Visualization of mineral-targeted alkaline phosphatase binding to sites of calcification in vivo.

A mineral-targeted form of recombinant tissue-nonspecific alkaline phosphatase (TNAP), asfotase alfa, was approved multi-nationally as an enzyme replacement therapy for hypophosphatasia in 2015. Two reports to-date have shown evidence of binding of this drug to mineralizing tissues using histochemistry and immunohistochemistry. Here, we sought to expand on those earlier studies by directly visualizing the in vivo binding of asfotase alfa conjugated with AnaTag HiLyte Fluor 750 or Alexa Fluor 647 fluorescent dye to sites of skeletal/dental mineralization and ectopic calcification. We utilized 40-day-old Tagln-Cre; Hprt(ALPL/Y) mice, a model of severe medial vascular calcification; Tie2-Cre; Hprt(ALPL/Y) mice, a model of severe intimal calcification; and sibling WT Hprt(ALPL/Y) mice, devoid of soft-tissue calcification. A single dose of 8 mg/kg labeled asfotase alfa was injected via the retro-orbital route. Skeletal tissues and soft organs were imaged ex-vivo two days after the injection. Strong fluorescence signal was observed in all skeletal tissues (calvaria, vertebra, long bones, jaw and mandibles) from mutant and WT mice. Fluorescence analysis of histological sections from bones revealed strong binding of asfotase alfa. Asfotase alfa binding to sites of ectopic calcification in the heart, aorta and renal artery were found in both the Tagln-Cre; Hprt(ALPL/Y) and Tie2-Cre; Hprt(ALPL/Y) mice but not in WT mice. In addition, asfotase alfa binding was also seen in the kidney stroma and brain of the Tie2-Cre; Hprt(ALPL/Y) mice. Our results show that fluorescence-labeled asfotase alfa administered in vivo binds not only to sites of skeletal and dental mineralization but also to sites of ectopic calcification in these animal models. The authors report no conflicts of interest.