title: Enhancement of drug delivery to bone: characterization of human tissue-nonspecific alkaline phosphatase tagged with an acidic oligopeptide.
abstract: Hypophosphatasia is caused by deficiency of activity of the tissue-nonspecific alkaline phosphatase (TNSALP), resulting in a defect of bone mineralization. Enzyme replacement therapy (ERT) with partially purified plasma enzyme was attempted but with little clinical improvement. Attaining clinical effectiveness with ERT for hypophosphatasia may require delivering functional TNSALP enzyme to bone. We tagged the C-terminal-anchorless TNSALP enzyme with an acidic oligopeptide (a six or eight residue stretch of L-Asp), and compared the biochemical properties of the purified tagged and untagged enzymes derived from Chinese hamster ovary cell lines. The specific activities of the purified enzymes tagged with the acidic oligopeptide were the same as the untagged enzyme. In vitro affinity experiments showed the tagged enzymes had 30-fold higher affinity for hydroxyapatite than the untagged enzyme. Lectin affinity chromatography for carbohydrate structure showed little difference among the three enzymes. Biodistribution pattern from single infusion of the fluorescence-labeled enzymes into mice showed delayed clearance from the plasma up to 18 h post infusion and the amount of tagged enzyme retained in bone was 4-fold greater than that of the untagged enzyme. In vitro mineralization assays with the bone marrow from a hypophosphatasia patient using each of the three enzymes in the presence of high concentrations of pyrophosphate provided evidence of bone mineralization. These results show the anchorless enzymes tagged with an acidic oligopeptide are delivered efficiently to bone and function bioactively in bone mineralization, at least in vitro. They suggest potential advantages for use of these tagged enzymes in ERT for hypophosphatasia, which should be explored.
authors.affiliation: Department of Pediatrics, Cardinal Glennon Children's Hospital, Saint Louis University, St. Louis, MO, USA.
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Nishioka
authors.forename: Shunji
authors.initials: S
authors.lastname: Tomatsu
authors.forename: Monica A
authors.initials: MA
authors.lastname: Gutierrez
authors.forename: Ken-ichi
authors.initials: K
authors.lastname: Miyamoto
authors.forename: Georgeta G
authors.initials: GG
authors.lastname: Trandafirescu
authors.forename: Patricia L C
authors.initials: PL
authors.lastname: Lopez
authors.forename: Jeffrey H
authors.initials: JH
authors.lastname: Grubb
authors.forename: Rie
authors.initials: R
authors.lastname: Kanai
authors.forename: Hironori
authors.initials: H
authors.lastname: Kobayashi
authors.forename: Seiji
authors.initials: S
authors.lastname: Yamaguchi
authors.forename: Gary S
authors.initials: GS
authors.lastname: Gottesman
authors.forename: Richard
authors.initials: R
authors.lastname: Cahill
authors.forename: Akihiko
authors.initials: A
authors.lastname: Noguchi
authors.forename: William S
authors.initials: WS
authors.lastname: Sly
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry / Q000493:pharmacokinetics*; D000818:Animals; D001216:Asparagine / Q000737:chemistry; D001854:Bone Marrow Cells / Q000187:drug effects* / Q000502:physiology; D002113:Calcification, Physiologic / Q000187:drug effects* / Q000502:physiology; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D017886:Durapatite / Q000737:chemistry; D006801:Humans; D007014:Hypophosphatasia / Q000473:pathology; D007223:Infant; D008099:Liver / Q000201:enzymology; D051379:Mice; D009842:Oligopeptides / Q000737:chemistry*; D014018:Tissue Distribution
chemical_list: D009842:Oligopeptides; D001216:Asparagine; D017886:Durapatite; D000469:Alkaline Phosphatase