title: Substantially improved pharmacokinetics of recombinant human butyrylcholinesterase by fusion to human serum albumin.
abstract: BACKGROUND
  Human butyrylcholinesterase (huBChE) has been shown to be an effective antidote against multiple LD50 of organophosphorus compounds. A prerequisite for such use of huBChE is a prolonged circulatory half-life. This study was undertaken to produce recombinant huBChE fused to human serum albumin (hSA) and characterize the fusion protein.
  
  
  RESULTS
  Secretion level of the fusion protein produced in vitro in BHK cells was approximately 30 mg/liter. Transgenic mice and goats generated with the fusion constructs expressed in their milk a bioactive protein at concentrations of 0.04-1.1 g/liter. BChE activity gel staining and a size exclusion chromatography (SEC)-HPLC revealed that the fusion protein consisted of predominant dimers and some monomers. The protein was confirmed to have expected molecular mass of approximately 150 kDa by Western blot. The purified fusion protein produced in vitro was injected intravenously into juvenile pigs for pharmacokinetic study. Analysis of a series of blood samples using the Ellman assay revealed a substantial enhancement of the plasma half-life of the fusion protein (approximately 32 h) when compared with a transgenically produced huBChE preparation containing >70% tetramer (approximately 3 h). In vitro nerve agent binding and inhibition experiments indicated that the fusion protein in the milk of transgenic mice had similar inhibition characteristics compared to human plasma BChE against the nerve agents tested.
  
  
  CONCLUSIONS
  Both the pharmacokinetic study and the in vitro nerve agent binding and inhibition assay suggested that a fusion protein retaining both properties of huBChE and hSA is produced in vitro and in vivo. The production of the fusion protein in the milk of transgenic goats provided further evidence that sufficient quantities of BChE/hSA can be produced to serve as a cost-effective and reliable source of BChE for prophylaxis and post-exposure treatment.
authors.affiliation: PharmAthene Canada Inc, (formally Nexia Biotechnologies Inc,), 7150 Alexander-Fleming, Montreal, QC H4S 2C8, Canada. yjhuang@videotron.ca
authors.forename: Yue-Jin
authors.initials: YJ
authors.lastname: Huang
authors.forename: Paul M
authors.initials: PM
authors.lastname: Lundy
authors.forename: Anthoula
authors.initials: A
authors.lastname: Lazaris
authors.forename: Yue
authors.initials: Y
authors.lastname: Huang
authors.forename: Hernan
authors.initials: H
authors.lastname: Baldassarre
authors.forename: Bin
authors.initials: B
authors.lastname: Wang
authors.forename: Carl
authors.initials: C
authors.lastname: Turcotte
authors.forename: Mélanie
authors.initials: M
authors.lastname: Côté
authors.forename: Annie
authors.initials: A
authors.lastname: Bellemare
authors.forename: Annie S
authors.initials: AS
authors.lastname: Bilodeau
authors.forename: Sandra
authors.initials: S
authors.lastname: Brouillard
authors.forename: Madjid
authors.initials: M
authors.lastname: Touati
authors.forename: Peter
authors.initials: P
authors.lastname: Herskovits
authors.forename: Isabelle
authors.initials: I
authors.lastname: Bégin
authors.forename: Nathalie
authors.initials: N
authors.lastname: Neveu
authors.forename: Eric
authors.initials: E
authors.lastname: Brochu
authors.forename: Janice
authors.initials: J
authors.lastname: Pierson
authors.forename: Duncan K
authors.initials: DK
authors.lastname: Hockley
authors.forename: Douglas M
authors.initials: DM
authors.lastname: Cerasoli
authors.forename: David E
authors.initials: DE
authors.lastname: Lenz
authors.forename: Harvey
authors.initials: H
authors.lastname: Wilgus
authors.forename: Costas N
authors.initials: CN
authors.lastname: Karatzas
authors.forename: Solomon
authors.initials: S
authors.lastname: Langermann
mesh_terms: D000818:Animals; D002091:Butyrylcholinesterase / Q000097:blood / Q000235:genetics / Q000493:pharmacokinetics*; D002460:Cell Line; D006224:Cricetinae; D006041:Goats; D006801:Humans; D007668:Kidney / Q000201:enzymology*; D008657:Metabolic Clearance Rate; D051379:Mice; D008822:Mice, Transgenic; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000493:pharmacokinetics*; D012709:Serum Albumin / Q000235:genetics / Q000493:pharmacokinetics*; D013552:Swine
chemical_list: D011993:Recombinant Fusion Proteins; D012709:Serum Albumin; D002091:Butyrylcholinesterase