In vitro and in vivo evaluation of native glucagon and glucagon analog (MAR-D28) during aging: Lack of cytotoxicity and preservation of hyperglycemic effect

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

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

Background: For automated prevention of hypoglycemia, there is a need for glucagon (or an analog) to be sufficiently stable so that it can be indwelled in a portable pump for at least 3 days. However, under some conditions, solutions of glucagon can form amyloid fibrils. Currently, the usage instructions for commercially available glucagon allow only for its immediate use. Methods: In NIH 3T3 fibroblasts, we tested amyloid formation and cytotoxicity of solutions of native glucagon and the glucagon analog MAR-D28 after aging under different conditions for 5 days. In addition, aged native glucagon was subjected to size-exclusion chromatography (SEC). We also studied whether subcutaneous aged Novo Nordisk GlucaGen® would have normal bioactivity in octreotide-treated, anesthetized, nondiabetic pigs. Results: We found no evidence of cytotoxicity from native glucagon or MAR-D28 (up to 2.5 mg/ml) at a pH of 10 in a glycine solvent. We found a mild cytotoxicity for both compounds in Tris buffer at pH 8.5. A high concentration of the commercial glucagon preparation (GlucaGen) caused marked cytotoxicity, but low pH and/or a high osmolarity probably accounted primarily for this effect. With SEC, the decline in monomeric glucagon over time was much lower when aged in glycine (pH 10) than when aged in Tris (pH 8.5) or in citrate (pH 3). Congo red staining for amyloid was very low with the glycine preparation (pH 10). In the pig studies, the hyperglycemic effect of commercially available glucagon was preserved despite aging conditions associated with marked amyloid formation. Conclusions: Under certain conditions, aqueous solutions of glucagon and MAR-D28 are stable for at least 5 days and are thus very likely to be safe in mammals. Glycine buffer at a pH of 10 appears to be optimal for avoiding cytotoxicity and amyloid fibril formation.

Original languageEnglish (US)
Pages (from-to)1311-1321
Number of pages11
JournalJournal of diabetes science and technology
Volume4
Issue number6
StatePublished - Nov 2010

Fingerprint

Cytotoxicity
Glucagon
Aging of materials
Amino acids
Size exclusion chromatography
Amyloid
Glycine
Mammals
Fibroblasts
Bioactivity
Gel Chromatography
In Vitro Techniques
Pumps
Swine
Congo Red
Tromethamine
Octreotide
Hypoglycemia
Citric Acid
Osmolar Concentration

Keywords

  • Amyloid
  • Cell culture
  • Cytotoxicity
  • Diabetes
  • Glucagon

ASJC Scopus subject areas

  • Endocrinology, Diabetes and Metabolism
  • Internal Medicine
  • Bioengineering
  • Biomedical Engineering

Cite this

In vitro and in vivo 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 J.; Engle, Julia M.; El Youssef, Joseph; Carroll, Julie M.; Roberts, Charles; DiMarchi, Richard D.

In: Journal of diabetes science and technology, Vol. 4, No. 6, 11.2010, p. 1311-1321.

Research output: Contribution to journalArticle

Ward, W. Kenneth ; Massoud, Ryan G. ; Szybala, Cory J. ; Engle, Julia M. ; El Youssef, Joseph ; Carroll, Julie M. ; Roberts, Charles ; DiMarchi, Richard D. / In vitro and in vivo evaluation of native glucagon and glucagon analog (MAR-D28) during aging : Lack of cytotoxicity and preservation of hyperglycemic effect. In: Journal of diabetes science and technology. 2010 ; Vol. 4, No. 6. pp. 1311-1321.
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abstract = "Background: For automated prevention of hypoglycemia, there is a need for glucagon (or an analog) to be sufficiently stable so that it can be indwelled in a portable pump for at least 3 days. However, under some conditions, solutions of glucagon can form amyloid fibrils. Currently, the usage instructions for commercially available glucagon allow only for its immediate use. Methods: In NIH 3T3 fibroblasts, we tested amyloid formation and cytotoxicity of solutions of native glucagon and the glucagon analog MAR-D28 after aging under different conditions for 5 days. In addition, aged native glucagon was subjected to size-exclusion chromatography (SEC). We also studied whether subcutaneous aged Novo Nordisk GlucaGen{\circledR} would have normal bioactivity in octreotide-treated, anesthetized, nondiabetic pigs. Results: We found no evidence of cytotoxicity from native glucagon or MAR-D28 (up to 2.5 mg/ml) at a pH of 10 in a glycine solvent. We found a mild cytotoxicity for both compounds in Tris buffer at pH 8.5. A high concentration of the commercial glucagon preparation (GlucaGen) caused marked cytotoxicity, but low pH and/or a high osmolarity probably accounted primarily for this effect. With SEC, the decline in monomeric glucagon over time was much lower when aged in glycine (pH 10) than when aged in Tris (pH 8.5) or in citrate (pH 3). Congo red staining for amyloid was very low with the glycine preparation (pH 10). In the pig studies, the hyperglycemic effect of commercially available glucagon was preserved despite aging conditions associated with marked amyloid formation. Conclusions: Under certain conditions, aqueous solutions of glucagon and MAR-D28 are stable for at least 5 days and are thus very likely to be safe in mammals. Glycine buffer at a pH of 10 appears to be optimal for avoiding cytotoxicity and amyloid fibril formation.",
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T1 - In vitro and in vivo evaluation of native glucagon and glucagon analog (MAR-D28) during aging

