NIH Public Access
`Author Manuscript
`Curr Diab Rep. Author manuscript; available in PMC 2014 March 31.
`Published in final edited form as:
`Curr Diab Rep. 2012 December ; 12(6): 705–710. doi:10.1007/s11892-012-0320-5.
`
`Stable liquid glucagon formulations for rescue treatment and bi-
`hormonal closed-loop pancreas
`
`Melanie A. Jackson, BS, Nicholas Caputo, MS, Jessica R. Castle, MD, Larry L. David, PhD,
`Charles T. Roberts Jr, PhD, and W. Kenneth Ward, MD
`Oregon Health and Science University (OHSU), 3181 SW Sam Jackson Park Road, OP05DC,
`Portland OR, and Legacy Health, 1225 NE 2nd Ave, Portland, OR 97232
`
`Abstract
`Small doses of glucagon given subcutaneously in the research setting by an automated system
`prevent most cases of hypoglycemia in persons with diabetes. However, glucagon is very unstable
`and cannot be kept in a portable pump. Glucagon rapidly forms amyloid fibrils, even within the
`first day after reconstitution. Aggregation eventually leads to insoluble gels, which occlude pump
`catheters. Fibrillation occurs rapidly at acid pH, but is absent or minimal at alkaline pH values of
`~10.
`
`Glucagon also degrades over time; this problem is greater at alkaline pH. Several studies suggest
`that its primary degradative pathway is deamidation, which results in a conversion of asparagine to
`aspartic acid.
`
`A cell-based assay for glucagon bioactivity that assesses glucagon receptor (GluR) activation can
`screen promising glucagon formulations. However, mammalian hepatocytes are usually
`problematic as they can lose GluR expression during culture. Assays for cyclic AMP (cAMP) or
`its downstream effector, protein kinase A (PKA), in engineered cell systems, are more reliable and
`suitable for inexpensive, high-throughput assessment of bioactivity.
`
`Keywords
`glucagon; cytotoxicity; amyloid; diabetes; assay
`
`Introduction and Background
`Glucagon, a 29-amino acid peptide, was discovered in 1922 by Kimball and Murlin during
`their efforts to purify insulin from pancreatic extracts. The discovery was accidental; they
`observed that after injection of insulin-containing extracts into animals, one of the
`preparations caused a rapid rise in blood glucose level before it caused the expected fall.
`Fortunately, they pursued this unexpected finding, verified it, and published the results in
`
`Contact Information- W. K. Ward (corresponding author): OHSU, 3181 SW Sam Jackson Park Road, OP05DC, Portland OR,
`wardk@ohsu.edu; Ph 503-494-1226; fax 503-494-4781.
`M. A. Jackson: OHSU, 3181 SW Sam Jackson Park Road, OP05DC, Portland OR 97239, jackmela@ohsu.edu; Ph 503 494 1226; fax
`503-494-4781
`N. Caputo: OHSU, 3181 SW Sam Jackson Park Road, OP05DC, Portland OR 97239, caputo@ohsu.edu; Ph 503-494-1226; fax
`503-494-4781
`J. R. Castle: OHSU, 3181 SW Sam Jackson Park Road, OP05DC, Portland OR 97239, castleje@ohsu.edu; Ph 503-494-1226; fax
`503-494-4781
`L. L. David: Proteomics Shared Resource, OHSU, 3181 SW Sam Jackson Park Road, Portland OR 97239, davidl@ohsu.edu; Ph
`503-418-1280
`C.T. Roberts: Oregon National Primate Research Center, 505 NW 185th Avenue, Beaverton, OR 97006-3448 and Department of
`Medicine, OHSU, robertsc@ohsu.edu; Ph 503-690-5259; fax 503-690-5532
`
`NIH-PA Author Manuscript
`
`NIH-PA Author Manuscript
`
`NIH-PA Author Manuscript
`
`Page 1
`
`NPS EX. 2045
`CFAD v. NPS
`IPR2015-00990
`
`

`
`Jackson et al.
`
`Page 2
`
`1923 [1]. It was not until the 1950’s that glucagon was purified and crystallized. In this era,
`glucagon was also found to exhibit unusual biochemical behavior; shortly after incubation in
`aqueous solution, many slender fibrillar structures were noted [2]. As the glucagon solutions
`were incubated over time, the concentration of these fibrils increased. The solutions
`eventually formed firm gels that contained high concentrations of packed fibrils.
