title: Pharmacokinetic and thrombolytic properties of cysteine-linked polyethylene glycol derivatives of staphylokinase.
abstract: Recombinant staphylokinase (SakSTAR) variants obtained by site-directed substitution with cysteine, in the core (lysine 96 [Lys96], Lys102, Lys109, and/or Lys135) or the NH(2)-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 5,000 (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 ((125)I-fibrin plasma clot), and injected into the jugular vein. A 50% clot lysis at 90 minutes required 110 microg/kg SakSTAR; 50 to 110 microg/kg of core-substitution derivatives with 1 P5; 25 microg/kg for NH(2)-terminal derivatives with 1 P5; 5 to 25 microg/kg with derivatives with 2 P5 or 1 P10; and 7 microg/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.
authors.affiliation: Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute of Biotechnology, Leuven, Belgium.
authors.forename: S
authors.initials: S
authors.lastname: Vanwetswinkel
authors.forename: S
authors.initials: S
authors.lastname: Plaisance
authors.forename: Z
authors.initials: Z
authors.lastname: Zhi-Yong
authors.forename: I
authors.initials: I
authors.lastname: Vanlinthout
authors.forename: K
authors.initials: K
authors.lastname: Brepoels
authors.forename: I
authors.initials: I
authors.lastname: Lasters
authors.forename: D
authors.initials: D
authors.lastname: Collen
authors.forename: L
authors.initials: L
authors.lastname: Jespers
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D006224:Cricetinae; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry*; D003553:Cystine / Q000737:chemistry; D004337:Drug Carriers; D004353:Drug Evaluation, Preclinical; D004789:Enzyme Activation / Q000187:drug effects; D005342:Fibrinolysis / Q000187:drug effects; D005343:Fibrinolytic Agents / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology* / Q000627:therapeutic use; D006207:Half-Life; D006801:Humans; D008298:Maleates / Q000737:chemistry; D008666:Metalloendopeptidases / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology* / Q000627:therapeutic use; D016297:Mutagenesis, Site-Directed; D010958:Plasminogen / Q000378:metabolism; D011092:Polyethylene Glycols / Q000737:chemistry*; D011499:Protein Processing, Post-Translational; D011655:Pulmonary Embolism / Q000188:drug therapy; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology / Q000627:therapeutic use; D013329:Structure-Activity Relationship
chemical_list: D003432:Cross-Linking Reagents; D004337:Drug Carriers; D005343:Fibrinolytic Agents; D008298:Maleates; D011993:Recombinant Fusion Proteins; D011092:Polyethylene Glycols; D003553:Cystine; D010958:Plasminogen; C030272:maleic acid; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus; D003545:Cysteine