Saturday, May 18, 2024
5/18/2024
PROJECT SUMMARY Therapeutics targeting the central nervous system (CNS) have a less than 10% likelihood of approval through the entire clinical trial process. This very low approval rating is a significant problem for both pharmaceutical companies (lost investment) and patients (fewer advanced treatment options), and it has often been tied to the difficulty of penetrating the blood-brain-barrier (BBB). It is generally reported that 98% of small molecules and all macromolecules and proteins are prevented from crossing the BBB. As a result, several key strategies have been developed in order to increase the likelihood of CNS entry. These range from chemical modification to increase lipophilicity (e.g. esterification), modulating the physical properties of the BBB, and to mimicking or conjugating to non-specific transporter substrates (e.g. nutrients such as sugars or amino acids). While these methods have shown promise in some cases, they all have significant drawbacks including a potential loss of activity, significant or severe side effects, and a lack of BBB targeting specificity resulting in possible systemic side effects, respectively. A fourth and more specific approach has therefore been developed, in which specific receptors on the BBB are targeted. One notable example has been the use of the transferrin receptor (TfR), which is responsible for iron uptake and transport. Conjugation of drugs such as chemotherapeutics to TfR targeting agents has been demonstrated to significantly increase CNS uptake and tumor targeting. However, targeting TfR, can also result in significant off-target localization and toxicity as it has been found with other widely distributed receptors (e.g. insulin receptor). We have therefore devised a new modification strategy by which large macromolecules and proteins may be modified to target the a7 subunit of the nicotinic acetylcholine receptor (a7nAChR) in order to efficiently cross the BBB. Our preliminary data demonstrates that through our modification strategy, we could successfully alter enhanced green fluorescent protein (eGFP) without affecting its fluorescent properties and without degrading its stability. Furthermore, this fluorescent acetylcholine receptor binding eGFP (FARB, Swiss German for color) localizes in primary neuronal cells through a7nAChR, is non-toxic, and localizes in mice brains (preliminary data). In this project, we therefore seek to confirm the efficiency of our FARB protein in crossing the BBB in vivo with both wild-type and a7nAChR knockout mice. This will be accomplished by tracking the distribution and fluorescent signals from both FARB and eGFP over time with an IVIS imaging system and then quantifying the concentration of both proteins in major organs (brain, heart, lungs, etc.), blood, and urine at various time points. Finally, immunohistochemical analysis of brain slices will allow us to determine the brain region(s) that are most targeted by FARB. Completing this project will demonstrate a new modification strategy that may be used to significantly increase the efficiency with which the CNS can be targeted by macromolecules and/or proteins.
HHS’ Tracking Accountability in Government Grants System (TAGGS) website provides access to detailed descriptions of grants, loans, aggregated direct payments and other types of financial assistance awarded by the HHS Operating Divisions (OPDIVs) and the Office of the Secretary Staff Divisions (STAFFDIVs).
