PROJECT SUMMARY Hypoglycemia, a decrease in blood sugar levels, is a common occurrence for the 30 million Americans with diabetes. In severe hypoglycemia, the individual is at risk for diabetic coma and even death. Although these events can be managed using a glucagon emergency kit, current kits require a multi-step procedure in which lyophilized glucagon is reconstituted and administered as an injection. Emergency kits are associated with a high rate of user error and frequently go unused. Additionally, kits are often not available when needed, as diabetic individuals find them cumbersome and rely on emergency room visits to treat severe hypoglycemia. The misuse and underutilization of glucagon emergency kits can be traced to the limited solubility and stability of glucagon. Glucagon is supplied as a powder in the kits because it is unstable in solution, rapidly forming amyloid fibrils. Additionally, glucagon has limited solubility at neutral pH and is solubilized in acidic pH in the kits. Monon Bioventures is developing glucagon derivatives with enhanced stability and solubility to provide a more versatile and user-friendly product. Monon Bioventures’ approach involves using reversible phosphorylation of glucagon to prevent the formation of amyloid fibrils, thus creating a solution-stable glucagon derivative, known as phospho-glucagon. Preliminary efforts have identified phospho-glucagons with improved solubility and stability and have shown increases in blood glucose comparable to native glucagon (in rats). These data support the continued development of lead phospho-glucagons through a Fast-Track program that is geared toward generating target product profiles (TPPs) for intranasal and self-injectable intramuscular phospho- glucagon formulations. This overall goal will be met through the execution of the following aims: Phase I Specific Aims are: 1) To execute formulation studies and structural modifications of lead phospho-glucagons to improve oxidative stability. 2) To develop and validate analytical methods for phospho-glucagon in plasma. The measures of success to advance to Phase II are 1) identification of at least two candidate phospho-glucagons formulated in solution at a neutral pH that have desired stability (<10% oxidation at 30oC for 3 months, no detectable fibrillation), solubility (>1 mg/mL), and functionality (increases blood glucose at rate and extent similar to native glucagon); and 2) a validated bioanalytical method with a limit of detection of 50 ng/mL or less. Phase II Specific Aims are: 1) To determine long-term stability of lead phospho-glucagon formulations. To assess the PK/pharmacodynamic (PD) properties of phospho-glucagon formulations. 3) To generate preliminary safety and immunogenicity profiles of the lead phospho-glucagon formulations. The measure of success for Phase II is to complete key pharmacological and safety assessments to construct the TPP.