`
`These fibrils are now known to exhibit a beta-pleated sheet amyloid protein configuration.
`Many subtypes of fibrils and protofibrils have been discovered; for excellent reviews, the
`reader is referred to the papers of Pedersen et al. [3, 4]. Though some compounds, including
`cyclodextrins, have been reported to reduce the tendency to form amyloid fibrils, there is no
`well-accepted additive or method that markedly prevents their formation.
`
`In 2004, a very interesting and widely cited report regarding the effect of fibrillated
`solutions of glucagon on mammalian cells was published [5]. The authors observed that,
`under certain conditions, fibrillated glucagon was toxic to mammalian cells in culture. They
`emphasized the potentially dangerous nature of glucagon fibrils and pointed out that there
`are several deadly diseases characterized by the formation of amyloid fibrils, including
`Alzheimer’s Disease, Parkinson’s Disease, and prion diseases. More recently, amyloid
`formation of lipoproteins has been reported to be associated with the pathogenesis of
`atherosclerosis [6].
`
`It should also be noted, however, that a recent comprehensive analysis of the conformation
`of secreted hormones in storage vesicles in vivo 7] demonstrated that most hormones are
`packed into amyloid fibrils prior to release, with dissociation to the monomeric form
`occurring after appearance in the circulation.
`
`Shortly after the report of potentially toxic effects of glucagon in vitro, several reports
`appeared which showed the benefit of closed-loop administration of glucagon in animals [8,
`9] and in humans [10, 11].
`
`These human reports emphasized the benefit of administering glucagon (with insulin) in the
`closed-loop setting. In our study, subjects with type 1 diabetes received insulin plus placebo
`on one occasion and insulin plus glucagon (as commanded by an automated algorithm) on
`another occasion. In terms of effectiveness, the duration of time in the hypoglycemic range
`for the bihormonal approach was less than half of the duration for the insulin-only
`experiments using doses of glucagon (given subcutaneously) that are generally quite small,
`and range from 60-200 μg over 5-10 min [11]. For these studies, commercially available
`lyophilized glucagon indwelled within a pump was frequently reconstituted with diluent in
`order to minimize the effect of fibrillation.
`
`These favorable clinical results suggest a definite need for a preparation of glucagon (or a
`suitable bioactive analog) that is sufficiently stable so that it can be indwelled in a portable
`pump for at least three days, the currently allowed maximum for portable pump use.
`Currently, the FDA-approved instructions for commercially available glucagon allow only
`for immediate usage of glucagon to treat severe hypoglycemia, after which the unused
`portion must be discarded.
`The effect of pH and other conditions on glucagon stability
`In a recent study [12], we investigated whether the pH values during aging would affect the
`degree of fibrillation and cytotoxicity. We were particularly interested in studying the
`alkaline range in addition to the more commonly utilized acidic range (the latter of which is
`used in both of the preparations that are commercially available in the US). Because of its
`
`Curr Diab Rep. Author manuscript; available in PMC 2014 March 31.
`
`NIH-PA Author Manuscript
`
`NIH-PA Author Manuscript
`
`NIH-PA Author Manuscript
`
`Page 2
`
`

`
`Jackson et al.
`
`Page 3
`
`low solubility based on an isoelectric point of approximately 7, the neutral range is generally
`not suitable for evaluation of glucagon.
`
`We were also interested in understanding the specific effects of pH and osmolarity on
`cytotoxicity, especially because we knew that some of the earlier studies did not report this
`information. The preparations were aged for various durations up to 7 days at 37° C.
`
`A pH study was carried out to assess the direct effect of pH on cytotoxicity in NIH-3T3
`cells. Glucagon aggregation and fibrillation were measured by the degree of reaction with
`the Congo red reagent.
