Cathespin K Radioligands for In Vivo Imaging

描述（由申请人提供）：据估计，骨质疏松症影响美国50岁以上成年人的一半以上，80岁以上妇女中有三分之一将遭受髋部骨折;在骨折的人中，近15-20%在1年内死亡。总体而言，2005年仅在美国与骨质疏松症相关的费用估计为190亿美元。该项目涉及一种新的分子成像放射性示踪剂的设计，开发和评估，用于骨质疏松症和其他肌肉骨骼疾病（例如，骨关节炎、类风湿性关节炎、骨质疏松症），涉及潜在的骨丢失。该项目将开发已知的组织蛋白酶K高亲和力抑制剂的放射性标记形式，组织蛋白酶K是一种在破骨细胞中高度表达的半胱氨酸蛋白酶，负责I型胶原降解和骨吸收。组织蛋白酶K在体内成像的潜力已被证明使用光学方法，该项目将扩展这一概念，临床上可翻译的成像方法，使用放射性核素标记。该资助将基于已发表的氰基嘧啶和吡咯并嘧啶支架，使用同位素（碳-11）取代或氟化类似物的合成（用于氟-18标记）制备一系列放射性标记的抑制剂。选择作为碳-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），对组织蛋白酶L和S具有优异的选择性（100-1000倍选择性），并且都容易使用[11 C]甲基碘标记。将使用荧光测定法在体外评价其中氟乙基取代甲基的新氟化类似物对组织蛋白酶K酶活性的抑制作用，并使用类似的[18 F]氟烷基化反应标记强效（<10 nM）抑制剂。将在大鼠中进行体内概念验证研究，以证明放射性示踪剂的破骨细胞依赖性定位，方法是局部骨骼注射RANKL（核因子B配体的受体激活剂，以诱导破骨细胞生成）或破骨细胞生成素（以抑制破骨细胞生成）。将通过microCT监测骨丢失的程度。将在RANKL给药大鼠中验证放射性定位具有组织蛋白酶特异性，并使用冷剂量的酶抑制剂进行阻断。成功的放射性示踪剂在RANKL处理的动物中表现出高摄取和保留，被冷抑制剂阻断，并且在骨保护素处理的动物中摄取减少，然后可以进一步评价为用于人类成像的潜在放射性配体。检测破骨细胞活性的增加可以提供新的诊断标准，除了结构完整性的标准测量之外，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，提供关于所选治疗方案的功效的即时反馈。. 公共卫生相关性：放射性标记形式的强效蛋白酶组织蛋白酶K的抑制剂将被制备为用于正电子发射断层扫描（PET）成像的放射性配体。这些放射性配体将作为破骨细胞数量和活性上调的生物标志物进行评价，破骨细胞是负责骨质疏松和相关肌肉骨骼疾病中骨降解的细胞。

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.