T2 - Lack of cytotoxicity and preservation of hyperglycemic effect

AU - Ward, W. Kenneth

AU - Massoud, Ryan G.

AU - Szybala, Cory J.

AU - Engle, Julia M.

AU - El Youssef, Joseph

AU - Carroll, Julie M.

AU - Roberts, Charles

AU - DiMarchi, Richard D.

PY - 2010/11

Y1 - 2010/11

N2 - Background: For automated prevention of hypoglycemia, there is a need for glucagon (or an analog) to be sufficiently stable so that it can be indwelled in a portable pump for at least 3 days. However, under some conditions, solutions of glucagon can form amyloid fibrils. Currently, the usage instructions for commercially available glucagon allow only for its immediate use. Methods: In NIH 3T3 fibroblasts, we tested amyloid formation and cytotoxicity of solutions of native glucagon and the glucagon analog MAR-D28 after aging under different conditions for 5 days. In addition, aged native glucagon was subjected to size-exclusion chromatography (SEC). We also studied whether subcutaneous aged Novo Nordisk GlucaGen® would have normal bioactivity in octreotide-treated, anesthetized, nondiabetic pigs. Results: We found no evidence of cytotoxicity from native glucagon or MAR-D28 (up to 2.5 mg/ml) at a pH of 10 in a glycine solvent. We found a mild cytotoxicity for both compounds in Tris buffer at pH 8.5. A high concentration of the commercial glucagon preparation (GlucaGen) caused marked cytotoxicity, but low pH and/or a high osmolarity probably accounted primarily for this effect. With SEC, the decline in monomeric glucagon over time was much lower when aged in glycine (pH 10) than when aged in Tris (pH 8.5) or in citrate (pH 3). Congo red staining for amyloid was very low with the glycine preparation (pH 10). In the pig studies, the hyperglycemic effect of commercially available glucagon was preserved despite aging conditions associated with marked amyloid formation. Conclusions: Under certain conditions, aqueous solutions of glucagon and MAR-D28 are stable for at least 5 days and are thus very likely to be safe in mammals. Glycine buffer at a pH of 10 appears to be optimal for avoiding cytotoxicity and amyloid fibril formation.

AB - Background: For automated prevention of hypoglycemia, there is a need for glucagon (or an analog) to be sufficiently stable so that it can be indwelled in a portable pump for at least 3 days. However, under some conditions, solutions of glucagon can form amyloid fibrils. Currently, the usage instructions for commercially available glucagon allow only for its immediate use. Methods: In NIH 3T3 fibroblasts, we tested amyloid formation and cytotoxicity of solutions of native glucagon and the glucagon analog MAR-D28 after aging under different conditions for 5 days. In addition, aged native glucagon was subjected to size-exclusion chromatography (SEC). We also studied whether subcutaneous aged Novo Nordisk GlucaGen® would have normal bioactivity in octreotide-treated, anesthetized, nondiabetic pigs. Results: We found no evidence of cytotoxicity from native glucagon or MAR-D28 (up to 2.5 mg/ml) at a pH of 10 in a glycine solvent. We found a mild cytotoxicity for both compounds in Tris buffer at pH 8.5. A high concentration of the commercial glucagon preparation (GlucaGen) caused marked cytotoxicity, but low pH and/or a high osmolarity probably accounted primarily for this effect. With SEC, the decline in monomeric glucagon over time was much lower when aged in glycine (pH 10) than when aged in Tris (pH 8.5) or in citrate (pH 3). Congo red staining for amyloid was very low with the glycine preparation (pH 10). In the pig studies, the hyperglycemic effect of commercially available glucagon was preserved despite aging conditions associated with marked amyloid formation. Conclusions: Under certain conditions, aqueous solutions of glucagon and MAR-D28 are stable for at least 5 days and are thus very likely to be safe in mammals. Glycine buffer at a pH of 10 appears to be optimal for avoiding cytotoxicity and amyloid fibril formation.

KW - Amyloid

KW - Cell culture

KW - Cytotoxicity

KW - Diabetes

KW - Glucagon

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