`
`In the pH study, we found that only those media pH values of 7.0-9.5 avoided cytotoxicity in
`NIH 3T3 cells. Similarly, we found that only the media osmolality range of 250-400 mOsm/
`l avoided cytotoxicity. Such findings are useful in interpreting studies in which compounds
`are tested for cytotoxicity. To the degree that the drug itself causes the cell culture media to
`fall out of these ranges, the appropriate interpretation would be that a toxic effect was at
`least partially attributable to the pH itself, quite apart from the compound being tested. For
`the commercially available glucagon, the pH of the cell culture media was approximately
`6.5. Not surprisingly, even without aging, a high concentration of this acid preparation (5
`mg/ml) caused immediate cytotoxicity, an effect likely due to pH or excipients.
`
`For glucagon prepared at a pH of 8.5 or 10 without aging, there was no significant
`cytotoxicity at any concentration. However, after aging for 5 days, there was some evidence
`of toxicity at pH 8.5 at a concentration of 2.5 mg/ml. Importantly, after aging for 5 days at a
`pH of 10 in glycine buffer, there was no evidence of cytotoxicity [12].
`
`Conditions that favor cytotoxicity also favor amyloid fibril formation and suggest that large
`amounts of fibrillar amyloid lead to cytotoxicity. Congo red amyloid intensity was greatest
`in glucagon prepared and aged at pH 3 in citrate buffer, indicating substantial amyloid fibril
`formation. At a mildly alkaline pH of 8.5, there was also substantial Congo red intensity
`after aging. In contrast, at a pH of 10, there was no Congo red staining for native glucagon,
`even after aging for 28 days. The results for size-exclusion chromatography (SEC)
`confirmed the Congo red findings. Using SEC, at pH values of 3, there was rapid loss of the
`monomeric glucagon peak over 1-3 days. At a pH of 8, the loss occurred over 1-3 weeks.
`However, at a pH of 10, there was remarkable preservation of the monomeric glucagon
`peak, with little if any loss even after 3-4 weeks [12]. Very recently, in unpublished studies,
`we sought to better understand the effect of differences in pH on glucagon fibrillation
`kinetics. Using very sensitive Thioflavin T and tryptophan fluorescence assays, we observed
`very rapid fibrillation between pH 8.6 and 9.6, starting within 24 hours of aging. However,
`with these sensitive tools, at higher pH values of 9.8-10, glucagon showed no evidence of
`amyloid fibril formation after one week [13].
`
`These experiments have clarified several key concepts regarding the stability of glucagon.
`
`1.
`
`In native glucagon, amyloid fibrils form very quickly (with substantial formation
`within 24 hours).
`
`2. Amyloid formation occurs much more rapidly at acid pH than at alkaline pH. At
`pH 10, formation of amyloid is almost undetectable.
`
`3.
`
`In studies in which compounds are investigated for potential cytotoxicity,
`experiments must be carried out within pH and osmolality ranges that are not
`inherently cytotoxic. (The cells commonly used for such assays, NIH-3T3 cells,
`require a pH of 7.0-9.5 and an osmolality of 250-400 mOsm/l).
`
`Curr Diab Rep. Author manuscript; available in PMC 2014 March 31.
`
`NIH-PA Author Manuscript
`
`NIH-PA Author Manuscript
`
`NIH-PA Author Manuscript
`
`Page 3
`
`

`
`Jackson et al.
`
`Page 4
`
`4. Even at high glucagon concentrations, there is no evidence of cytotoxicity when
`aged at a pH of 10, though there is some cytotoxicity at pH 8.5.
`
`5. The glycine buffer for pH 10 solutions need not be present in high concentration;
`20 mM is sufficient to hold glucagon at 1 mg/ml at a pH of 10. In fact, we believe
`such a low concentration to be desirable. A low concentration of buffer would
`allow faster equilibration down to normal neutral body pH after subcutaneous
`injection in humans.
`
`We also sought to address the activity of fresh and amyloid-rich, aged glucagon solutions
`after subcutaneous injection into non-diabetic pigs, since earlier data from our group and the
`Boston group suggested that aged solutions might have greater bioactivity than one might
`expect. The Yorkshire pigs were treated with octreotide to suppress their endogenous
`production of glucagon, in order to isolate the effect to exogenous glucagon. To understand
`the pharmacokinetic effect of glucagon, we also collected periodic serum samples for
`measurement of glucagon levels during these studies. We found that the pharmacodynamic
`(PD) hyperglycemic effect of fibrillated glucagon aged for one week was substantially
`similar to the PD effect of fresh glucagon. When the glucagon plasma levels were measured,
`we observed a tendency for the rise in glucagon to be slightly delayed (approximately 10-15
`minutes) in the aged vs freshly prepared formulation. This finding suggests that fibrillated
`glucagon, in which the glucagon monomers are held together by weak bonds, almost
`certainly dissociates over time to monomeric glucagon, which can be absorbed into the
`bloodstream [12]. Nonetheless, for safety and regulatory reasons, fibrillated glucagon is
`unsuitable for human drug use.
