Putrescine‐Modified Nerve Growth Factor: Bioactivity, Plasma Pharmacokinetics, Blood‐Brain/Nerve Barrier Permeability, and Nervous System Biodistribution

Abstract

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), spermidine, and spermine-dramatically increases their permeability coefficient-surface area product (PS) at the blood-brain and blood-nerve barriers after parenteral administration. In the present study, we have covalently modified nerve growth factor (NGF) with PUT by targeting carboxylic groups for their graded modification by controlling the ionization of these groups with pH. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, western, and isoelectric focusing analyses demonstrated conversion of NGF to its polyamine-modified derivatives at different pH values. Although the immunoreactivity of PUT-NGF determined by ELISA and western analysis decreased with decreasing pH, the biological activity of PUT-NGF was not affected at any pH as determined by survival and neurite extension of dorsal root ganglia and PC12 cultures. Plasma pharmacokinetics after a single intravenous bolus administration revealed intact PUT-NGF through 10 mm and 73-82% intact protein at 15 mm. The PS value for PUT-NGF was maximized and the residual plasma volume (Vp) 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 miii showed 22-33% intact protein in different brain regions, which represented 0.4-5.9 ng of PUT-NGF in different brain regions, a physiological dose that is capable of eliciting a bioresponse. The design of this polyamine-modifled NGF derivative that has enhanced permeability at the blood-brain and blood-nerve barriers with retained bioactivity may obviatethe necessity to create small-molecule mimics of NGF and may be applicable to neurotrophins, engineered multifunctional chimeric neurotrophins, antioxidant enzymes, and other therapeutic proteins with specific clinical application to neurological diseases. KeyWords: Bloodbrain/nerve barriers-Protein delivery-Nerve growth factor.

Nerve growth factor displays stimulatory effects on human skin and lung fibroblasts, demonstrating a direct role for this factor in tissue repair

“…This hypothesis is in line with previous studies showing that NGF is synthesized in cutaneous wound tissues and that its high levels may contribute to the efficient wound healing in the neonate (27). The observation that NGF contributes to healing after traumatic muscle injury (28,29), during diabetic conditions (30), and in corneal ulcers (7) is also consistent with this hypothesis.…”

Strategies for drug delivery to the central nervous system by systemic route

“…In general, due to net negative charge on cell membrane, a molecule should be preferably positively charged for effective adsorptivemediated transcytosis. Higher bioavailability of drugs such as anti-ras oncogene antibodies and nerve growth factors in brain has been achieved through cationization (Poduslo et al, 1998;Bickel et al, 2001). However, care should be taken since cationization in some cases could result in permanent loss in activity of molecules (further details are discussed under the section role of surface charge of nanoparticles) (Hervé et al, 2008).…”

Pathogenic Factors in Vascular Dementia and Alzheimer’s Disease

“…Polyamines are toxic to cultures of rat cerebellar granule cells [223]. Polyamines damage the BBB [224]. Thrombin stimulates ODC gene expression and polyamine synthesis [225].…”

“…BA interaction with blood vessel endothelial cells increases BBB permeability [270]. Polyamines damage the BBB [271]. Thrombin stimulates polyamine synthesis [272].…”