Strategies for enhancing enzyme delivery using the heparin/protamine-based drug delivery system

Enzymes are great drug candidates because they are highly specific and efficient in their catalytic activity, and they carry out many essential biological functions. Approaches such as polymer conjugation and the production of humanized enzymes have significantly improved the immunogenicity and half-life of enzymes making them more attractive as drug candidates. Unfortunately, enzymes' inability to differentiate between target and normal substrates, thus yielding systemic toxic effects, still stands as a major hurdle to their use as clinical drugs.; To overcome this major hurdle, a novel heparin/protamine system was investigated as a mechanism to enhance regional and intracellular enzyme delivery. The major goal of the delivery system is to create a pro-drug enzyme complex through the binding of a cationically modified enzyme with that of a heparin/antibody counterpart prior to administration. The heparin/antibody counterpart inhibits the modified enzyme's activity through the blockage of the enzyme's active site thereby alleviating systemic toxic effects. The attached antibody also brings the entire enzyme complex to the target site. Using competitive electrostatic binding, protamine is used to trigger release of the heparin/antibody blockage due to its higher binding affinity to heparin. As a result, the modified enzyme's activity could be released at the target site, yielding greater enzyme specificity while minimizing systemic toxic effects.; Through the use of asparaginase, in in vitro experiments, it was suggested that the activity of enzymes with small molecule substrate could not be inhibited upon heparin-macromolecule binding. In contrast, activity of enzymes with large molecule substrate such as tissue plasminogen activator was inhibited upon heparin-macromolecule binding and protamine was an effective trigger in releasing the inhibited activity in vitro. These results bring promise to the feasibility of the heparin/protamine delivery system in decreasing systemic enzyme toxic effects. Finally, the heparin/protamine delivery system was used to regulate TAT-mediated intracellular delivery. In vitro studies confirmed TAT's ability to mediated intracellular asparaginase delivery, heparin's ability to block TAT-mediated cell entry and protamine's ability to trigger release the heparin blockage. In vivo mice studies were also conducted to mimic the antibody targeting of TAT-Asp to tumor cells.