Neurotrophins, such as brain-derived neurotrophic factor (BDNF), are potential neuroprotective agents for the treatment of stroke, trauma, and neurodegenerative diseases. However, the therapeutic value of these new therapeutics may not be realized until effective strategies are developed for delivering neurotrophins through the brain capillary endothelial wall, which makes up the blood-brain barrier (BBB) in vivo. The present application develops a brain drug delivery strategy for the non-invasive transport of BDNF across the BBB for use as a neurotherapeutic in the ischemic brain. This work uses a combination of techniques derived from protein pegylation technology, avidin-biotin technology, and chimeric peptide technology. Chimeric peptides are formed when a non-transportable peptide therapeutic, e.g., BDNF, is conjugated to a BBB drug delivery vector. The latter is a receptor-specific monoclonal antibody (MAb) that undergoes receptor-mediated transcytosis through the BBB in vivo. The present studies used the murine OX26 MAb to the rat transferrin receptor. A conjugate of the OX26 MAb and streptavidin (SA) is prepared in parallel with the carboxyl-directed pegylation and mono-biotinylation of recombinant human BDNF. The biologic activity of the BDNF conjugate is demonstrated by trkB autophosphorolation assays in tissue culture and the formulation is characterized biochemically with electrophoresis, and Western blotting, as well as gel filtration chromatography. The model of global ischemia used in these studies is transient forebrain ischemia whereby an isoelectric electroencephalogram is induced for a 10 minute period, followed by resuscitation and recovery of the animals. A model of regional ischemia used in these studies is the middle cerebral artery occlusion (MCAO) model that is reversible to allow for reflow. Following recovery in either model, the therapeutic effects of BDNF are assessed by histology using Niss1 staining of the hippocampus with quantitative neuronal counting of neurons in the CA1 sector of the hippocampus. This brain-drug delivery strategy can be ultimately used in humans because recent studies have developed human- specific BBB transport vectors.
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