title: Improved pharmacokinetic and biodistribution properties of the selective urokinase inhibitor PAI-2 (SerpinB2) by site-specific PEGylation: implications for drug delivery.
abstract: OBJECTIVE
  Overexpression of the serine protease urokinase (uPA) is recognised as an important biomarker of metastatic disease and a druggable anticancer target. Plasminogen activator inhibitor type-2 (PAI-2/SerpinB2) is a specific uPA inhibitor with proven potential for use in targeted therapy. However, PAI-2 is rapidly cleared via the renal system which impairs tumor uptake and efficacy. Here we aimed to improve the pharmacological properties of PAI-2 by site-specific PEGylation.
  
  
  METHODS
  Several cysteine to serine substitution mutants were generated for PEGylation with PEG-maleimide (size range 12-30 kDa) and the physico-chemical and biochemical properties of the PEG-PAI-2 conjugates characterised. Radiolabeled proteins were used for evaluation of blood clearance and tissue uptake profiles in an orthotopic breast tumor xenograft mouse model.
  
  
  RESULTS
  PEGylation of the PAI-2(C161S) mutant gave a predominant mono-PEGylated-PAI-2 product (~90%) with full uPA inhibitory activity, despite a significant increase in hydrodynamic radius. Compared to un-PEGylated protein the plasma half-life and AUC for PEG20-PAI-2(C161S) were significantly increased. This translated to a 10-fold increase in tumor retention after 24 h compared to PAI-2(C161S), an effect not seen in non-target organs.
  
  
  CONCLUSIONS
  Our data underscores the potential for PEG20-PAI-2(C161S) drug conjugates to be further developed as anti-uPA targeted therapeutics with enhanced tumor retention.
authors.affiliation: School of Biological Sciences, Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, NSW, 2522, Australia.
authors.forename: Kara Lea
authors.initials: KL
authors.lastname: Vine
authors.forename: Sergei
authors.initials: S
authors.lastname: Lobov
authors.forename: Vineesh
authors.initials: V
authors.lastname: Indira Chandran
authors.forename: Nathanial Lachlan Ewart
authors.initials: NL
authors.lastname: Harris
authors.forename: Marie
authors.initials: M
authors.lastname: Ranson
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000008:administration & dosage / Q000097:blood / Q000737:chemistry / Q000493:pharmacokinetics*; D001943:Breast Neoplasms / Q000378:metabolism; D045744:Cell Line, Tumor; D002626:Chemistry, Pharmaceutical; D004337:Drug Carriers; D004355:Drug Stability; D005260:Female; D006801:Humans; D007275:Injections, Intravenous; D008657:Metabolic Clearance Rate; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D008958:Models, Molecular; D009154:Mutation; D017396:Plasminogen Activator Inhibitor 2 / Q000008:administration & dosage / Q000097:blood / Q000737:chemistry / Q000235:genetics / Q000493:pharmacokinetics*; D011092:Polyethylene Glycols / Q000737:chemistry*; D011487:Protein Conformation; D015842:Serine Proteinase Inhibitors / Q000008:administration & dosage / Q000097:blood / Q000737:chemistry / Q000493:pharmacokinetics*; D013678:Technology, Pharmaceutical / Q000379:methods; D014018:Tissue Distribution
chemical_list: D000970:Antineoplastic Agents; D004337:Drug Carriers; D017396:Plasminogen Activator Inhibitor 2; D015842:Serine Proteinase Inhibitors; D011092:Polyethylene Glycols