title: Prolonged half-life and preserved enzymatic properties of factor IX selectively PEGylated on native N-glycans in the activation peptide.
abstract: Current management of hemophilia B entails multiple weekly infusions of factor IX (FIX) to prevent bleeding episodes. In an attempt to make a longer acting recombinant FIX (rFIX), we have explored a new releasable protraction concept using the native N-glycans in the activation peptide as sites for attachment of polyethylene glycol (PEG). Release of the activation peptide by physiologic activators converted glycoPEGylated rFIX (N9-GP) to native rFIXa and proceeded with normal kinetics for FXIa, while the K(m) for activation by FVIIa-tissue factor (TF) was increased by 2-fold. Consistent with minimal perturbation of rFIX by the attached PEG, N9-GP retained 73%-100% specific activity in plasma and whole-blood-based assays and showed efficacy comparable with rFIX in stopping acute bleeds in hemophilia B mice. In animal models N9-GP exhibited up to 2-fold increased in vivo recovery and a markedly prolonged half-life in mini-pig (76 hours) and hemophilia B dog (113 hours) compared with rFIX (16 hours). The extended circulation time of N9-GP was reflected in prolonged correction of coagulation parameters in hemophilia B dog and duration of effect in hemophilia B mice. Collectively, these results suggest that N9-GP has the potential to offer efficacious prophylactic and acute treatment of hemophilia B patients at a reduced dosing frequency.
authors.affiliation: Haemostasis Biology, Novo Nordisk A/S, Maaloev, Denmark. hqsg@novonordisk.com
authors.forename: Henrik
authors.initials: H
authors.lastname: Østergaard
authors.forename: Jais R
authors.initials: JR
authors.lastname: Bjelke
authors.forename: Lene
authors.initials: L
authors.lastname: Hansen
authors.forename: Lars Christian
authors.initials: LC
authors.lastname: Petersen
authors.forename: Anette A
authors.initials: AA
authors.lastname: Pedersen
authors.forename: Torben
authors.initials: T
authors.lastname: Elm
authors.forename: Flemming
authors.initials: F
authors.lastname: Møller
authors.forename: Mette B
authors.initials: MB
authors.lastname: Hermit
authors.forename: Pernille K
authors.initials: PK
authors.lastname: Holm
authors.forename: Thomas N
authors.initials: TN
authors.lastname: Krogh
authors.forename: Jørn M
authors.initials: JM
authors.lastname: Petersen
authors.forename: Mirella
authors.initials: M
authors.lastname: Ezban
authors.forename: Brit B
authors.initials: BB
authors.lastname: Sørensen
authors.forename: Mette D
authors.initials: MD
authors.lastname: Andersen
authors.forename: Henrik
authors.initials: H
authors.lastname: Agersø
authors.forename: Haleh
authors.initials: H
authors.lastname: Ahmadian
authors.forename: Kristoffer W
authors.initials: KW
authors.lastname: Balling
authors.forename: Marie Louise S
authors.initials: ML
authors.lastname: Christiansen
authors.forename: Karin
authors.initials: K
authors.lastname: Knobe
authors.forename: Timothy C
authors.initials: TC
authors.lastname: Nichols
authors.forename: Søren E
authors.initials: SE
authors.lastname: Bjørn
authors.forename: Mikael
authors.initials: M
authors.lastname: Tranholm
mesh_terms: D000818:Animals; D001665:Binding Sites; D004195:Disease Models, Animal; D004285:Dogs; D005164:Factor IX / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005260:Female; D006207:Half-Life; D002836:Hemophilia B / Q000097:blood / Q000188:drug therapy / Q000235:genetics; D006490:Hemostatics / Q000097:blood / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D051379:Mice; D008817:Mice, Mutant Strains; D011092:Polyethylene Glycols / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D013552:Swine; D013556:Swine, Miniature
chemical_list: D006490:Hemostatics; D011134:Polysaccharides; D011994:Recombinant Proteins; D011092:Polyethylene Glycols; D005164:Factor IX