A novel, stable, aqueous glucagon formulation using ferulic acid as an excipient.

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. 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. 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. 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.

Effect of Repeated Glucagon Doses on Hepatic Glycogen in Type 1 Diabetes: Implications for a Bihormonal Closed-Loop System.

OBJECTIVE: To evaluate subjects with type 1 diabetes for hepatic glycogen depletion after repeated doses of glucagon, simulating delivery in a bihormonal closed-loop system. RESEARCH DESIGN AND METHODS: Eleven adult subjects with type 1 diabetes participated. Subjects underwent estimation of hepatic glycogen using (13)C MRS. MRS was performed at the following four time points: fasting and after a meal at baseline, and fasting and after a meal after eight doses of subcutaneously administered glucagon at a dose of 2 µg/kg, for a total mean dose of 1,126 µg over 16 h. The primary and secondary end points were, respectively, estimated hepatic glycogen by MRS and incremental area under the glucose curve for a 90-min interval after glucagon administration. RESULTS: In the eight subjects with complete data sets, estimated glycogen stores were similar at baseline and after repeated glucagon doses. In the fasting state, glycogen averaged 21 ± 3 g/L before glucagon administration and 25 ± 4 g/L after glucagon administration (mean ± SEM) (P = NS). In the fed state, glycogen averaged 40 ± 2 g/L before glucagon administration and 34 ± 4 g/L after glucagon administration (P = NS). With the use of an insulin action model, the rise in glucose after the last dose of glucagon was comparable to the rise after the first dose, as measured by the 90-min incremental area under the glucose curve. CONCLUSIONS: In adult subjects with well-controlled type 1 diabetes (mean A1C 7.2%), glycogen stores and the hyperglycemic response to glucagon administration are maintained even after receiving multiple doses of glucagon. This finding supports the safety of repeated glucagon delivery in the setting of a bihormonal closed-loop system.

Quantification of the glycemic response to microdoses of subcutaneous glucagon at varying insulin levels.

OBJECTIVE: Glucagon delivery in closed-loop control of type 1 diabetes is effective in minimizing hypoglycemia. However, high insulin concentration lowers the hyperglycemic effect of glucagon, and small doses of glucagon in this setting are ineffective. There are no studies clearly defining the relationship between insulin levels, subcutaneous glucagon, and blood glucose. RESEARCH DESIGN AND METHODS: Using a euglycemic clamp technique in 11 subjects with type 1 diabetes, we examined endogenous glucose production (EGP) of glucagon (25, 75, 125, and 175 µg) at three insulin infusion rates (0.016, 0.032, and 0.05 units/kg/h) in a randomized, crossover study. Infused 6,6-dideuterated glucose was measured every 10 min, and EGP was determined using a validated glucoregulatory model. Area under the curve (AUC) for glucose production was the primary outcome, estimated over 60 min. RESULTS: At low insulin levels, EGP rose proportionately with glucagon dose, from 5 ± 68 to 112 ± 152 mg/kg (P = 0.038 linear trend), whereas at high levels, there was no increase in glucose output (19 ± 53 to 26 ± 38 mg/kg, P = NS). Peak glucagon serum levels and AUC correlated well with dose (r2 = 0.63, P < 0.001), as did insulin levels with insulin infusion rates (r2 = 0.59, P < 0.001). CONCLUSIONS: EGP increases steeply with glucagon doses between 25 and 175 µg at lower insulin infusion rates. However, high insulin infusion rates prevent these doses of glucagon from significantly increasing glucose output and may reduce glucagon effectiveness in preventing hypoglycemia when used in the artificial pancreas.

Biochemical stabilization of glucagon at alkaline pH.

BACKGROUND: For patients with type 1 diabetes mellitus, a bihormonal artificial endocrine pancreas system utilizing glucagon and insulin has been found to stabilize glycemic control. However, commercially available formulations of glucagon cannot currently be used in such systems because of physical instability characterized by aggregation and chemical degradation. Storing glucagon at pH 10 blocks protein aggregation but results in chemical degradation. Reductions in pH minimize chemical degradation, but even small reductions increase protein aggregation. We hypothesized that common pharmaceutical excipients accompanied by a new excipient would inhibit glucagon aggregation at an alkaline pH. METHODS AND RESULTS: As measured by tryptophan intrinsic fluorescence shift and optical density at 630 nm, protein aggregation was indeed minimized when glucagon was formulated with curcumin and albumin. This formulation also reduced chemical degradation, measured by liquid chromatography with mass spectrometry. Biological activity was retained after aging for 7 days in an in vitro cell-based bioassay and also in Yorkshire swine. CONCLUSIONS: 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.

Mechanisms of glucagon degradation at alkaline pH.

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.