Mechanisms of glucagon degradation at alkaline pH

Abstract

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.

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

“…This finding is consistent with what has been shown previously, that is, that some minor modifications do not produce any major potency shifts. 17 PD properties in swine showed no significant difference in the maximal rise in venous blood glucose after glucagon injection when comparing aged FAFG with either unaged FAFG or unaged synthetic glucagon. Time to early half max was significantly different comparing unaged synthetic glucagon vs unaged FAFG with the latter being faster.…”

“…In addition to its tendency to aggregate, glucagon spontaneously degrades through chemical mechanisms such as deamidation and oxidation. 16,17 For these reasons, it is not feasible to use current preparations of glucagon in a portable pump at room or body temperatures over the several days necessary for automated delivery. To avoid these issues, when used in the research setting during closed-loop studies, glucagon is freshly reconstituted from the lyophilized form using commercial glucagon preparations at the point of care.…”

Closed loop insulin delivery in diabetes

“…Despite the clear theoretical advantage of having the possibility of preventing imminent hypoglycaemia with a glucagon bolus, dual-hormone closed loop does not completely eliminate the need of rescue carbohydrates administered by the patients upon a closed loop triggered alarm [51,64]. Additionally, the stability [65] and consequences of long-term subcutaneous glucagon administration are not resolved. It seems, therefore, more likely that an insulin-only closed loop system with possible co-treatment with adjunct injectable, oral medications, or both, will be the first to get the market approval from the regulatory bodies.…”

Biochemical Stabilization of Glucagon at Alkaline pH

“…Native human glucagon was synthesized by AmbioPharm, Inc. (Beech Island, SC) as reported previously. 16 The peptide was purified to > 99% and shipped as a lyophilized solid. Reagents were of highest purity available.…”

“…15 Our previous work examined chemical degradation of glucagon at alkaline pH, which include deamidation and oxidation. 16 At pH 9, glucagon aggregates more but degrades much less than at pH 10; therefore, the use of pH 9 would require the addition of anti-aggregation excipients. To address this issue, we report here on the effects of commonly used excipients and the novel use of curcumin.…”