Stable Liquid Glucagon Formulations for Rescue Treatment and Bi-Hormonal Closed-Loop Pancreas
Small doses of glucagon given subcutaneously in the research setting by an automated system prevent most cases of hypoglycemia in persons with diabetes. However, glucagon is very unstable and cannot be kept in a portable pump. Glucagon rapidly forms amyloid fibrils, even within the first day after reconstitution. Aggregation eventually leads to insoluble gels, which occlude pump catheters. Fibrillation occurs rapidly at acid pH, but is absent or minimal at alkaline pH values of ~10. Glucagon also degrades over time; this problem is greater at alkaline pH. Several studies suggest that its primary degradative pathway is deamidation, which results in a conversion of asparagine to aspartic acid. A cell-based assay for glucagon bioactivity that assesses glucagon receptor (GluR) activation can screen promising glucagon formulations. However, mammalian hepatocytes are usually problematic as they can lose GluR expression during culture. Assays for cyclic AMP (cAMP) or its downstream effector, protein kinase A (PKA), in engineered cell systems, are more reliable and suitable for inexpensive, high-throughput assessment of bioactivity.
KeywordsGlucagon Cytotoxicity Amyloid Diabetes Assay Rescue treatment Bi-hormonal closed-loop pancreas
This work was financially supported in part by grants from Juvenile Diabetes Research Foundation (#17-2012-15), the Legacy Good Samaritan Foundation, and by NIH (NCRR,ORIP) grant # P51OD011092.
Conflicts of interest: MA Jackson: none N Caputo: has received grant support from the Juvenile Diabetes Research Foundation; and Novo Nordisk provided glucagon for research purposes as an in kind donation; JR Castle: has received grant support from the Juvenile Diabetes Research Foundation; and is on the Speaker's Bureau for Amylin; LL David: none; CT Roberts Jr: has received grant support from the Juvenile Diabetes Research Foundation; WK Ward: has received grant support from the Juvenile Diabetes Research Foundation; has received support for travel to meetings for the study or otherwise from the Juvenile Diabetes Research Foundation; Novo Nordisk provided glucagon for research purposes as an in kind donation; is on the scientific advisory board of Xerix Pharmaceuticals; some of the authors filed patents regarding methods of chemically stabilizing glucagon.
Papers of particular interest, published recently, have been highlighted as: • Of importance
- 1.Kimball CP, Murlin JR. Aqueous extracts of pancreas: some precipitation reactions of insulin. J Biol Chem. 1923;66:337.Google Scholar
- 7.• Maji SK, Perrin MH, Sawaya MR, Jessberger S, Vadodaria K, Rissman RA, et al. Functional amyloids as natural storage of peptide hormones in pituitary secretory granules. Science. 2009;325:328–32. Very interesting article demonstrating that packing of hormones into amyloid is the natural way that hormones are stored in secretory granules prior to their secretion..PubMedCrossRefGoogle Scholar
- 12.• Ward WK, Massoud RG, Szybala CJ, Engle JM, El Youssef J, Carroll JM, et al. In vitro and in vivo evaluation of native glucagon and glucagon analog (MAR-D28) during aging: lack of cytotoxicity and preservation of hyperglycemic effect. J Diabetes Sci Technol. 2010;4:1311–21. An article that demonstrates the near absence of glucagon fibrillation at pH 10..PubMedGoogle Scholar
- 13.Jackson M, Stonex T, Caputo N, El Youssef J, Branigan D, Castle JR, et al. Development of a stable formulation of liquid glucagon for use in a bihormonal pump. Presented at 2012 Scientific Session, American Diabetes Association, Philadelphia, PA; 2012.Google Scholar
- 17.Steiner SS, Li M, Hauser R, Pohl R. Stabilized glucagon formulation for bihormonal pump use. J Diabetes Sci Technol. 2010;4:1332–7.Google Scholar
- 18.Steiner SS, Hauser R, Li M, Feldstein R, Pohl, R. Stabilized glucagon solutions. US Patent Application # 12/891,240.Google Scholar
- 20.DiMarchi MA, Smiley DL, DiMarchi M, Chabenne JR, Day J. Glucagon analogs exhibiting enhanced solubility and stability. US Patent Application # 12/999,284.Google Scholar
- 21.Prestrelski S, Fang WJ, Carpenter JF, Kinzell J. Glucagon formulations for the treatment of hypoglycemia. US Patent Application # 13/186,275.Google Scholar
- 22.Jezek J. Stable aqueous systems comprising proteins. US Patent Application 11/994,408.Google Scholar