Blood, Vol. 95 No. 3 (February 1), 2000:
pp. 936-942
Pharmacokinetic and thrombolytic properties of cysteine-linked
polyethylene glycol derivatives of staphylokinase
Sophie Vanwetswinkel,
Stephane Plaisance,
Zhang Zhi-yong,
Ingrid Vanlinthout,
Kathleen Brepoels,
Ignace Lasters,
Désiré Collen, and
Laurent Jespers
From the Center for Transgene Technology and Gene Therapy, Flanders
Interuniversity Institute of Biotechnology, Leuven, Belgium.
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-pyridyl-disulfide 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 µg/kg SakSTAR; 50 to 110 µg/kg of core-substitution derivatives with 1 P5; 25 µg/kg
for NH2-terminal derivatives with 1 P5; 5 to 25 µg/kg
with derivatives with 2 P5 or 1 P10; and 7 µg/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
mono-PEGylated staphylokinase variants may be used for single
intravenous bolus injection.
