Neurotrophins, such as brain derived neurotrophic factor (BDNF), are potential therapeutic agents for the treatment of stroke, trauma, and neurodegenerative diseases. However, the therapeutic value of these agents may not be realized until effective strategies are devised for delivering neurotrophins through the brain capillary endothelial wall, which makes up the blood-brain barrier (BBB) in vivo. The present application proposes a series of experiments that will develop a brain drug delivery vehicle for the transportation of BDNF across the BBB in control and ischemic brain. The experiments will be subdivided into four specific aims. First, recombinant human BDNF will be monobiotinylated and coupled to a BBB transport vector comprised of streptavidin (SA) and the murine OX26 monoclonal antibody to the rat transferrin receptor. Owing to the presence of high concentrations of transferrin receptor on the BBB, the OX26 monoclonal antibody is enabled to transcytose through the BBB via a receptor-mediated process. Secondly, the biologic activity of the BDNF coupled to the OX26/SA vector will be examined using BDNF radioreceptor assays. These assays will determine whether a cleavable or non-cleavable linker is required for conjugating the BDNF to the OX26/SA vector. Third, the pharmacokinetics in anesthetized rats will be determined for the unconjugated BDNF and the BDNF conjugated to the OX26/SDA vector. These pharmacokinetic studies will be performed at the doses of BDNF to be administered in the therapeutic studies pertaining to a rat ischemia model. In addition, the BDNF will be conjugated to polyethylene glycol (PEG) and the """"""""pegylated"""""""" BDNF will be examined in parallel with the non-pegylated BDNF. The pegylation is intended to optimize the plasma pharmacokinetics, which will further augment the brain delivery of the neurotrophin therapeutic. Fourth, the effects of the neurotrophin in protecting hippocampal pyramidal cells in the CA1 sector will be examined in a transient cerebral ischemia model. Anesthetized rats will be subjected to 10 minutes of cerebral ischemia; these rats will recover and be treated over a 7 day period with three different doses of BDNF coupled to the OX26/SA vector. This brain drug delivery strategy is applicable to humans with existing human BBB-specific vectors. These results could provide the basis for delivering neurotrophin through the BBB in humans afflicted with stroke.
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