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; 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.

项目摘要 滥用酗酒和肥胖会导致肝感染，脂肪变性和纤维化。肝细胞 损伤导致肝细胞和库普弗细胞（KCS）释放炎症细胞因子，这会导致引起 肝星状细胞的激活（HSC）。如果未解决，则可能导致肝纤维化和发展 肝硬化。刺猬（HH）信号传导调节肝纤维化的多个途径，包括上皮到 - 间充质转变（EMT），HSC激活和炎症。此外，慢性肝病与 随着miRNA的失调。特别是，miR-96在肝损伤后进行更新并促进脂肪肝 疾病（FLD）。 miR-96引起胰岛素受体（INSR）和胰岛素受体底物（IRS）-1的功能故障 导致胰岛素信号传导和糖原合成受损。此外，miR-96下调了Smad7和 FOXO1-3并促进转化生长因子-BETA 1（TGF-β1）介导的肝纤维化。在我们的初步中 研究，我们证明了HH信号配体在内的上调，包括PTCH1，SHH和GLI2原因 在5％乙醇和高脂饮食（HFD）的小鼠中，胶原蛋白和脂肪沉积显着增加。我们 合成了一种新型的HH途径抑制剂2-氯-N 1- [4-氯-3-（2-吡啶基）苯基] -n4，N4-双 - 吡啶基甲基）-1,4-苯甲酰辅酰胺（MDB5），具有GLI1/2抑制活性在纳摩尔（NM）中 专注。用MDB5治疗HFD和酒精诱导的肝病（ALD）小鼠导致 天冬氨酸氨基转移酶（AST）和丙氨酸的肝损伤标记水平显着降低 氨基转移酶（ALT）和进一步的消融GLI2及其靶基因。我们在ALD和HFD中的miRNA分析 识别的miR-96始终更新的鼠标肝脏。目标扫描分析表明miR-96目标 几种抗炎和抗纤维化基因。肝细胞中miR-96表达的敲低 具有抗MIR-96的HSC导致受影响的基因SMAD7和FOXO3的恢复。我们合成 糖酸（GA）共轭GA-PEG-P（ASP）-G-DC-G-TEPA共聚物用于肝特异性体内递送 分别为MDB5和抗MIR-96。纤维化中MDB5浓度显着增加 MDB5加载的GA-NP的系统给药后1H肝脏。 TGF-β和HH信号传导串扰 TGF-β1通过RAS信号传导的下游诱导GLI-1。因此，我们假设 使用GA-NP的MDB5和抗MIR-96组合疗法可以防止酒精和脂肪诱导的肝损伤， 和纤维化。我们的具体目的是i）建立HH抑制剂MDB5对酒精的治疗效率 高脂饮食诱发肝损伤； ii）建立miR-96在HFD和ALD小鼠模型中的纤维化作用， 和iii）确定用MDB5和抗MIR-96的肝脏靶向NP的治疗效率用于治疗 HFD和ALD鼠标模型。我们的长期目标是了解ALD和 NAFLD至肝纤维化，并建立新的肝脏特异性治疗方法。