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 18h 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.

Bone-Targeting Endogenous Secretory Receptor for Advanced Glycation End Products Rescues Rheumatoid Arthritis

“…Pathological lesions in CIA appear not only in bone but also in synovial tissue, which does not contain HA. Consistent with the previous studies (27,28), acidic oligopeptide-tagged esRAGEs, including D 6 -esRAGE, were localized to a mineralized region in bone, where they were retained for over 1 week. The principle of bone-targeting by using an acidic oligopeptide is based on the affinity between negatively charged acidic oligopeptide and positively charged calcium in HA.…”

“…A significant difference between acidic oligopeptide-tagged esRAGEs was not observed in the biodistribution. In addition, it is likely that a longer stretch of acidic oligopeptide affects physiological properties, except for biodistribution of esRAGE, because Nishioka et al (27) demonstrated that the longer stretch of acidic oligopeptide more largely changed the N-linked oligosaccharides structure of glycoprotein. The ligand-binding V-domain of RAGE contains two potential N-glycosylation sites, and the N-linked oligosaccharides on RAGE are suggested to mediate the interaction with its ligands (20,21).…”

“…It was indicated that the antibiotics fluoroquinolones, tagged with an acidic oligopeptide, were successfully targeted to bone and could be effective in treating osteomyelitis if the appropriate dose was given (24). We and other groups have applied this bonetargeting system to large molecules, namely, enzymes (tissue-nonspecific alkaline phosphatase, β-glucuronidase and N-acetylgalactosamine-6-sulfate sulfatase), showing that the tagged enzymes were delivered more efficiently to bone and that the clinical and pathological improvement in bone was observed in murine models of hypophosphatasia, mucopolysaccharidosis VII and mucopolysaccharidosis IVA, respectively (27)(28)(29)(30). The clinical trial for the patients with hypophosphatasia is now in progress with a favorable clinical consequence rescuing the devastating skeletal bone disease (31).…”

Therapies for the bone in mucopolysaccharidoses

“…Kasugai et al, 2000 showed that they could enhance estradiol uptake by bone and prevent osteoporosis by tagging the hormone to Glu 6 (E6) [75,76]. We and others have recently applied this new bone-targeting system to a large molecule, an enzyme (tissue nonspecific alkaline phosphatase), showing that the tagged enzymes are delivered more efficiently to bone [77][78][79] and that the tagged enzyme improves clinical and pathological effects of a mouse model of a systemic bone disease, hypophosphatasia, better than untagged native enzyme [80]. Human Nacetylgalactosamine-6-sulfate sulfatase (GALNS) and ß-glucuronidase (GUSB) have also been bioengineered to add E6 and D6.…”

Current and emerging treatments and surgical interventions for Morquio A syndrome: A review

“…63,64 The authors of this present paper and others have recently attached this novel bone-targeting peptide to an enzyme (tissue nonspecific alkaline phosphatase) and shown that the tagged enzyme is delivered more specifically to bone than unmodified enzyme, improving the clinical and pathological consequence of the systemic bone disease, hypophosphatasia. 65,66 Human GALNS has also been bioengineered to add a hexa-glutamate sequence (E6) to its N-terminus (E6-GALNS). This tagged enzyme has a markedly reduced rate of clearance from the circulation, increasing blood levels 20 times higher than that of the untagged enzyme.…”