title: Polyethylene glycol-conjugated adenosine phosphorylase: development of alternative enzyme therapy for adenosine deaminase deficiency.
abstract: Purine nucleoside phosphorylase had previously been engineered to accept 6-amino substituted purine nucleosides by two active site substitutions, Asn243Asp; Lys244Gln. In the present study, recombinant adenosine phosphorylase (AP) has been conjugated to branched polyethylene glycol (PEG) polymers of approximately 42.5 kDa. Matrix-assisted laser desorption/ionization analysis and SDS acrylamide electrophoresis analysis indicated a subunit composition of greater than 205 kDa consistent with the conjugation of as many as four PEG molecules per AP subunit. The PEG-conjugated enzyme retained greater than 90% of the native catalytic activity. Administration of the enzyme to mice demonstrated the PEG-AP to have a 67-fold increased plasma half-life compared to the native enzyme, 65.1+/-2.9 h versus 57.8+/-1.1 min, respectively. PEG-AP was principally confined to the plasma with minimal activity detected in tissues and of these spleen had the greatest activity and essentially no activity was found in urine. PEG-AP has retained activity with inosine and its injection into PNP-deficient mice resulted in a 2.7-fold increase in urine urate. AP was also shown to protect human CEM cells in culture from the toxic effects of 2'-deoxyadenosine. These studies provide evidence for consideration of PEG-AP as an alternative enzyme therapy for the inherited deficiency of adenosine deaminase.
authors.affiliation: Department of Medical Genetics, Faculty of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1.
authors.forename: Lee J
authors.initials: LJ
authors.lastname: Brewerton
authors.forename: Ernest
authors.initials: E
authors.lastname: Fung
authors.forename: Floyd F
authors.initials: FF
authors.lastname: Snyder
mesh_terms: D000243:Adenosine Deaminase / Q000172:deficiency*; D000818:Animals; D002478:Cells, Cultured; D015195:Drug Design; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006207:Half-Life; D051379:Mice; D011092:Polyethylene Glycols / Q000737:chemistry*; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry* / Q000493:pharmacokinetics / Q000627:therapeutic use; D011994:Recombinant Proteins / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014018:Tissue Distribution
chemical_list: D011994:Recombinant Proteins; D011092:Polyethylene Glycols; C019603:adenosine phosphorylase; D011683:Purine-Nucleoside Phosphorylase; D000243:Adenosine Deaminase