PROLONGED HALF-LIFE IN THE CIRCULATION OF A CHEMICAL CONJUGATE BETWEEN A PROUROKINASE DERIVATIVE AND HUMAN SERUM-ALBUMIN

Pro-urokinase is a natural plasminogen activator that displays a clot- lysis activity through a fibrin-dependent mechanism. It seems to be a promising agent for the treatment of coronary thrombosis. Like tissue- type plasminogen activator and two-chain urokinase-type plasminogen ac tivator, pro-urokinase has a very short half-life in circulation. It h as been described that conjugation of serum albumin with pro-urokinase in plasma may occur that could protect this protein from degradation. In this study we describe the insertion of an extra cysteine residue in the N-terminal end of des-(C11 -K135)-pro-urokinase (Delta 125-proU K), a pro-urokinase deletion mutant lacking amino acids 11-135. We hav e expressed and purified the new mutein [H5K, S9C, N10T]des-(C11-K135) -pro-urokinase (Cys-Delta 125-pro-urokinase) and chemically conjugated it with serum albumin via the extra cysteine of Cys-Delta 125-pro-uro kinase. The purified conjugate obtained has a lower specific amidolyti c activity (72000 U/mg) than unconjugated Cys-Delta 125-pro-urokinase (240000 U/mg) due to its higher molecular mass and has a similar fibri nolytic activity in a clot lysis test to that of Delta 125-pro-urokina se. We established an ELISA to measure the concentration of the conjug ate in plasma and to follow the pharmacokinetics of the conjugate in m onkeys after bolus injection. The conjugate displays significant lysis of human plasma clots in vivo and a dramatic increase of the half-lif e in the circulation, with respect to pro-urokinase and Delta 125-pro- urokinase. Therefore, preliminary biological characterisation of this conjugate indicates that it could be a good candidate to inject as a b olus, compared with the infusion regimen needed with pro-urokinase.