Development and Preclinical Evaluation of Nanoformulations in Liver Fibrotic Mice

肝纤维化小鼠纳米制剂的开发和临床前评价

• 批准号: 10639037
• 负责人: Ram I. Mahato
• 金额: $ 42.08万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2028-02-29
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10639037
• 关键词: ADD-1 protein; Ablation; Adrenal Cortex Hormones; Adverse effects; Affect; Alanine Transaminase; Alcohol abuse; Alcoholic Liver Diseases; Alcoholic steatohepatitis; Alcohols; Animal Model; Anti-Inflammatory Agents; Antioxidants; Apoptosis; Aspartate Transaminase; B-Lymphocytes; Binding; Biodistribution; Cells; Cessation of life; Chemicals; Cirrhosis; Collagen; Combined Modality Therapy; Deposition; Development; Diet; Disease; Disease Progression; Drug Kinetics; Economic Burden; Erinaceidae; Ethanol; Etiology; Evaluation; Extracellular Matrix; FOXO1A gene; FOXO3A gene; Fatty acid glycerol esters; Fibrosis; Formulation; GLI gene; GLI-1; GLI2 gene; Genes; Glycogen; Glycyrrhetinic Acid; Goals; Health; Hepatic; Hepatic Stellate Cell; Hepatocyte; High Fat Diet; Immunosuppressive Agents; Impairment; In Vitro; Inflammation; Inflammatory; Inflammatory Response; Insulin Receptor; Kupffer Cells; Ligands; Liquid substance; Liver; Liver Fibrosis; Liver diseases; MADH7 gene; Macrophage; Mediating; Metabolic Diseases; MicroRNAs; Minor; Mus; Myofibroblast; Obesity; Pathogenesis; Pathway interactions; Patients; Pharmaceutical Preparations; Play; Production; Regulatory Pathway; Role; SHH gene; Sampling; Scanning; Signal Induction; Signal Pathway; Signal Transduction; TGFB1 gene; Therapeutic; Toxic effect; Transfection; Transforming Growth Factor beta; Transforming Growth Factors; Treatment Efficacy; Up-Regulation; Validation; alcohol prevention; cell injury; chronic liver disease; copolymer; cytokine; diacylglycerol O-acyltransferase; effective therapy; epithelial to mesenchymal transition; fatty liver disease; global health; hepatoprotective; immunoreaction; in vivo; inhibitor; innovation; insulin receptor substrate 1 protein; insulin signaling; knock-down; lipid biosynthesis; liver inflammation; liver injury; liver repair; mRNA Expression; mRNA sequencing; mortality; mouse model; nanoformulation; nanomedicine; nanomolar; nanoparticle; non-alcoholic fatty liver disease; nonalcoholic steatohepatitis; novel; preclinical evaluation; prevent; progenitor; repaired; restoration; side effect

PROJECT SUMMARY Liver injury from alcohol abuse, and obesity can lead to liver inflammation, steatosis, and fibrosis. Liver cell damage results in the release of inflammatory cytokines from hepatocytes and Kupffer cells (KCs), which cause the activation of hepatic stellate cells (HSCs). If unresolved, it may result in liver fibrosis and progression to cirrhosis. Hedgehog (Hh) signaling regulates multiple pathways in liver fibrosis, including epithelial-to- mesenchymal transition (EMT), HSC activation, and inflammation. Further, chronic liver diseases are associated with the dysregulation of miRNAs. Specifically, miR-96 is upregulated after liver damage and promotes fatty liver disease (FLD). miR-96 caused malfunctioning of insulin receptor (INSR) and insulin receptor substrate (IRS)-1 results in impaired insulin signaling and glycogen synthesis. Further, miR-96 downregulates SMAD7 and FOXO1-3 and promotes transforming growth factor-beta 1 (TGF-β1) mediated liver fibrosis. In our preliminary studies, we demonstrated the upregulation of Hh signaling ligands including PTCH1, SHH, and GLI2 cause significant increase in collagen and fat deposition in 5% ethanol and high-fat diet (HFD) fed mouse. We synthesized a novel Hh pathway inhibitor 2-chloro-N 1-[4-chloro-3-(2-pyridinyl) phenyl]-N4, N4-bis(2- pyridinylmethyl)-1,4-benzenedicarboxamide (MDB5) with GLI1/2 inhibitory activity in nanomolar (nM) concentration. Treatment of both HFD and alcohol-induced liver disease (ALD) mice with MDB5 resulted in a significant decrease in the levels of liver injury markers aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and further ablate GLI2, and its target genes. Our miRNA profiling in ALD and HFD mouse liver identified miR-96 was consistently upregulated. Target Scan analysis revealed that miR-96 targets several anti-inflammatory and anti-fibrogenic genes. The knockdown of miR-96 expression in hepatocytes and HSCs with anti-miR-96 resulted in the restoration of affected genes SMAD7 and FOXO3. We synthesized glycyrrhetinic acid (GA) conjugated GA-PEG-P(Asp)-g-DC-g-TEPA copolymers for liver-specific in vivo delivery of MDB5 and anti-miR-96, respectively. There was a significant increase in MDB5 concentration in the fibrotic liver at 1h post systemic administration of MDB5 loaded GA-NPs. TGF-β and Hh signaling crosstalk whereby TGF-β1 induces GLI-1 through downstream consequence of RAS signaling. Therefore, we hypothesize that the combination therapy of MDB5 and anti-miR-96 using GA-NPs could prevent alcohol and fat induced liver injury, and fibrosis. Our specific aims are to i) establish the therapeutic efficacy of the Hh inhibitor MDB5 on alcohol and high fat diet induced liver injury; ii) establish the profibrotic role of miR-96 in in HFD and ALD mouse models, and iii) determine the therapeutic efficacy of liver targeted NPs loaded with MDB5 and anti-miR-96 for treating HFD and ALD mouse models. Our long-term goal is to understand the progressive mechanisms of ALD and NAFLD to liver fibrosis, and establish new, liver-specific treatments.

