A prodrug approach for delivery of t-PA: construction of the cationic t-PA prodrug by a recombinant method and preliminary in vitro evaluation of the construct.

Abstract

Previously, we reported a novel prodrug approach, that could lead to targeted thrombolysis without the risk of bleeding. The approach consists of a protein conjugate made of two components: a fibrin targeting antibody (Ab) linked to an anionic heparin, and a plasminogen activator (PA) derivatized with cationic species. These two components are linked by means of an electrostatic interaction. Because the cationic species are small, the modified PA would retain its thrombolytic activity. However, this activity would be inhibited after binding to the counterpart due to the blockage of the PA active site by the appended macromolecules. Because protamine is a clinical antagonist to heparin, it can be used in humans to dissociate the modified PA from its counterpart. Thus, the approach would permit the administration of a fibrin targeting but inactive thrombolytic drug (thereby alleviating the bleeding risk by avoiding systemic generation of plasmin), and subsequently a triggered release of the active drug to the fibrin deposit. In our previous work, we demonstrated the feasibility of the approach by producing a positively charged PA by means of chemical conjugation of a cationic CRRRRRRR peptide with urokinase. In this study, we further extended our work and produced a similar cationic t-PA by means of a recombinant DNA approach; i.e., by fusion of a poly(Arg)7 peptide to the kringle-1 domain of t-PA. Results obtained from the restriction enzyme analysis and the Western blot yielded full identification of this recombinant protein. This recombinant poly(Arg)7-modified-t-PA protein conjugate (termed "rmt-PA" hereafter) completely retained the fibrinolytic activity of the original recombinant, unmodified t-PA (termed "rt-PA" hereafter), as measured by the chromogenic assay and fibrin agar lysis assay. The prodrug and triggered release features of the proposed approach were confirmed by partial inhibition of the plasminogen activating activity of this protein by heparin, and the partial reversal of such inhibition by protamine.