SiRNAs in the Treatment of Alcoholic Liver Disease

描述（由申请人提供）：慢性肝损伤通常导致纤维化，可进展为肝硬化。在美国，肝硬化是肝衰竭、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，但它容易出现采样错误，具有侵入性，并且风险较大。重要的是，不存在允许非侵入性测量纤维化进展的方法，即，纤维化这种缺乏已经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。我们已经开发了三聚化的小分子配体，当与99 mTc-放射性示踪剂连接时，特异性靶向激活的肝星状细胞（通过PDGF-β受体）和激活的胆管细胞（通过整合素α v β 6）。在啮齿动物肝纤维化模型中的体外和体内研究证实了它们的高亲和力（在低纳摩尔/皮摩尔）范围内，以及它们在检测远高于背景信号的肝纤维化方面的特异性。在这项提案中，我们计划：1）提高三聚体av 6和PDGF-3的亲和力，生物分布和清除率。通过使用具有更长和更刚性的接头的金刚烷骨架，用于基于99 mTc的SPECT/CT成像的受体靶向配体，2）通过添加惰性填充结构域优化体内生物分布和清除，3）将靶向配体再合成为用于PET/CT成像的18F衍生物，并验证它们非侵入性地定量纤维化发生的能力，4）在用抗纤维化剂治疗之前和之后的两种肝纤维化发生的体内模型中验证靶向构建体，5）降低肝脏中的高非特异性背景信号，以及6）将联合收割机microSPECT和microPET与MRI结合以改善解剖配准。特定目标的完成将产生一种或多种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，它将创建第一个用于检测和定量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，和2）通过测量单次或仅几次剂量的抗纤维化剂后的信号抑制程度，在一小组个体化患者中快速定量检测抗纤维化剂及其组合。公共卫生相关性：慢性肝病通常会导致肝纤维化（瘢痕组织的形成），这反过来又会发展为肝硬化和终末期肝衰竭。侵入性活组织检查是目前评估患者纤维化进展的唯一方法（即，纤维化）和抗纤维化药物的有效性。我们已经开发了两种靶向小分子配体，其特异性针对纤维化过程中不受调节的细胞表面标志物，并在初步研究中显示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，我们描述了一组系统的实验，旨在优化SPECT/MRI和PET/MRI成像的纤维化特异性放射性示踪剂，目标是将其快速转化为临床。

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.