<i>In Vitro</i> and <i>in Vivo</i> Evaluation of Native Glucagon and Glucagon Analog (MAR-D28) during Aging: Lack of Cytotoxicity and Preservation of Hyperglycemic Effect

Ward, W. Kenneth; Massoud, Ryan G.; Szybala, Cory; Engle, J. Michael; Youssef, Joseph El; Carroll, Julie M.; Roberts, Charles T.; DiMarchi, Richard D.

doi:10.1177/193229681000400604

Cited by 20 publications

(40 citation statements)

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“…In aqueous environments, glucagon forms polymerized gels and fibrils, a process affected by temperature, concentration, and ionic strength [2, 8, 25, 26]. While fibrillation occurs rapidly at acidic pH, it is greatly minimized in alkaline solutions [14, 33]. Although fibrillation is minimized at a pH of 10, degradative processes such as deamidation tend to be prevalent at alkaline pH [15, 22].…”

Section: Introductionmentioning

confidence: 99%

Mechanisms of glucagon degradation at alkaline pH

et al. 2013

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Glucagon is unstable and undergoes degradation and aggregation in aqueous solution. For this reason, its use in portable pumps for closed loop management of diabetes is limited to very short periods. In this study, we sought to identify the degradation mechanisms and the bioactivity of specific degradation products. We studied degradation in the alkaline range, a range at which aggregation is minimized. Native glucagon and analogs identical to glucagon degradation products were synthesized. To quantify biological activity in glucagon and in the degradation peptides, a protein kinase A-based bioassay was used. Aged, fresh, and modified peptides were analyzed by liquid chromatography with mass spectrometry (LCMS). Oxidation of glucagon at the Met residue was common but did not reduce bioactivity. Deamidation and isomerization were also common and were more prevalent at pH 10 than 9. The biological effects of deamidation and isomerization were unpredictable; deamidation at some sites did not reduce bioactivity. Deamidation of Gln 3, isomerization of Asp 9, and deamidation with isomerization at Asn 28 all caused marked potency loss. Studies with molecular-weight-cutoff membranes and LCMS revealed much greater fibrillation at pH 9 than 10. Further work is necessary to determine formulations of glucagon that minimize degradation and fibrillation.

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Section: Introductionmentioning

confidence: 99%

Mechanisms of glucagon degradation at alkaline pH

et al. 2013

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“…[6][7][8][9][10] However, glucagon is an unstable peptide and forms amyloid fibrils when aged in acidic aqueous solutions. [11][12][13][14] These fibrils, which can form gels, have been reported to be cytotoxic 13,15 and could occlude pump delivery catheters. In addition to its tendency to aggregate, glucagon spontaneously degrades through chemical mechanisms such as deamidation and oxidation.…”

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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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“…glucagon formulations form a variety of fibrillar polymorphisms, 10 but glucagon formulated at pH 10 remains monomeric. 12 Although glucagon, like other hormones, is stored in amyloid fibrils in its membrane-bound secretory granules, 41 aggregation of the drug must be minimized for exogenous administration via a pump.…”

Section: Stabilized Glucagon Is Bioactive In Vitro and In Vivomentioning

confidence: 99%

“…[3][4][5][6][7] However, commercially available glucagon, a lyophilized solid, rapidly aggregates after reconstitution with water, forming high-molecular-weight complexes, [8][9][10] making it unsuitable for extended use in a closed-loop system. Aggregated glucagon is cytotoxic at high concentrations, 11,12 and its action in vivo is delayed compared with fresh glucagon. 12 Methods that show promise for stabilizing glucagon include formulation in alkaline media, 12 creation of mutant glucagon analogs, 13 the use of surfactants to increase solubility at neutral pH, 14 and the use of anhydrous solvents to minimize chemical interactions with water.…”

Section: Introductionmentioning

confidence: 99%

“…Aggregated glucagon is cytotoxic at high concentrations, 11,12 and its action in vivo is delayed compared with fresh glucagon. 12 Methods that show promise for stabilizing glucagon include formulation in alkaline media, 12 creation of mutant glucagon analogs, 13 the use of surfactants to increase solubility at neutral pH, 14 and the use of anhydrous solvents to minimize chemical interactions with water. 15 Our previous work examined chemical degradation of glucagon at alkaline pH, which include deamidation and oxidation.…”

Section: Introductionmentioning

confidence: 99%

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