`Degradation of glucagon
`Many proteins degrade over time, and deamidation of asparagine and glutamine residues are
`a known degradation mechanism in mammals. Robinson and Robinson have suggested that
`this process is a normal mechanism that appropriately regulates protein activity lifetime in
`animals [14]. Deamidation is typically pH-dependent, with the lowest rates occurring around
`pH 4-6, with marked rate increases occurring at both high and low pH extremes [14].
`Asparagine deamidation is more common than glutamine deamidation, and is better
`understood. At acidic pH, direct hydrolysis is the major mechanism for deamidation. At
`neutral and alkaline pH, succinimide ring formation contributes to deamidation, which is
`inhibited by steric hindrance from neighboring amino acid side chains. Glutamine
`deamidation is less well understood, but kinetic evidence points to direct hydrolysis [15].
`The intermediate formations in asparagine involve a five-membered ring, while glutamine
`intermediates involve a less-stable six-membered ring [16].
`
`Experimentally, low pH glucagon has demonstrated deamidation at glutamine and
`asparagine residues and peptide cleavage at aspartate residues [16]. Glucagon has three
`glutamine residues and one asparagine, making four of its 29 amino acids susceptible to
`deamidation. Our preliminary work, presented in abstract form [13], showed a pH-
`dependent degradation of native glucagon, which increased with rising pH from pH 8.6 to
`10. The degradation products were analyzed by liquid chromatography-mass spectrometry
`(LCMS), which showed deamidation as the major degradative pathway, accompanied by
`some oxidation and chain cleavage. Deamidated glucagon appears on LC-MS analysis as a
`separate, well-defined peak with a retention time 2-3 minutes shorter than that of native
`glucagon. Its mass is increased by 1 Dalton over native glucagon. We observed that, by day
`3 of aging at 370C at pH 10, 35% of the glucagon at pH 10 has been deamidated, compared
`to only 20% at pH 9 at the same time point [13].
`
`Curr Diab Rep. Author manuscript; available in PMC 2014 March 31.
`
`NIH-PA Author Manuscript
`
`NIH-PA Author Manuscript
`
`NIH-PA Author Manuscript
`
`Page 4
`
`

`
`Jackson et al.
`
`Page 5
`
`These data confirm that both the aggregation and degradation of glucagon are strongly pH-
`dependent. Because there is no one pH value that avoids both processes, it is likely that
`stabilizing agents will be needed to optimize glucagon formulations.
`
`There are other approaches that show promise in achieving a stable, liquid glucagon
`formulation. First, a team at Biodel, Inc., found a way to stabilize glucagon at neutral pH by
`the use of a surfactant accompanied by a sugar. Like the alkaline preparation discussed
`above and the other approaches discussed below, the formulation has not been approved by
`the FDA. The Biodel formulation was found to be reasonably stable by reverse-phase HPLC
`over several weeks [17]. For more details regarding the Biodel approach, the reader is
`referred to a published patent application [18].
`
`Another approach is to create a mutant form of glucagon by substituting non-native amino
`acids into the peptide chain. Chabenne and DiMarchi et al. reported that such a substitution
`near the C-terminal end of the glucagon molecule achieved a substantial degree of
`biostability [19]. For a summary of glucagon analogs proposed by this group, whose
`technology is now owned by Roche, see patent application [20]. The group from Xeris
`Pharmaceuticals presented preliminary evidence at the 2012 ADA Scientific Sessions
`(http://xerispharma.com/ADA_Poster_FINAL_5_30.pdf) showing that native glucagon can
`be stabilized in liquid form with the use of a non-aqueous solvent (dimethyl sulfoxide). In
`their presentation, they reported that this formulation largely avoids glucagon degradation
`and fibrillation. For their patent application, see [21]. Other approaches designed to stabilize
`glucagon include the method of Arecor, Inc., which uses excipients that exchange protons
`with proteins [22].
