Cathespin K Radioligands for In Vivo Imaging

描述(申请人提供)：据估计，美国50岁以上的成年人中有一半以上患有骨质疏松症，80岁以上的女性中有1/3将遭受髋部骨折；在骨折的人中，近15%-20%的人在1年内死亡。总体而言，仅在美国，2005年与骨质疏松症相关的成本估计为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肌肉骨骼疾病(如骨关节炎、类风湿性关节炎、骨质疏松症)的正电子发射断层扫描(PET)研究，涉及潜在的骨丢失。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，这是一种在破骨细胞中高表达的胞苷蛋白酶，它负责I型胶原降解和骨吸收。组织蛋白酶K的体内成像的潜力已经被证明使用光学方法，本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的成像方法。这笔赠款将在已发表的氰基嘧啶和吡咯嘧啶支架的基础上，使用同位素(碳-11)取代或合成氟化类似物(用于氟-18标记)来制备一系列放射性标记的抑制剂。被选为碳-11标记起始靶点的化合物都对人组织蛋白酶K有很高的亲和力(~lt；10纳米分子)，比组织蛋白酶L和S有很好的选择性(选择性100-1000倍)，而且都很容易用[11C]甲基碘标记。新的氟乙基取代甲基的类似物将在体外用荧光分析法评估对组织蛋白酶K酶活性的抑制作用，并用类似的[18F]氟烷基化反应标记有效的(&lt；10 nm)抑制剂。体内概念验证研究表明，放射性示踪剂依赖于破骨细胞的定位将通过局部骨骼注射RANKL(核因子B受体激活剂B配体，诱导破骨生成)或骨原激活素(抑制破骨生成)来完成。骨丢失的程度将通过微型CT进行监测。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进行阻断。成功的放射性示踪剂在RANKL治疗的动物中表现出高摄取和滞留，而在冷抑制剂阻断的动物中表现出高摄取和滞留，而在骨前整合素治疗的动物中表现出较低的摄取，然后可以进一步评估为潜在的用于人类成像的放射性配基。对破骨细胞活性增加的检测可以提供新的诊断标准，除了结构完整性的标准测量之外，还可以考虑破骨细胞的活性，从而在等待进一步的下游骨获得或丢失之前提供关于所选治疗方案的有效性的即时反馈。。 公共卫生相关性：将制备放射性标记形式的有效蛋白酶组织蛋白酶K抑制剂，作为正电子发射断层扫描(PET)成像的放射性配基。这些放射性配基将被评估为破骨细胞数量和活性上调的生物标志物，破骨细胞是导致骨质疏松症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.