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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，从而提高血糖水平。正常情况下会释放胰高血糖素内生性的。然而，对于一些疾病，如I型糖尿病，低血糖定义为血糖水平低于70毫克/分升的情况出现在大量患者中。低血糖有一系列副作用，包括头晕和颤抖，视力模糊和癫痫发作，昏迷和死亡。为了治疗这种潜在的致命疾病，胰高血糖素是通过注射给药的。然而，胰高血糖素通常在几个小时内在溶液中失活，形成淀粉样蛋白- 比如有毒的纤维，对患者有潜在的危险。这就需要一个复杂的注射系统其中该肽以干粉的形式保存，并在使用前在酸性pH中溶解。白酒的酸度配方会导致注射部位疼痛。最近批准的一种配方绕过了这个问题，但使用有机溶剂，这是不受欢迎的。最近，PI发现了具有生物响应性的纳米凝胶，胰升糖素在中性溶液中至少维持三周，并保持胰升糖素生物活性。纳米凝胶含有一种以高血糖素为交联剂，合成了自然界中发现的糖稳定剂海藻糖。温和地下还原条件胰高血糖素被释放。释放的胰升糖素和胰升糖素纳米粒生物活性。这项工作很重要，因为它表明海藻糖纳米凝胶可以用来稳定高血糖素在中性溶液中，需要时释放活性多肽。在此，建议在此基础上建立制备稳定和递送高血糖素的新剂型。具体地说，假设海藻糖纳米凝胶将稳定水溶液中的高血糖素，并在室温下释放活性在体内是安全和非免疫原性的。为了检验这一假设并达到目标，有三个提出了具体的目标。首先是开发尺寸均匀的纳米凝胶，并评价其长期稳定性。纳米颗粒配方中的胰高血糖素。实现这一目标的是聚甲基丙烯酸酯海藻糖-吡啶二硫化物将合成甲基丙烯酸酯)，并与二硫代化聚乙二醇交联制得。胰高血糖素会通过共价和物理包埋来负载，形成均匀的凝胶。由此产生的胰升糖素纳米凝胶将是对其长期溶解和固相稳定性进行了表征和研究。观察聚合的标准测试将进行胰升糖素和纳米颗粒的化学变化和胰高血糖素的化学变化。第二个将是研究胰升糖素纳米凝胶的体内外有效性和生物利用度。标准的细胞分析将是用来确定活动。然后，原位肝脏灌注法将评估灌流液中的葡萄糖浓度。一旦暴露在纳米凝胶中，就可以进行分子信号验证。过程中的血糖逆调节胰岛素耐量测试将验证整个动物的活动。第三个目标将是确定在体内的安全性纳米颗粒配方。具体来说，急性毒性、生物分布、排泄、药代动力学、抗体对纳米凝胶的生成和细胞因子反应将在小鼠身上进行测试。这项工作是与长期的目的为I型糖尿病患者安全有效地控制血糖水平提供新的治疗手段。