`
`The foregoing methods mentioned here also have the potential to stabilize liquid glucagon
`for use as a rescue injection for persons with diabetes who suffer from marked symptomatic
`hypoglycemia in the setting of an emergency. Currently, it is necessary for the companions
`of such individuals to quickly mix a lyophilized powder with an aqueous diluent and then
`inject the solution subcutaneously or intramuscularly into the patient. Such a procedure is
`very difficult for a lay person to accomplish, especially in the heat of an emergency. If a
`stable liquid solution were available, the procedure would be simpler, safer, and less
`anxiety-provoking.
`
`In order to avoid the need for manual mixing, Xeris is developing a glucagon pen that uses a
`non-aqueous liquid solvent that requires no mixing. In addition, Enject, Inc., has developed
`a pen device that, upon injection, automatically mixes the diluent with the lyophilized
`glucagon powder. Though not yet commercially available, this device is summarized in the
`following document (http://www.enject.com/uploads/Enject_article_BioCentury.pdf).
`Tolerability of subcutaneously-administered of alkaline proteins in humans
`There is a paucity of information about tolerability of alkaline drug solutions that are
`administered subcutaneously. In an off-label fashion, furosemide, a highly alkaline solution,
`is sometimes given by the subcutaneous route, but the parenteral formulation is approved
`only for intravenous use [23]. Because of the question of tolerability of glucagon at a high
`pH, we carried out a study in which we administered a common, well-tolerated protein
`(human albumin) to normal human subjects. In a double-blind fashion, on separate
`occasions, a physician administered albumin (1 mg/ml) buffered at pH 7.4 (with phosphate
`buffer) and albumin buffered at pH 10 (with glycine buffer). Each subject received 4
`injections: albumin at each pH given fast (over 10 seconds) or slow (over 60 seconds).
`Subjects rated their experience of discomfort on a 6-point scale, from 0-5, with 0
`representing no discomfort. The results [24] demonstrated that:
`
`Curr Diab Rep. Author manuscript; available in PMC 2014 March 31.
`
`NIH-PA Author Manuscript
`
`NIH-PA Author Manuscript
`
`NIH-PA Author Manuscript
`
`Page 5
`
`

`
`Jackson et al.
`
`Page 6
`
`1. Slow injections caused slightly more discomfort than fast injections.
`
`2. Albumin at pH 10 caused slightly more discomfort than albumin at pH 7.4.
`
`3. However, even at pH 10, the discomfort was rated as minimal or slight,
`approximately 1 on a scale of 0-5.
`
`4. Using the Draize scale, there was no more visible inflammation at the subcutaneous
`site with the alkaline injection vs. the neutral injection.
`Methods for assessment of glucagon bioactivity in vitro
`As outlined above, many preparations of glucagon are chemically unstable and lose
`bioactivity over time. For this reason, it is important that glucagon analogs or formulations
`be demonstrated to be bioactive using in vitro assays before initiation of in vivo animal or
`clinical studies. In contrast to in vivo studies, cellular assays of glucagon action represent a
`high-throughput, inexpensive assay that can be used to quickly screen different glucagon
`formulations. Cell-based assays are based on the fact that the major biological actions of
`glucagon, such as inhibition of hepatic glucose production, result from activation of the cell-
`surface glucagon receptor (GluR) in hepatocytes. The GluR is a classical 7-transmembrane
`G protein-coupled receptor that is linked to adenylate cyclase, which generates the second
`messenger cAMP after ligand binding [25]. The increased intracellular cAMP then binds to
`the regulatory subunit of PKA, resulting in the release of the PKA catalytic subunit, which
`then is able to phosphorylate downstream effectors of the GluR signaling cascade [26]. The
`unactivated PKA complex is localized in a punctate pattern due to association with
`cytoskeletal cellular structures, while activated PKA catalytic subunits become dispersed in
`the cytoplasm as they interact with target substrates. Thus, GluR activation can be assessed
`by determination of glucagon-stimulated dispersion of PKA.
