Pre-clinical Validation of A Novel Protein Drug Candidate for ASH and NASH Treatment

总结 酒精性脂肪性肝炎（ASH）和非酒精性脂肪性肝炎（NASH）影响了大量的 在美国和世界各地的人口。目前，主要未满足的医疗需求包括缺乏方法或 特异性耗竭活化HSC和毛细血管化LSEC的试剂以及非侵入性 可视化胶原蛋白积聚、HSC活化和LSEC的方法和试剂 纤维化肝脏中的毛细血管化。我们已经开发了一种蛋白质药物候选物（称为 “ProAgio”），其在新位点靶向整合素αvβ3以诱导表达整合素αvβ3的细胞凋亡。 一种新的机制。ProAgio特异性诱导表达整合素β 3的细胞凋亡 通过募集和激活caspase 8在细胞质结构域的细胞具有高功效。我们 我们的初步研究表明，对携带肝纤维化/肝硬化的小鼠进行治疗， TAA/酒精CCl_4和高脂饮食诱导NASH小鼠ProAgio逆转肝脏 纤维化/肝硬化。此外，我们还开发了新型蛋白质MRI造影剂（ProCA）， 使我们能够评估纤维化肝脏中的胶原蛋白含量和整合素αvβ3阳性HSC和LSEC。 纤维化小鼠的磁共振成像显示了我们开发的造影剂的上级特性 用于胶原蛋白和整合素β 1/β 3阳性细胞评估。健康人的初步毒性分析 小鼠表明ProAgio和我们开发的MRI造影剂在非常低的剂量下对小鼠没有毒性， 高剂量该项目的目标是大力进行ProAgio作为药物的临床前验证 适合ASH/NASH患者治疗。我们将通过三个具体目标实现我们的目标。 目的1是检测ProAgio逆转高脂饮食肝纤维化的有效性 以及多次酗酒和慢性酗酒诱导的ASH模型。调查 ProAgio在ASH小鼠模型中的作用将进一步临床前验证ProAgio作为 ASH/NASH治疗药物。目的2是监测和验证ProAgio对胶原的影响 用我们研制的MRI造影剂对纤维化肝进行MRI成像。我们的MR 成像辅助验证不仅将验证ProAgio作为ASH/NASH的有效性， 治疗剂，而且还验证药物作用的靶点和机制。目标3是临床前 通过毒理学（TOX）验证ProAgio作为ASH/NASH治疗候选药物， 药代动力学（PK）分析。我们的研究将为ASH/NASH治疗开辟一条新的途径， 通过蛋白质设计进行诊断/预后。我们研究的成功不仅能开发出一种新的蛋白质 用于肝纤维化/肝硬化治疗的药物，而且还测试高效的MRI造影剂， 使我们能够准确和非侵入性地监测纤维化的进展和消退， 评估治疗效果。这种发展有望填补主要的医疗空白 并有助于设计治疗策略以逆转纤维化和随访高危患者。

Summary Alcoholic Steatohepatitis (ASH) and Nonalcoholic Steatohepatitis (NASH) affects a large population in US and worldwide. Currently, major unmet medical needs include lack of method or agent to specifically deplete activated HSC and capillarized LSEC as well as noninvasive methodology and reagents to visualize collagen build-up, HSC activation, and LSEC capillarization in fibrotic liver. We have developed a protein drug candidate (referred to as “ProAgio”) that targets integrin αvβ3 at a novel site to induce apoptosis of integrin αvβ3 expressing cells by a novel mechanism. ProAgio specifically induces apoptosis of integrin v3 expressing cells with a high efficacy by recruiting & activating caspase 8 at cytoplasmic domain of. We demonstrated in our preliminary studies that treatment of mice that carries liver fibrosis/cirrhosis induced by TAA/alcohol CCl4 and the high-fat diet induce NASH mice with ProAgio reversed liver fibrosis/cirrhosis. In addition, we have developed novel protein MRI contrast agents (ProCAs) that allow us to assess collagen contents and integrin αvβ3 positive HSCs & LSECs in fibrotic liver. MR imaging of fibrotic mice demonstrated superior properties of our developed contrast agents for collagen and integrin v3 positive cell assessment. Preliminary toxicity analyses with healthy mice indicate that ProAgio and our developed MRI contrast agents are not toxic to mice at very high dose. The goal of this project is to vigorously pre-clinical validation of ProAgio as a drug candidate for ASH/NASH patient treatment. We will achieve our objective by three specific aims. Aim 1 is to examine the effectiveness of ProAgio in reversal of liver fibrosis using high-fat diet plus multiple binge alcohol and chronic alcohol binge induced ASH models. Investigation of the effects of ProAgio in ASH mouse models will further pre-clinical validation of ProAgio as an ASH/NASH treatment drug. Aim 2 is to monitor and validate the effects of ProAgio on collagen and HSC in fibrotic liver by MR imaging using our developed MRI contrast agents. Our MR imaging aided validation will not only validate the effectiveness of ProAgio as an ASH/NASH treatment agent, but also validate the target and mechanism of drug action. Aim 3 is Pre-clinical validations of ProAgio as an ASH/NASH treatment drug candidate via toxicology (TOX) and pharmacokinetic (PK) analyses. Our study will open a new avenue for ASH/NASH treatment and diagnosis/prognosis by protein design. Success in our studies will not only develop a new protein drug for liver fibrosis/cirrhosis treatment but also test highly effective MRI contrast agents that allow us to accurately and non-invasively monitor fibrosis progression and regression for assessment of treatment effects. Such development is expected to fill in the major medical gaps and to facilitate to devise treatment strategy to reverse fibrosis and follow high risk patients.