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.
Get full access to this article
View all access options for this article.
References
1.
JiangG, ZhangBB. Glucagon and regulation of glucose metabolism. Am J Physiol Endocrinol Metab, 2003; 284:E671–E678.
2.
CryerPE: Minireview: glucagon in the pathogenesis of hypoglycemia and hyperglycemia in diabetes. Endocrinology, 2012; 153:1039–1048.
3.
El-KhatibFH, JiangJ, GerrityRG, DamianoER: Pharmacodynamics and stability of subcutaneously infused glucagon in a type 1 diabetic swine model in vivo. Diabetes Technol Ther, 2007; 9:135–144.
4.
WardWK, EngleJ, DumanHM, BergstromCP, KimSF, FederiukIF: The benefit of subcutaneous glucagon during closed-loop glycemic control in rats with type 1 diabetes. IEEE Sensors J, 2008; 8:89–96.
5.
CastleJR, EngleJM, El YoussefJ, et al.: Novel use of glucagon in a closed-loop system for prevention of hypoglycemia in type 1 diabetes. Diabetes Care, 2010; 33:1282–1287.
6.
El-KhatibFH, RussellSJ, NathanDM, SutherlinRG, DamianoER: A bihormonal closed-loop artificial pancreas for type 1 diabetes. Sci Transl Med, 2010; 2:27ra.
7.
HaidarA, LegaultL, DallaireM, et al.: Glucose-responsive insulin and glucagon delivery (dual-hormone artificial pancreas) in adults with type 1 diabetes: a randomized crossover controlled trial. Can Med Assoc J, 2013; 185:297–305.
8.
PedersenJS: The nature of amyloid-like glucagon fibrils. J Diabetes Sci Technol, 2010; 4:1357–1367.
9.
PedersenJS, DikovD, FlinkJL, HjulerHA, ChristiansenG, OtzenDE: The changing face of glucagon fibrillation: structural polymorphism and conformational imprinting. J Mol Biol, 2006; 355:501–523.
10.
PedersenJS, OtzenDE: Amyloid—a state in many guises: survival of the fittest fibril fold. Protein Sci, 2008; 17:2–10.
11.
OnoueS, OhshimaK, DebariK, et al.: Mishandling of the therapeutic peptide glucagon generates cytotoxic amyloidogenic fibrils. Pharm Res, 2004; 21:1274–1283.
12.
WardWK, MassoudRG, SzybalaCJ, et al.: In vitro and in vivo evaluation of native glucagon and glucagon analog (MAR-D28) during aging: lack of cytotoxicity and preservation of hyperglycemic effect. J Diabetes Sci Technol, 2010; 4:1311–1321.
13.
ChabenneJR, DiMarchiMA, GelfanovVM, DiMarchiRD: Optimization of the native glucagon sequence for medicinal purposes. J Diabetes Sci Technol, 2010; 4:1322–1331.
PrestrelskiS, FangW-J, CarpenterJF, KinzellJ, inventors: Stable glucagon formulations for the treatment of hypoglycemia. U.S. Patent Application 20120046225. February23, 2012.
16.
CaputoN, CastleJR, BergstromCP, et al.: Mechanisms of glucagon degradation at alkaline pH. Peptides, 2013; 45:40–47.
17.
MaheshwariRK, SinghAK, GaddipatiJ, SrimalRC: Multiple biological activities of curcumin: a short review. Life Sci, 2006; 78:2081–2087.
18.
AnandP, ThomasSG, KunnumakkaraAB, et al.: Biological activities of curcumin and its analogues (congeners) made by man and Mother Nature. Biochem Pharmacol, 2008; 76:1590–1611.
19.
Lopez-LazaroM: Anticancer and carcinogenic properties of curcumin: considerations for its clinical development as a cancer chemopreventive and chemotherapeutic agent. Mol Nutr Food Res, 2008; 52(Suppl 1):S103–S127.
20.
AggarwalBB, SungB: Pharmacological basis for the role of curcumin in chronic diseases: an age-old spice with modern targets. Trends Pharmacol Sci, 2009; 30:85–94.
21.
GuptaSC, PrasadS, KimJH, et al.: Multitargeting by curcumin as revealed by molecular interaction studies. Nat Prod Rep, 2011; 28:1937–1955.
22.
