Human Genetics Program, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA.
J Bone Miner Res. 2020 Sep;35(9):1765-1771. doi: 10.1002/jbmr.4038. Epub 2020 May 8.
A mineral-targeted form of recombinant tissue-nonspecific alkaline phosphatase (TNAP), asfotase alfa, was approved multinationally 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 mice, a model of severe medial vascular calcification; Tie2-Cre; Hprt mice, a model of severe intimal calcification; and sibling WT Hprt 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 2 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 and Tie2-Cre; Hprt mice but not in WT mice. In addition, asfotase alfa binding was also found in the kidney stroma and brain of the Tie2-Cre; Hprt 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. © 2020 American Society for Bone and Mineral Research.
一种针对矿物质的重组组织非特异性碱性磷酸酶（TNAP）形式，阿法特司，于 2015 年被多国批准为成釉细胞蛋白缺乏症的酶替代疗法。迄今为止，已有两项报告表明，该药物通过组织化学和免疫组织化学与矿化组织结合。在这里，我们试图通过直接观察 AnaTag HiLyte Fluor 750 或 Alexa Fluor 647 荧光染料标记的阿法特司与骨骼/牙齿矿化和异位钙化部位的体内结合，来扩展这些早期研究。我们利用 40 天大的 Tagln-Cre；Hprt 小鼠，一种严重的中膜血管钙化模型；Tie2-Cre；Hprt 小鼠，一种严重的内膜钙化模型；以及没有软组织钙化的同胞 WT Hprt 小鼠。通过眶后途径注射单剂量 8mg/kg 标记的阿法特司。注射后 2 天，对骨骼组织和软组织进行离体成像。突变体和 WT 小鼠的所有骨骼组织（颅骨、椎骨、长骨、颌骨和下颌骨）均观察到强烈的荧光信号。对骨骼组织切片的荧光分析显示阿法特司的结合很强。在 Tagln-Cre；Hprt 和 Tie2-Cre；Hprt 小鼠中均发现阿法特司与心脏、主动脉和肾动脉的异位钙化部位结合，但在 WT 小鼠中未发现。此外，在 Tie2-Cre；Hprt 小鼠的肾脏基质和大脑中也发现了阿法特司的结合。我们的研究结果表明，体内给予的荧光标记的阿法特司不仅与骨骼和牙齿矿化部位结合，还与这些动物模型中异位钙化部位结合。
