Modification of insulin molecule for controlled delivery at basal level from triblock copolymers

 描述(申请人提供)：健康人有两种类型的胰岛素分泌-基础和刺激。基础胰岛素在两餐之间和夜间以0.5-1单位/小时的速度持续分泌，虽然基础胰岛素水平较低，但它调节过夜肝脏葡萄糖的速率和两餐之间较长时间的葡萄糖输出。这使得睡前有足够的葡萄糖水平来产生大脑能量。目前还没有一种单一的控释系统可以在单次皮下注射后持续较长时间地释放胰岛素，以满足基础胰岛素的需求。突释是聚合物制剂治疗药物控释中的一个主要问题。在这一应用中，我们建议对胰岛素分子进行修饰，以消除猝发释放。我们提出了一种新的修饰胰岛素分子的方法，利用胰岛素的独特性质(在锌存在下自结合，以及与阳离子聚合物如壳聚糖相互作用的能力)，然后将络合物添加到温敏聚合物的水溶液中，以控制胰岛素的释放。温敏聚合物在室温下溶解于水中，在注射部位形成植入体温。由于锌-胰岛素-壳聚糖复合体的尺寸较大，因此它将通过减少锌-胰岛素-壳聚糖复合体从聚合物水凝胶基质中的扩散来避免与胰岛素猝发释放相关的问题，并有助于稳定传递系统中的蛋白质，同时提供胰岛素在基础水平的受控释放。该项目的长期目标是开发新型控释给药系统，该系统可以在1型糖尿病患者单次皮下注射后，以构象和化学稳定且具有生物活性的形式将胰岛素释放到基础水平，持续时间更长(~3个月)。我们提出了三个具体的研究目标：(1)合成具有不同聚乳酸和聚乙二醇聚乳酸链长的温度敏感型聚乳酸-聚乙二醇-聚乳酸三嵌段共聚物，并用凝胶渗透色谱(GPC)和1H核磁共振表征其临界凝胶浓度、凝胶转变温度、重均相对分子质量和数均相对分子质量。用上述共聚物和锌-胰岛素-壳聚糖复合体制备胰岛素原位凝胶控释系统，并对胰岛素的体外释放进行研究。此外，胰岛素在释放样品和释放系统中的稳定性将使用傅里叶变换红外光谱(FTIR)、圆二色谱(CD)、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS PAGE)、天然PAGE和基质辅助激光解吸/电离飞行时间(MALDI-TOF)质谱仪技术进行研究。(2)。采用四甲基偶氮唑盐(3-(4，5-dimethylthiazol-2-yl)-2，5-diphenyltetrazolium))比色法评价聚合物缓释系统的体外生物相容性，通过组织学分析评价其体内生物相容性。(3)。通过测定血清胰岛素和血糖水平，研究链脲佐菌素(STZ)诱导的糖尿病大鼠体内胰岛素在体内的吸收和生物活性。采用酶联免疫吸附试验检测血清中的抗胰岛素抗体，以排除异物反应的可能性。链脲佐菌素诱导的糖尿病导致体重减轻。因此，将评估糖尿病大鼠在糖尿病诱导前后以及胰岛素治疗后体重的变化。这项拟议的研究将大大有助于开发一种新的给药系统，在单次皮下注射后以受控的速度给药三个月，以提高患者的生活质量，并减少与糖尿病相关的长期并发症。

 DESCRIPTION (provided by applicant): There are two types of insulin secretion in healthy people- basal and stimulated. The basal insulin is secreted continuously between meals and throughout the night at a rate of 0.5-1 unit/h. Although the basal insulin level is low, it modulats the rate of overnight hepatic glucose and glucose output during prolonged periods between meals. This allows for sufficient glucose level for cerebral energy production at bedtime. There is not a single controlled release delivery system available to deliver insulin continuously for longer duration after a single subcutaneous injection to meet the needs of the basal insulin. Burst release is a major issue in controlled delivery of therapeutics from polymeric formulations. In this application, we are proposing to modify the insulin molecule in order to eliminate the burst release. We propose a novel approach of modifying insulin molecule by utilizing the combination of distinctive properties of insulin (self-association in presence of zinc, and abilityto interact with cationic polymers such as chitosan) and subsequent addition of the complex into aqueous solution of thermo sensitive polymer for controlled delivery of insulin. Thermo sensitive polymer is solution in water at room temperature and turns into implant at body temperature at the site of injection. The proposed delivery system would circumvent the problems associated with insulin burst release by reducing the diffusion of zinc insulin-chitosan complex from the polymeric hydrogel matrix due to its larger size and help to stabilize the protein inside the delivery system, while providing controlled release of insulin at basal level. The long-term goal of this project is to develop novel controlled release delivery systems which can deliver insulin a basal level in a conformationally and chemically stable and biologically active form for longer duration (~3 months) after a single subcutaneous injection in patients with type 1 diabetes. We propose to study three specific aims: (1)To synthesize temperature sensitive poly (lactic acid)-poly (ethylene glycol)- poly(lactic acid) (PLAPEG-PLA) triblock copolymers with varying PLA and PEG chain lengths and characterize their critical gel concentration, gel transition temperature, weight average molecular weight by gel permeation chromatography (GPC), and number average molecular weight by 1H NMR. In situ gel-forming controlled delivery systems of insulin will be prepared, using the above copolymers and zinc-insulin-chitosan complex, and will be studied for the in vitro release of insulin. Further, the stability of insulin in the released samples and in the delivery systems will be investigated using Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE), native PAGE and Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry techniques. (2). To evaluate the biocompatibility of polymeric delivery systems in vitro by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and in vivo in rats by histological analysis. (3). To study in vivo absorption and bioactivity of insulin from the delivery systems in the streptozoticin (STZ) induced diabetic rats by measuring serum insulin and glucose levels, respectively. The anti-insulin antibody in serum will be assessed by ELISA in order to rule out the possibility of foreign body response. STZ induced diabetes leads to reduction in the body weight. Therefore the change in body weight in diabetic rats before and after induction of diabetes, as well as after insulin treatment will be evaluated. The proposed study will contribute significantly to the development of a novel delivery system to deliver insulin at a controlled rate for three months after a single subcutaneous injection in order to improve patients' quality of life, and decrease the long term complications associated with diabetes.