GuptaSC, PatchvaS, KohW, AggarwalBB: Discovery of curcumin, a component of golden spice, and its miraculous biological activities. Clin Exp Pharmacol Physiol, 2012; 39:283–299.
23.
LimGP, ChuT, YangF, BeechW, FrautschySA, ColeGM: The curry spice curcumin reduces oxidative damage and amyloid pathology in an Alzheimer transgenic mouse. J Neurosci, 2001; 21:8370–8377.
24.
OnoK, YoshiikeY, TakashimaA, HasegawaK, NaikiH, YamadaM: Potent anti-amyloidogenic and fibril-destabilizing effects of polyphenols in vitro: implications for the prevention and therapeutics of Alzheimer's disease. J Neurochem, 2003; 87:172–181.
25.
OnoK, HasegawaK, NaikiH, YamadaM: Curcumin has potent anti-amyloidogenic effects for Alzheimer's β-amyloid fibrils in vitro. J Neurosci Res, 2004; 75:742–750.
26.
YangF, LimGP, BegumAN, et al.: Curcumin inhibits formation of amyloid beta oligomers and fibrils, binds plaques, and reduces amyloid in vivo. J Biol Chem, 2005; 280:5892–5901.
27.
PoratY, AbramowitzA, GazitE: Inhibition of amyloid fibril formation by polyphenols: structural similarity and aromatic interactions as a common inhibition mechanism. Chem Biol Drug Des, 2006; 67:27–37.
28.
MishraS, PalaniveluK: The effect of curcumin (turmeric) on Alzheimer's disease: an overview. Ann Indian Acad Neurol, 2008; 11:13–19.
29.
AhmadB, LapidusLJ: Curcumin prevents aggregation in alpha-synuclein by increasing reconfiguration rate. J Biol Chem, 2012; 287:9193–9199.
KurienBT, SinghA, MatsumotoH, ScofieldRH: Improving the solubility and pharmacological efficacy of curcumin by heat treatment. Assay Drug Dev Technol, 2007; 5:567–576.
32.
NilssonMR: Techniques to study amyloid fibril formation in vitro. Methods, 2004; 34:151–160.
33.
HudsonSA, EcroydH, KeeTW, CarverJA: The thioflavin T fluorescence assay for amyloid fibril detection can be biased by the presence of exogenous compounds. FEBS J, 2009; 276:5960–5972.
34.
NoormagiA, PrimarK, TouguV, PalumaaP: Interference of low-molecular substances with the thioflavin-T fluorescence assay of amyloid fibrils. J Pept Sci, 2012; 18:59–64.
35.
ZaccoloM, De GiorgiF, ChoCY, et al.: A genetically encoded, fluorescent indicator for cyclic AMP in living cells. Nat Cell Biol, 2000; 2:25–29.
LeungMH, KeeTW: Effective stabilization of curcumin by association to plasma proteins: human serum albumin and fibrinogen. Langmuir, 2009; 25:5773–5777.
38.
WangYJ, PanMH, ChengAL, et al.: Stability of curcumin in buffer solutions and characterization of its degradation products. J Pharm Biomed Anal, 1997; 15:1867–1876.
39.
PedersenJS, DikovD, OtzenDE: N- and C-terminal hydrophobic patches are involved in fibrillation of glucagon. Biochemistry, 2006; 45:14503–14512.
40.
ReinkeAA, GestwickiJE: Structure-activity relationships of amyloid beta-aggregation inhibitors based on curcumin: influence of linker length and flexibility. Chem Biol Drug Des, 2007; 70:206–215.
41.
MajiSK, PerrinMH, SawayaMR, et al.: Functional amyloids as natural storage of peptide hormones in pituitary secretory granules. Science, 2009; 325:328–332.
42.
AmundsenI, OiestadÅM, EkebergD, KristoffersenL: Quantitative determination of fifteen basic pharmaceuticals in ante- and post-mortem whole blood by high pH mobile phase reversed phase ultra high performance liquid chromatography-tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci, 2013; 927:112–123.
43.
WardWK, CastleJR, BraniganDL, MassoudRG, El YoussefJ: Discomfort from an alkaline formulation delivered subcutaneously in humans: albumin at pH 7 versus pH 10. Clin Drug Investig, 2012; 32:433–438.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
