected at any pH as determined by surv ival and neurite extension of dorsal root ganglia and PC12 cultures. P lasma pharmacokinetics after a single intravenous bolus administration revealed intact PUT-NGF through 10 min and 73-82% intact protein at 1 5 min. The PS value for PUT-NGF was maximized and the residual plasma volume (V-p) of the protein in the blood vessels minimized when the pH of the modification reaction was >6.4. The biodistribution of PUT-NGF at 15 min showed 22-33% intact protein in different brain regions, wh ich represented 0.4-5.9 ng of PUT-NGF in different brain regions, a ph ysiological dose that is capable of eliciting a bioresponse. The desig n of this polyamine-modified NGF derivative that has enhanced permeabi lity at the blood-brain and blood-nerve barriers with retained bioacti vity may obviate the necessity to create small-molecule mimics of NGF and may be applicable to neurotrophins, engineered multifunctional chi meric neurotrophins, antioxidant enzymes, and other therapeutic protei ns with specific clinical application to neurological diseases.