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.

项目总结