A Novel Heparin/Protamine‐Based Pro‐Drug Type Delivery System for Protease Drugs

Previously we proposed a heparin/protamine‐based system for delivery of protease drugs such as tissue‐specific plasminogen activator (tPA). To demonstrate the feasibility of this approach as well as its pro‐drug and triggered release features, positively charged peptides [(Arg)7Cys] were successfully linked to tissue‐specific plasminogen activator (tPA) using the crosslinking agent N‐succinimidyl‐3‐(2‐pyridyldithio)‐ propionate. This cation‐modified tPA showed much stronger heparin affinity than the parent tPA. The complex formed by mtPA and heparin was stable in human plasma, and the activity of mtPA in such a complex was inhibited by the appended heparin. Similarly, the activity of mtPA could also be inhibited by a heparin‐antifibrin IgG conjugate in which heparin was linked, via endpoint attachment, to the sugar moieties in the Fc region of anti‐fibrin IgG. Aside from this pro‐drug feature exhibited by the binding of the macromolecule heparin to mtPA, results from chromogenic and in vitro clot lysis assay demonstrated that the heparin‐induced inhibition of the mtPA activity could be easily reversed by the addition of an adequate amount of protamine. These findings suggest the applicability of the heparin/protamine delivery system to abort the potential bleeding risks associated with clinical use of tPA. In addition to the chemical conjugation method, modified tPA could also be produced by the recombinant DNA method. The expressed modified tPA (EmtPA) thus prepared retained the full catalytic activity of the parent tPA, and this activity could also be inhibited by heparin, and the heparin‐induced inhibition could be reversed following the addition of protamine. © 2000 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 89: 664–673, 2000