Prolonged Half‐Life in the Circulation of a Chemical Conjugate Between a Pro‐Urokinase Derivative and Human Serum Albumin

Abstract

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 activator, pro‐urokinase has a very short half‐life in circulation.It has 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 (Δ125‐proUK), a pro‐urokinase deletion mutant lacking amino acids 11–135. We have expressed and purified the new mutein [H5K, S9C, N10T]des‐(C11‐K135)‐pro‐urokinase (Cys‐Δ125‐pro‐urokinase) and chemically conjugated it with serum albumin via the extra cysteine of Cys‐Δ125‐pro‐urokinase.The purified conjugate obtained has a lower specific amidolytic activity (72000 U/mg) than unconjugated Cys‐Δ125‐pro‐urikinase (240000 U/mg) due to its higher molecular mass and has a similar fibrinolytic activity in a clot lysis test to that of Δ125‐pro‐urokinase.We established an ELISA to measure the concentration of the conjugate in plasma and to follow the pharmacokinetics of the conjugate in monkeys after bolus injection.The conjugate displays significant lysis of human plasma clots in vivo and a dramatic increase of the half‐life in the circulation, with respect to pro‐urokinase and Δ125‐pro‐urokinase. Therefore, preliminary biological characterisation of this conjugate indicates that it could be a good candidate to inject as a bolus, compared with the infusion regimen needed with pro‐urokinase.