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

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 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.