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

基本信息

批准号：
10558471
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
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-04-01 至 2025-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10558471
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 Dryness Encapsulated Endocrinology Excretory function Exposure to FDA approved Fiber Formulation Foundations Free Radicals 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 Polymers 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 nanosized novel therapeutics peptide drug peptide hormone polymerization professor receptor response side effect success sugar tool

项目摘要

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.

项目摘要胰高血糖素与肝脏中的受体相互作用，以提高葡萄糖水平。通常释放胰高血糖素内源性。然而，对于某些I型糖尿病等疾病，低血糖定义为血糖水平低于70 mg/dl的患者发生。低血糖的副作用从头晕摇摇欲坠，使视力和癫痫发作模糊，昏迷和死亡。为了治疗这种潜在的致命状况，胰高血糖素通过注射给药。然而，胰高血糖素通常在数小时内在溶液中灭活，形成淀粉样蛋白就像对患者有毒且潜在危险的纤维一样。这需要复杂的注射系统其中，肽作为干粉保存，并在使用前溶解在酸性pH中。酸度配方会在注射部位引起疼痛。最近经过批准的配方绕过了这个问题，但是利用有机溶剂，这是不可取的。最近，PI发现了稳定的生物措施纳米凝胶中性溶液中的胰高血糖素至少三周，并保持胰高血糖生物活性。纳米凝胶在大自然中发现的糖稳定剂，黄核，利用胰高血糖素作为交叉链接的合成。在温和的下还原条件释放了胰高血糖素。释放的胰高血糖素和胰高血糖素纳米颗粒为生物活性。这项工作很重要，因为它表明可以利用海藻糖纳米凝胶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，建议建立在这个基础上准备一种新的配方，以稳定和送给胰高血糖素。具体来说，假设海藻糖纳米凝胶将在水溶液中稳定胰高血糖素，在室温下，将释放活跃胰高血糖素，并在体内保持安全和非免疫原性。为了检验这一假设并达到目标，三个提出了具体目标。首先是开发均匀尺寸的纳米凝胶并评估长期稳定性纳米颗粒配方中的胰高血糖素的。实现这一目标，聚（甲基丙烯酸甲丙烯酸三核-CO-吡啶基二硫基甲基丙烯酸酯）将与双硫化聚乙二醇合成并交联。胰高血糖素会通过共价和物理封装加载以形成均匀的凝胶。由此产生的胰高血糖素纳米凝胶将是表征并研究了长期溶液和固相稳定性。观察聚合的标准测试将进行胰高血糖素和纳米颗粒以及胰高血糖素的化学变化。第二是研究体外和体内功效和胰葡萄糖纳米凝胶的生物利用度。标准的蜂窝测定将是用于确定活动。然后，原位肝灌注分析将评估灌注葡萄糖浓度暴露于纳米凝胶并允许分子信号验证。葡萄糖反调节胰岛素耐受性测试将验证全动物的活性。第三个目的是确定体内安全纳米颗粒配方。具体而言，急性毒性，生物分布，排泄，药代动力学，抗体在小鼠中将测试对纳米凝胶的产生和细胞因子反应。这项工作与长期提供新的治疗工具，以安全有效地管理I型糖尿病患者的葡萄糖水平。