SiRNAs in the Treatment of Alcoholic Liver Disease

描述(申请人提供)：慢性肝损伤通常会导致纤维化，进而发展为肝硬变。在美国，肝硬变是导致肝功能衰竭、肝细胞癌和肝脏相关死亡的主要原因。目前，肝活检是评估纤维化的唯一方法，但它容易出现抽样错误，具有侵入性，而且有风险。重要的是，目前还没有一种方法可以非侵入性地测量纤维化的进展，即纤维化形成。这种缺乏排除了个体风险评估和临床研究以测试抗纤维化药物。我们的初步数据表明，肝纤维化的分子成像是可能的。我们已经开发出三聚小分子配体，当连接到99mTC-放射性示踪剂时，可以特异性地靶向激活的肝星状细胞(通过PDGF受体)和激活的胆管细胞(通过整合素av？6)。在体外和体内对啮齿动物肝纤维化模型的研究证实了它们的高亲和力(在低纳摩尔/皮摩尔范围内)和它们检测肝纤维化的特异性远远高于背景信号。在这个方案中，我们计划：1)通过使用具有更长和更坚硬的连接基的金刚烷骨架来提高99mTc-SPECT/CT显像的三聚化的av？6和PDGF-β受体靶向配体的亲和力、生物分布和清除性；2)通过增加惰性的块状结构域来优化体内的生物分布和清除性；3)重新合成靶向配体作为18F衍生物用于PET/CT成像并验证其无创定量纤维化的能力；4)在两个体内肝纤维化模型中验证靶向结构体在抗纤维化药物治疗前后的作用；5)减少肝脏中的高非特异性背景信号，6)将microSPECT和microPET与MRI相结合，提高解剖配准的准确性。完成特定目标将产生一个或多个优化的放射性示踪剂，以及SPECT、PET、CT和MRI成像的优化工作流程。因此，它将创建第一个检测和量化肝纤维化的集成系统，为该技术快速转化为临床奠定基础。这项技术将允许：1)纤维化进展的个体风险评估，以及2)通过测量单一剂量或仅几剂抗纤维化药物后信号抑制的程度，在小群个体化患者中快速定量测试抗纤维化药物及其组合。公共卫生相关性：慢性肝病通常导致肝纤维化(形成疤痕组织)，进而可发展为肝硬化和终末期肝功能衰竭。目前，侵入性活检是评估患者纤维化进展(即纤维化形成)和抗纤维化药物有效性的唯一方法。我们已经开发了两种针对纤维化形成过程中不受调控的细胞表面标志物的靶向小分子配体，并在初步研究中表明，这些试剂在体外和体内作为SPECT诊断试剂表现良好。在这项研究中，我们描述了一套系统的实验，旨在优化SPECT/MRI和PET/MRI成像的纤维化特异性放射性示踪剂，目标是将其快速转移到临床。

DESCRIPTION (provided by applicant): Chronic liver damage usually leads to fibrosis, which can progress to cirrhosis. Cirrhosis is the primary cause of hepatic failure, hepatocellular cancer, and liver-related death in the United States. Currently, liver biopsy is the only method to assess fibrosis, but it is prone to sampling error, is invasive, and is risky. Importantly, there exists no method that permits non-invasive measurement of the progression of fibrosis, i.e., fibrogenesis. This lack has precluded individual risk assessment and clinical studies to test antifibrotic agents. Our preliminary data show that molecular imaging of hepatic fibrogenesis is possible. We have developed trimerized small molecule ligands that, when linked to a 99mTc-radiotracer, specifically target activated hepatic stellate cells (via the PDGF-¿ receptor) and activated cholangiocytes (via integrin av¿6). In vitro and in vivo studies in rodent models of liver fibrosis confirmed their high affinity (in the low nanomolar/picomolar) range and their specificity to detect hepatic fibrogenesis well above the background signal. In this proposal we plan to: 1) improve the affinity, biodistribution, and clearance of trimerized av¿6 and PDGF- ¿ receptor targeting ligands for 99mTc-based SPECT/CT imaging by use of adamantane backbones with longer and more rigid linkers, 2) optimize in vivo biodistribution and clearance by addition of an inert bulking domain, 3) resynthesis of the targeting ligands as 18F derivatives for PET/CT imaging and validation of their ability to quantify fibrogenesis non-invasively, 4) validate the targeting constructs in two in vivo models of hepatic fibrogenesis before and after treatment with antifibrotic agents, 5) reduce the high nonspecific background signal in liver, and 6) combine microSPECT and microPET with MRI to improve anatomical co-registration. Completion of the Specific Aims will result in one or more optimized radiotracers, and an optimized workflow for SPECT, PET, CT, and MRI imaging. As such, it will create the first integrated system for detection and quantitation of hepatic fibrogenesis, laying the foundation for rapid translation of the technology to the clinic. This technology will permit: 1) individual risk assessment of fibrosis progression, and 2) the rapid quantitative testing of antifibrotic agents and their combinations in small groups of individualized patients by measuring the extent of signal suppression after a single or only a few doses of these agents. Public Health Relevance: Chronic liver disease often leads to liver fibrosis (formation of scar tissue), which in turn can progress to cirrhosis and end-stage liver failure. Invasive biopsy is presently the only way to assess patients for the progression of fibrosis (i.e., fibrogenesis) and the effectiveness of antifibrotic drugs. We have developed two targeted small molecule ligands specific for cell surface markers unregulated during fibrogenesis and show in preliminary studies that these agents perform well as SPECT diagnostic agents in vitro and in vivo. In this study, we describe a systematic set of experiments aimed at optimizing fibrogenesis-specific radiotracers for SPECT/MRI and PET/MRI imaging, with the goal being their rapid translation to the clinic.