Optimization of the Native Glucagon Sequence for Medicinal Purposes

Abstract

Background: Glucagon is a life-saving medication used in the treatment of hypoglycemia. It possesses poor solubility in aqueous buffers at or near physiological pH values. At low and high pH, at which the peptide can be formulated to concentrations of a milligram or more per milliliter, the chemical integrity of the hormone is limited, as evidenced by the formation of multiple degradation-related peptides. Consequently, the commercial preparation is provided as a lyophilized solid with an acidic diluent and directions for rendering it soluble at the time of use. Any unused material is recommended for disposal immediately after initial use. Methods: A set of glucagon analogs was prepared by solid-phase peptide synthesis to explore the identification of a glucagon analog with enhanced solubility and chemical stability at physiological pH. The physical properties of the peptide analogs were studied by solubility determination, high-performance chromatography, and mass spectral analysis. The biochemical properties were determined in engineered human embryonic kidney cell line 293 (HEK293) cells that overexpressed either the human glucagon or glucagon-like peptide-1 (GLP-1) receptors linked to a luciferase reporter gene. Results: We observed the previously characterized formation of glucagon degradation products upon incubation of the peptide in dilute acid for extended periods or elevated temperature. Lowering the isoelectric point of the hormone through the substitution of asparagine-28 with aspartic acid significantly increased the solubility at physiological pH. Similarly, the C-terminal extension (Cex) of the hormone with an exendin-based, 10-residue, C-terminal sequence yielded a peptide of dramatically enhanced solubility. These two glucagon analogs, D28 and Cex, maintained high potency and selectivity for the glucagon receptor relative to GLP-1 receptor. Conclusions: Glucagon presents unique structural challenges to the identification of an analog of high biological activity and selectivity that also possesses sufficient aqueous solubility and stability such that it might be developed as a ready-to-use medicine. The glucagon analogs D28 and Cex demonstrated all of the chemical, physical, and biochemical properties supportive of further study as potential clinical candidates for treatment of hypoglycemia.

Analysis of the glucagon receptor first extracellular loop by the substituted cysteine accessibility method

“…Investigation of chimeric receptor activation by hybrid peptides showed that, like Glucagon Like Peptide-1 (GLP-1) and Gastric Inhibitory Peptide (GIP) [22,31,42], the glucagon carboxyl-terminal half is anchored on the receptor extracellular Nterminal domain, and the receptor is activated by the interaction of the amino-terminal half of the peptide with the transmembrane domain [30,32,39]. This conclusion has been supported by several other studies: glutamate 68 in the GLP-1 receptor N-terminal domain (replaced by K64 in the glucagon receptor) reduces the GLP-1 receptor affinity for glucagon by charge repulsion with its' C-terminal carboxyl [4,8]; an aspartate (D385, replaced by E387 in the GLP-1 receptor) in TM7 is responsible for preferential recognition of the glucagon S2 over GLP-1 A2; K187 and I194 side chains in TM2 participate in the preference for glucagon Q3 [24,30] over GLP-1 E3; and unidentified side chains in ECL2, for glucagon K12 [30].…”

Anti-Obesity Therapy: from Rainbow Pills to Polyagonists

“…Collectively, these nonglycemic effects render glucagon an interesting candidate for pharmacological management of body weight. However, beyond the central dilemma of its diabetogenic liability resides the fact that glucagon is poorly suited as a drug substance, given its short duration of action, poor aqueous solubility, and chemical stability at physiologic pH (Gratzer et al, 1972;Chabenne et al, 2010). The solubility of glucagon in physiologic buffer can be dramatically improved by extension of its sequence at the C terminus with the CEX terminal end of exendin-4 Chabenne et al, 2010).…”

“…However, beyond the central dilemma of its diabetogenic liability resides the fact that glucagon is poorly suited as a drug substance, given its short duration of action, poor aqueous solubility, and chemical stability at physiologic pH (Gratzer et al, 1972;Chabenne et al, 2010). The solubility of glucagon in physiologic buffer can be dramatically improved by extension of its sequence at the C terminus with the CEX terminal end of exendin-4 Chabenne et al, 2010). Chronic s.c. infusion of DIO mice with glucagon-CEX in physiologic buffer improved body weight and glycemic control with equal efficacy when compared with equimolar administration of exendin-4 .…”

Innovations in Technology for the Treatment of Diabetes: Clinical Development of the Artificial Pancreas (an Autonomous System)

“…It should be noted that prolonged glucagon infusion might require use of a new formulation or analog molecule to avoid fibrillation. 16 The use of such a drug in a clinical trial would have to be cleared by a joint process involving two FDA centers, the CDRH and the CDER. Although the panel endorsed the idea of "one size will not fit all," which means that various patients will require various protocols and outcomes, this diversity of outcomes might be achievable with a limited number of products or tools that can be calibrated to the needs of the individual patient.…”