Stabilization of Glucagon by Trehalose Gylcopolymer Nanogels

海藻糖乙二醇聚合物纳米凝胶对胰高血糖素的稳定性

• 批准号: 10372210
• 负责人: Heather D Maynard
• 金额: $ 38.05万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10372210
• 关键词: Acidity; Address; Amyloid; Animals; Antibodies; Biochemistry; Biodistribution; Biological Assay; Biological Availability; Biophysics; Blood Glucose; Cell Culture Techniques; Cellular Assay; Cessation of life; Charge; Chemicals; Chemistry; Coma; Crosslinker; Dangerousness; Detection; Diabetes Mellitus; Disease; Disulfides; Dizziness; Dose; Drug Delivery Systems; Drug Kinetics; Endocrinology; Excretory function; Exposure to; FDA approved; Fiber; Formulation; Foundations; Free Radicals; Freezing; Gel; Generations; Glucagon; Glucose; Goals; Hepatocyte; Hormones; Hour; Human; Hypertension; Hypoglycemia; Immune response; In Situ; In Vitro; Inflammatory Response; Injections; Insulin; Insulin-Dependent Diabetes Mellitus; Investigation; Lipopolysaccharides; Liver; Longitudinal Studies; Measures; Medicine; Methacrylates; Molecular; Mus; NanoGel; Nature; Organic solvent product; Ovalbumin; Pain; Pathway interactions; Patients; Peptides; Perfusion; Phase; Positron-Emission Tomography; Powder dose form; Proteins; Reagent; Refrigeration; Safety; Saline; Seizures; Signal Transduction; Site; Solid; System; Techniques; Temperature; Testing; Therapeutic; Time; Tissues; Trehalose; Validation; Vision; Work; acute toxicity; aged; aqueous; biomaterial compatibility; counterregulation; crosslink; cytokine; diabetic patient; ethylene glycol; experience; experimental study; in vivo; insulin tolerance; nanoparticle; novel therapeutics; peptide drug; peptide hormone; polymerization; professor; receptor; response; side effect; success; sugar; tool

Project Summary Glucagon interacts with receptors in the liver to raise glucose levels. Normally glucagon is released endogenously. Yet for some diseases such as Type I diabetes, hypoglycemia defined as blood glucose level below 70 mg/dL occurs in a large number of patients. Hypoglycemia has side effects ranging from dizziness and shakiness, to blurry vision and seizures, to coma and death. To treat this potentially fatal condition, glucagon is administered via injection. However, glucagon is typically inactivated in solution within hours, forming amyloid- like fibers that are toxic and potentially dangerous for patients. This necessitates a complicated injection system wherein the peptide is kept as a dry powder and dissolved in acidic pH right before use. The acidity of the formulation causes pain at the injection site. A recently approved formulation circumvents this issue, but instead utilizes organic solvent, which is undesirable. Recently the PI discovered bioresponsive nanogels that stabilize glucagon in neutral solution for at least three weeks and maintain glucagon bioactivity. Nanogels containing a sugar stabilizer found in Nature, trehalose, were synthesized utilizing glucagon as the cross-linker. Under mildly reductive conditions the glucagon was released. Released glucagon and the glucagon nanoparticles were bioactive. This work is important because it suggests that trehalose nanogels can be utilized to stabilize glucagon in neutral solution and release active peptide when needed. Herein, it is proposed to build upon this foundation to prepare a new formulation for the stabilization and delivery of glucagon. Specifically, it is hypothesized that trehalose nanogels will stabilize glucagon in aqueous solutions and at room temperature, will release active glucagon and be safe and non-immunogenic in vivo. To test this hypothesis and meet the objectives, three specific aims are proposed. The first is to develop uniform sized nanogels and evaluate the long term stability of glucagon in the nanoparticle formulation. The achieve this, poly(methacrylate trehalose-co-pyridyl disulfide methacrylate) will be synthesized and cross-linked with bisthiolated poly(ethylene glycol). Glucagon will be loaded by covalent and physical encapsulation to form uniform gels. The resulting glucagon nanogels will be characterized and studied for long term solution and solid phase stability. Standard tests to observe aggregation of the glucagon and nanoparticles and chemical changes of the glucagon will be undertaken. Second will be to investigate in vitro and in vivo efficacy and bioavailability of glucagon nanogels. Standard cellular assays will be utilized to determine activity. Then an in situ liver perfusion assay will assess perfusate glucose concentrations upon exposure to the nanogels and allow for molecular signaling validation. Glucose counterregulation during insulin tolerance test will validate activity in whole animals. The third aim will be to determine in vivo safety of the nanoparticle formulation. Specifically, acute toxicity, biodistribution, excretion, pharmacokinetics, antibody generation and cytokine response to the nanogels will be tested in mice. This work is aligned with the long-term goal to provide new therapeutic tools to safely and effectively manage glucose levels for Type I diabetes patients.

