Design, Synthesis, and Evaluation of GUNW-3 as a Brain Targeting Agent

Abstract

The blood brain barrier (BBB) is a barrier in the brain that separates the peripheral blood circulation system from the central nerve system (CNS). The barrier effectively protects the brain from xenobiotics. The BBB serves as a physical barrier through the tight junction of endothelial cells that were found to be 50-100 times tighter than that of normal endothelial cells. Different drug efflux pumps such as P-glycoproteins and multidrug-resistance proteins are found to be overdistributed on the BBB. These drug efflux pumps help pump xenobiotics out if they enter the cells and serv as an additional mechanism to prevent xenobiotics from entering the CNS. The tight junction, drug efflux pumps, and other features of the BBB prevent almost 98% of small molecules, such as most therapeutics, and almost all large molecules such as biologics, recombinant genes and proteins from entering the brain. The inability to reach the therapeutic concentration caused by the barrier is often the major cause of treatment failure for brain diseases. Although the BBB blocks foreign compounds from entering the CNS, endogenous compounds, such as glucose, amino acids, peptides, neurotransmitters, and glutathione (GSH), enter the CNS readily through their corresponding receptors or transporters present in the BBB. Some of these receptors or transporters have been targeted for facilitating therapeutics, diagnostics, and other compounds to cross the BBB to reach the CNS. GSH is an endogenous three amino acid peptide. It plays various roles in the body: as a major antioxidant, a compound that removes toxic compounds, and a compound involved in other cellular functions. GSH crosses the BBB through a Nadependent GSH transporter. Recently, GSH transporters have been found effective in facilitating crossing of compounds through the BBB to reach the CNS. To achieve GSH transporter-mediated BBB crossing, GSH has been linked to a therapeutic agent (GSHDrug) to form a prodrug. The prodrug crosses the BBB by binding the GSH part to a GSH transporter followed by internalization of the prodrug. GSH has also been linked to polyethylene glycol (PEG) which is connected to a phospholipid (P) to form GSH-PEG-P or polyethylene glycol connected to vitamin E to form GSH-PEG-E. GSH-PEG-P and GSH-PEG-E have been coated on the surface of liposomes (GSH-PEGylated liposomes) to facilitate crossing of the liposomes through the BBB using the mechanism of binding the GSH moiety to a GSH transporter followed by internalization of the liposomes through endocytosis or transcytosis. The GSH-PEGylated liposomes have been shown to safely enhance the delivery to the brain by approximately 3-folds. We have developed GUNW-3 as a GSH-transporter dependent brain targeting agent. GUNW-3 was designed by connecting a hydrophilic GSH molecule to a hydrophobic cholesterol molecule through a two ehthylene glycol unit linker with a hope that the GSH part can serve as a brain-targeting structure through binding to the GSH transporter and facilitate the entry into the brain. This dissertation describes the design, synthesis, and fully characterization of GUNW-3. The dissertation also describes the ability of GUNW-3 to form micelles by itself (GUNW-3 micelles), the ability of GUNW- 3 micelles to cross the BBB to reach the brain, and the ability of GUNW-3 micelles to carry a dye (DiR) to the brain. Further, the dissertation shows the ability of GUNW-3 helps guide liposomes to the brain by forming GUNW-3 liposomes and the ability of GUNW-3 liposomes to deliver a dye (DiR) to the brain. Below is a brief description of the findings in this dissertation. GUNW-3 was synthesized in 4 steps from cholesterol and other commercially available reagents. GUNW-3 was found to be relatively stable. A cytotoxicity study of GUNW-3 revealed IC50 values of 0.65 mM and 0.47 mM for CV-1 cells (monkey kidney cells) and NCI-H226 cells (human lung cancer cells) respectively. GUNW-3 was found to form micelles by itself with a CMC value of 3.9 μM. CMC is a critical micelle parameter to reflect the stability of micelles and is also a parameter to determine if the micelles are stable enough to be used for a clinical application. The CMC of micelles need to be in μM concentration so that the micelles are stable enough to remain as micelles once being diluted in the blood stream. The CMC in low μM (3.9 μM) of GUNW-3 suggests that GUNW-3 micelles can be used for a therapeutic application. Further, the CMC of GUNW-3 is much lower than the IC50 values of the molecule indicating that GUNW-3 is not cytotoxic. For brain targeting, our data with ex-vivo imaging of the brains shows that the brain uptake of DiR, a dye, delivered by GUNW-3 micelles were 5 times higher than that of the control liposome and 12 times higher than that of free DiR at the first hour. After 48 h, the brain uptake of DiR delivered by GUNW-3 micelles was 6.5 times higher than that of the control liposome and 14 times higher than that of free DiR. GUNW-3 was also found to help deliver liposomes to the brain most likely by embedding the hydrophobic cholesterol part into the liposome double lipid layer and the hydrophilic GSH part floating on the surface of the liposomes for brain targeting. Our data from ex-vivo imaging of the brains demonstrate that GUNW-3 liposomes were able to significantly (\u3e3 folds) improve the delivery of DiR to the brain and retain in the brain well when compared with the control liposomes. Liposomes and micelles are known effective drug carriers that can be used to deliver various drugs or compounds such as small molecule therapeutics, DNA, RNA, and proteins (e.g., antibodies). Liposomes and micelles can encapsulate drugs and protect them from in vivo/in vitro degradation. They can also help reduce drug clearance, increase in vivo drug half-life, enhance the drug payload, control drug release, and improve drug solubility. The abilities to deliver DiR to the brain by GUNW-3 micelles and GUNW-3 liposomes warrantee further investigation of these two brain targeting delivery systems for delivering compounds to the brain for brain disease treatment or prevention. In summary, we have synthesized and characterized the rationally designed GUNW-3 as a brain targeting agent. GUNW-3 micelles and GUNW-3 liposomes showed promising brain targeting abilities. GUNW-3 micelles and GUNW-3 liposomes will be promising delivery systems for therapeutic and diagnostic molecules