Biochemical Stabilization of Glucagon at Alkaline pH

Caputo, Nicholas; Jackson, Melanie; Castle, Jessica R.; Youssef, Joseph El; Bakhtiani, Parkash A.; Bergstrom, Colin P.; Carroll, Julie M.; Breen, Matthew; Leonard, Gerald; David, Larry L.; Roberts, Charles T.; Ward, W. Kenneth

doi:10.1089/dia.2014.0047

Cited by 15 publications

(15 citation statements)

References 42 publications

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“…Aggregated glucagon as a result of fibril formation not only loses its function in vivo but can also be cytotoxic at elevated concentrations . Several options are being investigated to find a stable formulation that is compatible with use in infusion pumps over several days including the use of alkaline solutions, glucagon analogues and excipients that stabilize glucagon at neutral pH . The safety profile of new formulations as they become available will also need to be tested according to their potential long‐term side effects.…”

Section: Safety Profile Of Glucagon Formulationsmentioning

confidence: 99%

Glucagon in artificial pancreas systems: Potential benefits and safety profile of future chronic use

Taleb

Haidar

Messier

et al. 2016

Diabetes Obesity Metabolism

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The role of glucagon in the pathophysiology of diabetes has long been recognized, although its approved clinical use has so far been limited to the emergency treatment of severe hypoglycaemia. A novel use of glucagon as intermittent mini-boluses is proposed in the dual-hormone version (insulin and glucagon) of the external artificial pancreas. Short-term studies suggest that the incorporation of glucagon into artificial pancreas systems has the potential to further decrease hypoglycaemic risk and improve overall glucose control; however, the potential long-term safety and benefits also need to be investigated given the recognized systemic effects of glucagon. In the present report, we review the available animal and human data on the physiological functions of glucagon, as well as its pharmacological use, according to dosing and duration (acute and chronic). Along with its main role in hepatic glucose metabolism, glucagon affects the cardiovascular, renal, pulmonary and gastrointestinal systems. It has a potential role in weight reduction through its central satiety function and its role in increasing energy expenditure. Most of the pharmacological studies investigating the effects of glucagon have used doses exceeding 1 mg, in contrast to the mini-boluses used in the artificial pancreas. The available data are reassuring but comprehensive human studies using small but chronic glucagon doses that are close to the physiological ranges are lacking. We propose a list of variables that could be monitored during long-term trials of the artificial pancreas. Such trials should address the questions about the risk-benefit ratio of chronic glucagon use.

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Section: Safety Profile Of Glucagon Formulationsmentioning

confidence: 99%

Glucagon in artificial pancreas systems: Potential benefits and safety profile of future chronic use

Taleb

Haidar

Messier

et al. 2016

Diabetes Obesity Metabolism

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“…We have previously investigated the effects of curcumin, an active ingredient of turmeric, for its effects on glucagon aggregation, and demonstrated that curcumin reduced glucagon aggregation; 18 however, due to its shorter half-life, it is not useful over prolonged periods of time. Although the presence of glucagon and human serum albumin (HSA) improves the half-life of curcumin, it still may not be effective enough over longer intervals.…”

mentioning

confidence: 99%

A Novel, Stable, Aqueous Glucagon Formulation Using Ferulic Acid as an Excipient

Bakhtiani

Caputo

Castle

et al. 2014

J Diabetes Sci Technol

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Background: Commercial glucagon is unstable due to aggregation and degradation. In closed-loop studies, it must be reconstituted frequently. For use in a portable pump for 3 days, a more stable preparation is required. At alkaline pH, curcumin inhibited glucagon aggregation. However, curcumin is not sufficiently stable for long-term use. Here, we evaluated ferulic acid, a stable breakdown product of curcumin, for its ability to stabilize glucagon.Methods: Ferulic acid-formulated glucagon (FAFG), composed of ferulic acid, glucagon, L-methionine, polysorbate-80, and human serum albumin in glycine buffer at pH 9, was aged for 7 days at 37°C. Glucagon aggregation was assessed by transmission electron microscopy (TEM) and degradation by high-performance liquid chromatography (HPLC). A cell-based protein kinase A (PKA) assay was used to assess in vitro bioactivity. Pharmacodynamics (PD) of unaged FAFG, 7-day aged FAFG, and unaged synthetic glucagon was determined in octreotide-treated swine. Results:No fibrils were observed in TEM images of fresh or aged FAFG. Aged FAFG was 94% intact based on HPLC analysis and there was no loss of bioactivity. In the PD swine analysis, the rise over baseline of glucose with unaged FAFG, aged FAFG, and synthetic native glucagon (unmodified human sequence) was similar.Conclusions: After 7 days of aging at 37°C, an alkaline ferulic acid formulation of glucagon exhibited significantly less aggregation and degradation than that seen with native glucagon and was bioactive in vitro and in vivo. Thus, this formulation may be stable for 3-7 days in a portable pump for bihormonal closed-loop treatment of T1D.

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“…It is known that amphipathic peptides are inclined to form oligomers that are held together by noncovalent interactions and are in equilibrium with the monomer [ 42 , 48 , 49 ]. The organization of peptides in oligomers or aggregates is often associated with the induction of toxicity and immunogenicity, as well as a reduction in their activity [ 42 , 50 , 51 , 52 , 53 ]. However, for other groups of peptides, oligomerization or aggregation is an intrinsic part of the peptide’s natural mode of action.…”

Section: Discussionmentioning

confidence: 99%

Concentration- and pH-Dependent Oligomerization of the Thrombin-Derived C-Terminal Peptide TCP-25

et al. 2020

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Peptide oligomerization dynamics affects peptide structure, activity, and pharmacodynamic properties. The thrombin C-terminal peptide, TCP-25 (GKYGFYTHVFRLKKWIQKVIDQFGE), is currently in preclinical development for improved wound healing and infection prevention. It exhibits turbidity when formulated at pH 7.4, particularly at concentrations of 0.3 mM or more. We used biochemical and biophysical approaches to explore whether the peptide self-associates and forms oligomers. The peptide showed a dose-dependent increase in turbidity as well as α-helical structure at pH 7.4, a phenomenon not observed at pH 5.0. By analyzing the intrinsic tryptophan fluorescence, we demonstrate that TCP-25 is more stable at high concentrations (0.3 mM) when exposed to high temperatures or a high concentration of denaturant agents, which is compatible with oligomer formation. The denaturation process was reversible above 100 µM of peptide. Dynamic light scattering demonstrated that TCP-25 oligomerization is sensitive to changes in pH, time, and temperature. Computational modeling with an active 18-mer region of TCP-25 showed that the peptide can form pH-dependent higher-order end-to-end oligomers and micelle-like structures, which is in agreement with the experimental data. Thus, TCP-25 exhibits pH- and temperature-dependent dynamic changes involving helical induction and reversible oligomerization, which explains the observed turbidity of the pharmacologically developed formulation.

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Biochemical Stabilization of Glucagon at Alkaline pH

Abstract: Based on these findings, a formulation of glucagon stabilized with curcumin, polysorbate-80, l-methionine, and albumin at alkaline pH in glycine buffer may be suitable for extended use in a portable pump in the setting of a bihormonal artificial endocrine pancreas.

Cited by 15 publications

References 42 publications

Glucagon in artificial pancreas systems: Potential benefits and safety profile of future chronic use

Glucagon in artificial pancreas systems: Potential benefits and safety profile of future chronic use

A Novel, Stable, Aqueous Glucagon Formulation Using Ferulic Acid as an Excipient

Concentration- and pH-Dependent Oligomerization of the Thrombin-Derived C-Terminal Peptide TCP-25

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