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

• Published in: Blood Vol. 95; no. 3; pp. 936 - 942
• Main Authors: Vanwetswinkel, Sophie, Plaisance, Stephane, Zhi-yong, Zhang, Vanlinthout, Ingrid, Brepoels, Kathleen, Lasters, Ignace, Collen, Désiré, Jespers, Laurent
• Format: Journal Article
• Language: English
• Published: Washington, DC Elsevier Inc 01-02-2000; The Americain Society of Hematology
• Subjects: Amino Acid Substitution; Animals; Biological and medical sciences; Blood. Blood coagulation. Reticuloendothelial system; Cricetinae; Cross-Linking Reagents - pharmacology; Cysteine - chemistry; Cystine - chemistry; Drug Carriers; Drug Evaluation, Preclinical; Enzyme Activation - drug effects; Fibrinolysis - drug effects; Fibrinolytic Agents - chemistry; Fibrinolytic Agents - pharmacokinetics; Fibrinolytic Agents - pharmacology; Fibrinolytic Agents - therapeutic use; Half-Life; Humans; Maleates - chemistry; Medical sciences; Metalloendopeptidases - chemistry; Metalloendopeptidases - pharmacokinetics; Metalloendopeptidases - pharmacology; Metalloendopeptidases - therapeutic use; Mutagenesis, Site-Directed; Pharmacology. Drug treatments; Plasminogen - metabolism; Polyethylene Glycols - chemistry; Protein Processing, Post-Translational; Pulmonary Embolism - drug therapy; Rabbits; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - pharmacokinetics; Recombinant Fusion Proteins - pharmacology; Recombinant Fusion Proteins - therapeutic use; Structure-Activity Relationship; Ethylene oxide polymer; Human; Cysteine; Immunogenicity; Aminoacid; Animal; Recombinant protein; Pharmacokinetics; Biological activity; Fibrinolytic; Molecular weight
• DOI: 10.1182/blood.V95.3.936.003k20_936_942
• EISSN: 1528-0020
• ISSN: 0006-4971

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.