项目概要 胰高血糖素与肝脏中的受体相互作用以提高血糖水平。正常情况下胰高血糖素会被释放 内生的。然而，对于某些疾病，例如 I 型糖尿病，低血糖定义为血糖水平 大量患者的血浆浓度低于 70 mg/dL。低血糖有头晕等副作用 颤抖、视力模糊和癫痫发作、昏迷和死亡。为了治疗这种可能致命的疾病，胰高血糖素 通过注射施用。然而，胰高血糖素通常在溶液中数小时内失活，形成淀粉样蛋白 例如有毒且对患者有潜在危险的纤维。这需要复杂的注射系统 其中肽以干粉形式保存并在使用前溶解在酸性pH中。的酸度 制剂会引起注射部位疼痛。最近批准的配方规避了这个问题，但取而代之的是 使用有机溶剂，这是不希望的。最近，PI 发现了生物响应纳米凝胶，可以稳定 胰高血糖素在中性溶液中保存至少三周并保持胰高血糖素生物活性。纳米凝胶含有 自然界中发现的糖稳定剂海藻糖是利用胰高血糖素作为交联剂合成的。轻轻地下 还原条件下胰高血糖素被释放。释放的胰高血糖素和胰高血糖素纳米颗粒 生物活性。这项工作很重要，因为它表明海藻糖纳米凝胶可用于稳定胰高血糖素 在中性溶液中并在需要时释放活性肽。在此，建议在此基础上 制备用于稳定和递送胰高血糖素的新制剂。具体来说，假设 海藻糖纳米凝胶将稳定水溶液中的胰高血糖素，并在室温下释放活性物质 胰高血糖素，并且在体内安全且无免疫原性。为了检验这一假设并实现目标，三个 提出了具体目标。首先是开发尺寸均匀的纳米凝胶并评估其长期稳定性 纳米颗粒制剂中的胰高血糖素。为了实现这一目标，聚(甲基丙烯酸酯海藻糖-共-吡啶二硫化物 甲基丙烯酸酯）将被合成并与双硫醇化聚（乙二醇）交联。胰高血糖素将会 通过共价和物理封装形成均匀的凝胶。所得胰高血糖素纳米凝胶将是 对其长期溶液和固相稳定性进行了表征和研究。观察聚集的标准测试 将进行胰高血糖素和纳米颗粒的分析以及胰高血糖素的化学变化。第二个将是 研究胰高血糖素纳米凝胶的体外和体内功效和生物利用度。标准细胞测定将是 用于确定活性。然后原位肝脏灌注测定将评估灌注液葡萄糖浓度 暴露于纳米凝胶后并允许分子信号传导验证。期间的血糖反调节 胰岛素耐量测试将验证整个动物的活性。第三个目标是确定体内安全性 纳米粒子配方。具体来说，急性毒性、生物分布、排泄、药代动力学、抗体 纳米凝胶的产生和细胞因子对纳米凝胶的反应将在小鼠中进行测试。这项工作符合长期目标 目标是提供新的治疗工具来安全有效地管理 I 型糖尿病患者的血糖水平。