HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY Pharmacokinetic and thrombolytic properties of cysteine-linked polyethylene glycol derivatives of staphylokinase

Recombinant staphylokinase (SakSTAR) variants obtained by site-directed substitution with cysteine, in the core (lysine 96 [Lys96], Lys102, Lys109, and/or Lys135) or the NH2-terminal region that is released during activation of SakSTAR (serine 2 [Ser2] and/or Ser3), were derivatized with thiol-specific (ortho-pyridyldisulfide or maleimide) polyethylene glycol (PEG) molecules with molecular weights of 5000 (P5), 10 000 (P10), or 20 000 (P20). The specific activities and thrombolytic potencies in human plasma were unaltered for most variants derivatized with PEG (PEGylates), but maleimide PEG derivatives 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 injection induced dose-related lysis of a plasma clot, fibrin labeled with 125 iodine (125I-fibrin plasma clot), and injected into the jugular vein. A 50% clot lysis at 90 minutes required 110 mg/kg SakSTAR; 50 to 110 mg/kg of core-substitution derivatives with 1 P5; 25 mg/kg for NH2-terminal derivatives with 1 P5; 5 to 25 mg/kg with derivatives with 2 P5 or 1 P10; and 7 mg/kg with P20 derivatives. Core substitution with 1 or 2 P5 molecules did not significantly reduce the immunogenicity of SakSTAR in rabbits. Derivatization of staphylokinase with a single PEG molecule allows controllable reduction of the clearance while maintaining thrombolytic potency at a reduced dose. This indicates that monoPEGylated staphylokinase variants may be used for single intravenous bolus injection. (Blood. 2000;95:936-942)