Pharmacokinetic and thrombolytic properties of cysteine-linked polyethylene glycol derivatives of staphylokinase

Recombinant staphylokinase (SakSTAR) variants obtained by site-directed sub stitution with cysteine, in the core (lysine 96 [Lys96], Lys102, Lys109, an d/or Lys135) or the NH2-terminal region that is released during activation of SakSTAR (serine 2 [Ser2] and/or Ser3), were derivatized with thiol-speci fic (ortho-pyridyl-disulfide or maleimide) polyethylene glycol (PEG) molecu les with molecular weights of 5000 (P5), 10 000 (P10), or 20 000 (P20), The specific activities and thrombolytic potencies in human plasma were unalte red for most variants derivatized with PEG (PEGylates), but maleimide PEG d erivatives had a better temperature stability profile. In hamsters, SakSTAR was cleared at 2.2 mL/min; variants with 1 P5 molecule were cleared 2- to 5-fold; variants with 2 P5 or 1 P10 molecules were cleared 10- to 30-fold; and variants with 1 P20 molecule were cleared 35-fold slower. A bolus injec tion Induced dose-related lysis of a plasma clot, fibrin labeled with 125 i odine (I-125-fibrin plasma clot), and injected into the jugular vein. A 50% clot lysis at 90 minutes required 110 mu g/kg SakSTAR; 50 to 110 mu g/kg o f core-substitution derivatives with 1 P5; 25 mu g/kg for NH2-terminal deri vatives with 1 P5; 5 to 25 mu g/kg with derivatives with 2 P5 or 1 P10; and 7 mu g/kg with P20 derivatives, Core substitution with 1 or 2 P5 molecules did not significantly reduce the Immunogenicity of SakSTAR in rabbits, Der ivatization of staphylokinase with a single PEG molecule allows controllabl e reduction of the clearance while maintaining thrombolytic potency at a re duced dose. This indicates that mono-PEGylated staphylokinase variants may be used for single intravenous bolus injection. (C) 2000 by The American Society of Hematology.