Cathespin K Radioligands for In Vivo Imaging

描述（由申请人提供）：骨质疏松症估计影响美国50岁以上成年人的一半以上，80岁以上女性中有三分之一会患髋部骨折；在骨折患者中，近15-20%的人在一年内死亡。总的来说，2005年仅在美国，与骨质疏松症相关的费用估计为190亿美元。该项目涉及设计、开发和评估一种新的分子成像放射性示踪剂，用于骨质疏松症和其他涉及潜在骨质流失的肌肉骨骼疾病（如骨关节炎、风湿性关节炎、骨质疏松症）的正电子发射断层扫描（PET）研究。该项目将开发已知的高亲和力组织蛋白酶K抑制剂的放射性标记形式，组织蛋白酶K是一种半胱氨酸蛋白酶，在破骨细胞中高度表达，负责I型胶原蛋白降解和骨吸收。组织蛋白酶K在体内成像的潜力已经通过光学方法得到了证明，该项目将把这一概念扩展到使用放射性核素标记的临床可翻译成像方法。该资助将基于已发表的氰嘧啶和吡罗嘧啶支架，使用同位素（碳-11）替代或氟化类似物合成（用于氟-18标记）制备一系列放射性标记抑制剂。作为碳-11标记的初始靶标的化合物都对人组织蛋白酶K具有高亲和力（< 10纳摩尔），比组织蛋白酶L和S具有出色的选择性（100-1000倍选择性），并且都很容易使用[11C]甲基碘标记。新的氟化类似物，其中氟乙基取代甲基，将在体外评估对组织蛋白酶K酶活性的抑制作用，使用荧光测定，并使用类似的[18F]氟烷基化反应标记强效（<10 nM）抑制剂。在体内证明破骨细胞依赖的放射性示踪剂定位的概念研究将在大鼠中进行，使用局灶性骨骼注射RANKL（核因子b配体受体激活剂，诱导破骨细胞生成）或骨原蛋白（抑制破骨细胞生成）。显微ct监测骨质流失程度。将在RANKL处理的大鼠中验证放射性定位是组织蛋白酶特异性的，并用冷剂量的酶抑制剂阻断。成功的放射性示踪剂在被冷抑制剂阻断的rankl处理的动物中表现出高吸收和保留，并且在骨保护素处理的动物中表现出低吸收，然后可以进一步评估作为潜在的人类成像放射性配体。检测破骨细胞活性增加可能提供新的诊断标准，除了标准的结构完整性测量外，还考虑破骨细胞活性，从而在等待进一步的下游骨增加或损失发生之前，对所选择的治疗方案的有效性提供即时反馈。

DESCRIPTION (provided by applicant): Osteoporosis is estimated to affect over half of adults in the United States over the age of 50, and 1 in 3 women over the age of 80 will suffer a hip fracture; of those who fracture, nearly 15-20% die within 1 year. Overall, the costs associated with osteoporosis in the United States alone were estimated at $19 billion in 2005. This project involves the design, development and evaluation of an new molecular imaging radiotracers for Positron Emission Tomography (PET) studies of osteoporosis and other musculoskeletal diseases (e.g., osteoarthritis, rheumatoid arthritis, osteoporosis) involving potential bone loss. The project will develop radiolabeled forms of known high affinity inhibitors of cathepsin K, a cyteine protease that in highly expressed in osteoclasts and that is responsible for type I collagen degradation and bone resorption. The potential for in vivo imaging of cathepsin K has been demonstrated using optical methods, and this project will extend this concept to a clinically translatable imaging method using radionuclide labeling. This grant will prepare series of radiolabeled inhibitors based on the published cyanopyrimidine and pyrrolopyrimidine scaffolds, using isotopic (carbon-11) substitution or synthesis of fluorinated analogs (for fluorine-18 labeling). The compounds selected as initial targets for carbon-11 labeling all have high affinity for human cathepsin K (< 10 nanomolar), excellent selectivity over cathepsins L and S (100-1000 fold selective) and all are readily labeled using [11C]methyl iodide. New fluorinated analogs where fluoroethyl groups replace methyl groups will be evaluated in vitro for inhibitory action on cathepsin K enzymatic activity, using a fluorescent assay, and potent (<10 nM) inhibitors labeled using analogous [18F]fluoroalkylation reactions. In vivo proof of concept studies to demonstrate osteoclast-dependent localization of radiotracers will be done in rats using focal skeletal injections of RANKL (receptor activator for nuclear facktorB ligand, to induce osteoclastogenesis) or osteoprotogerin (to inhibit osteoclastogenesis). Extent of bone loss will be monitored by microCT. Verification that radioactivity localization is cathepsin-specific will be done in RANKL treated rats and blocking with cold doses of enzyme inhibitor. Successful radiotracers that exhibit high uptake and retention in RANKL-treated animals that is blocked by cold inhibitor, and reduced uptake in osteoprotegrin-treated animals, can then be further evaluated as potential radioligands for human imaging. Detection of increased osteoclastic cellular activity may provide new diagnostic criteria that take osteoclast activity into account in addition to standard measures of structural integrity, and thus provide immediate feedback on the efficacy of a chosen treatment protocol before waiting for further downstream gain or loss of bone to occur. . PUBLIC HEALTH RELEVANCE: Radiolabeled forms of inhibitors of the potent protease cathepsin K will be prepared as radioligands for positron emission tomography (PET) imaging. These radioligands will be evaluated as biomarkers of the up-regulation of numbers and activity of osteoclasts, the cells responsible for the degradation of bone in osteoprorosis and related musculoskeletal diseases.