P4‐450: A new therapeutic strategy against Alzheimer's disease (AD) and other brain diseases: intracellular neurotransport delivery (i.nt.) of CB‐NGF and CB‐cargo conjugates may rescue AD and other brain diseases

Background: The blood-brain barrier (BBB) excludes neurotrophic factors and/or other therapeutic proteins with large molecular weight from the brain parenchyma, so that these potential drugs against Alzheimer’s disease(AD) cannot be used with any non-invasive measures by clinicians or patients themselves. The well-known central effect of the nerve growth factor (NGF) is that it rescues cholinergic neurons in the basal forebrain from toxicity or trauma. Although the first neurotrophin NGF won the Nobel prize for medicine and physiology in 1986, and is widely used in basic research with invasive measures, but still sleeping outside the clinical field of brain diseases. Methods: CB-NGF and CB-cargo were prepared according our lab’s modifications of di-sulfide (S-S) method or NaIO4 method. Hippocampus destroyed rats and/or beta-Amyloid peptide 25-35 induced amnesic mice were used as AD-like models. Expts in vitro were performed in PC12 cell culture conditioning with CB-NGF, native NGF and CB. Western blotting tests of NGF and CB-NGF receptor combining on TrkA and pTrkA (post binding phosphorylation of TrkA) were also performed with PC12 cells. Morris water maze (MWM) tests were performed in AD-like models. Results: Both conjugation procedures(S-S and/or NaIO4) have no influence on the bioactivity of CB and its cargos. The time windows in Western blotting of NGF and CB-NGF on pTrkA binding were different, the CB-NGF-pTrkA combining time was postponed almost 30 min later than NGF-pTrkA , it indicates the TrkA non-combined CB-NGF has been occupied the GM1 binding site and internalized in the first period, or GM1-CB-NGF combining is more powerful than NGF-TrkA’s and can push the CB-NGF-pTrkA binding to a later phase, this makes the TrkA non-combined CB-NGF possessing a secondary NGF transneuronal effects in vivo. In both rat and mouse AD model MWM tests showed in the CB-NGF treated group, the spatial learning and memory were significantly improved with comparison of the NGF administered ones. The ChAT positive neurons in the nucleus basalis of Meynert and diagonal band of Broca were almost fully rescued in CB-NGF treated AD models. Furthermore, the stem cell/progenitor stream in the forebrain of the CB-NGF treated mice was significantly enhanced in the core of the olfactory bulb (BrdU labeled cell counting). All these transneuronal effects indicate that CB-NGF nasally administered may rescue AD via i.nt. (partly published in Neuroscience 2008, 155:234) Conclusions: CB-cargo targeting i.nt. provides a proof-of-principle milestone in the therapeutic approach of neurodegenerative brain diseases with CB conjugated macromolecular weight polypeptides or immunoglobulins etc. With its specific powerful GM1-CB neurotransport and secondary transneuronal effects, CB-cargo provides a novel route of entrance into the brain parenchyma and a new therapeutic strategy bypassing BBB for targeting into the CNS. These stably covalent new compounds (CB-NGF etc) are deserved as a patent for industrialization.