项目概要 酗酒和肥胖引起的肝损伤可导致肝脏炎症、脂肪变性和肝细胞纤维化。 损伤导致肝细胞和库普弗细胞 (KC) 释放炎症细胞因子，从而导致 肝星状细胞（HSC）的激活如果得不到解决，可能会导致肝纤维化并进展为肝纤维化。 肝硬化 Hedgehog (Hh) 信号调节肝纤维化的多种途径，包括上皮细胞到肝纤维化。 此外，间质转化（EMT）、HSC 激活和炎症也与慢性肝病相关。 具体而言，miR-96 在肝损伤后表达上调，并促进脂肪肝。 疾病（FLD）。miR-96 导致胰岛素受体（INSR）和胰岛素受体底物（IRS）-1 功能障碍。 导致胰岛素信号传导和糖原合成受损。此外，miR-96 下调 SMAD7 和 SMAD7。 FOXO1-3 并促进转化生长因子-β1 (TGF-β1) 介导的肝纤维化。 研究中，我们证明了 Hh 信号配体（包括 PTCH1、SHH 和 GLI2）的上调导致 5%乙醇和高脂饮食（HFD）喂养的小鼠的胶原蛋白和脂肪沉积显着增加。 合成了一种新型 Hh 通路抑制剂 2-氯-N 1-[4-氯-3-(2-吡啶基)苯基]-N4, N4-bis(2- 吡啶基甲基)-1,4-苯二甲酰胺 (MDB5)，具有纳摩尔 (nM) 水平的 GLI1/2 抑制活性 用 MDB5 治疗 HFD 和酒精诱导的肝病 (ALD) 小鼠，结果显示： 肝损伤标志物天冬氨酸转氨酶 (AST) 和丙氨酸水平显着降低 转氨酶 (ALT)，并进一步消除 GLI2 及其靶基因在 ALD 和 HFD 中的 miRNA 分析。 小鼠肝脏发现 miR-96 持续上调 靶标扫描分析显示 miR-96 的靶标。 肝细胞中多种抗炎和抗纤维化基因的表达下调。 具有抗 miR-96 的 HSC 导致受影响的基因 SMAD7 和 FOXO3 的恢复。 甘草次酸 (GA) 缀合 GA-PEG-P(Asp)-g-DC-g-TEPA 共聚物，用于肝脏特异性体内递送 纤维化组织中MDB5和抗miR-96的浓度显着增加。 因此，在全身施用装载 MDB5 的 GA-NP 后 1 小时，肝脏中的 TGF-β 和 Hh 信号串扰。 TGF-β1 通过 RAS 信号传导的下游结果诱导 GLI-1。 使用 GA-NPs 联合治疗 MDB5 和抗 miR-96 可以预防酒精和脂肪引起的肝损伤， 我们的具体目标是 i) 确定 Hh 抑制剂 MDB5 对酒精的治疗功效。 和高脂肪饮食诱导的肝损伤；ii) 建立 miR-96 在 HFD 和 ALD 小鼠模型中的促纤维化作用， iii) 确定负载 MDB5 和抗 miR-96 的肝脏靶向 NP 用于治疗的治疗效果 HFD 和 ALD 小鼠模型我们的长期目标是了解 ALD 和 ALD 的进展机制。 NAFLD 到肝纤维化，并建立新的肝脏特异性治疗方法。