`
`Glucagon activity in vitro can be assessed in hepatocytes by directly measuring rises in
`glucose levels in the cell media or reduction in intracellular glycogen stores. Such direct
`measurements require the use of hepatocytes that are able to store glycogen and retain
`responsiveness to glucagon. However, it is important to note that both the storage of
`glycogen and the expression of the GluR are differentiation-dependent processes that may
`be lost in transformed hepatocyte-derived cell lines. Thus, cell line choice is critical. HepG2
`cells, a commonly used hepatoma line that retains many hepatocyte-specific characteristics,
`did not respond to glucagon in our hands or in the experience of others [27]. Though some
`investigators have successfully used primary rat or human hepatocytes to assay the glucagon
`effect [28, 29], primary hepatocytes are expensive, inconvenient, and their performance, in
`our experience, is inconsistent.
`
`The alternative is to generate GluR-expressing lines by transient [27, 30, 31] or stable [32]
`expression of a human or rodent GluR cDNA. Stable expression generally produces lower
`effects of GluR than transient expression. The lower levels may be adequate for binding
`studies but are not sufficient for generation of sufficiently detectable levels of cAMP; thus, it
`can be difficult to select clones that express the appropriate level of GluR without resulting
`in ligand-induced desensitization [33].
`
`An alternative approach, favored by the authors of this review, involves the use of a Chinese
`hamster ovary cell line available from Thermo-Scientific in Lafayette, CO (http://
`www.thermoscientific.com/ecomm/servlet/productsdetail_11152 11961931-1) that, although
`not expressing endogenous GluR, has been stably transfected with a cDNA encoding the
`human GluR as well as a cDNA encoding a fluorescent version of PKA comprised of a
`fusion protein of the catalytic subunit of PKA and green fluorescent protein (GFP). GluR
`activation is assessed by measurement of the disappearance of highly fluorescent PKA
`
`Curr Diab Rep. Author manuscript; available in PMC 2014 March 31.
`
`NIH-PA Author Manuscript
`
`NIH-PA Author Manuscript
`
`NIH-PA Author Manuscript
`
`Page 6
`
`

`
`Jackson et al.
`
`Page 7
`
`aggregates after ligand treatment with the use of fluorescence microscopy. This approach is
`suitable for relatively high-throughput screening in a multi-well format and allows time-
`course and dose-response studies using different ligands to activate PKA.
`
`A figure of this glucagon assay using PKA-GFP activation is shown as Figure 1. It can be
`seen that fluorescence (expressed as normalized activity on the Y-axis) declines with
`increasing glucagon effect. Note that the region over which fluorescence declines
`corresponds to the physiological range in mammals (30-300 pg/ml) over which glucagon
`exerts its biologic effects.
`
`Conclusions
`In summary, automated delivery of low-dose glucagon is proving useful in preventing
`hypoglycemia, a feared complication of insulin-treated diabetes. However, glucagon is a
`highly unstable peptide prone to spontaneous polymerization to an amyloid form (which can
`be minimized at alkaline pH) and spontaneous degradation. A cell-based PKA-based
`fluorescent bioassay is a convenient way of assessing glucagon formulations in vitro.
`
`Cited References
`1. Kimball CP, Murlin JR. Aqueous extracts of pancreas: some precipitation reactions of insulin. J.
`Biol. Chem. 1923; 66:337.
`2. Staub A, Behrens OK. The glucagon content of crystalline insulin preparations. J. Clin. Invest.
`1954; 33:1629–1633. [PubMed: 13211819]
`3. Pedersen JS. The nature of amyloid-like glucagon fibrils. J Diabetes Sci Technol. 2010; 4:1357–
`1367. [PubMed: 21129330]
`4. Pedersen JS, Dikov D, Flink JL, Hjuler HA, Christiansen G, Otzen DE. The changing face of
`glucagon fibrillation: structural polymorphism and conformational imprinting. J Mol Biol. 2006;
`355:501–523. [PubMed: 16321400]
`5. Onoue S, Ohshima K, Debari K, Koh K, Shioda S, Iwasa S, Kashimoto K, Yajima T. Mishandling
`of the therapeutic peptide glucagon generates cytotoxic amyloidogenic fibrils. Pharm Res. 2004;
`21:1274–1283. [PubMed: 15290870]
`6. Teoh CL, Griffin MD, Howlett GJ. Apolipoproteins and amyloid fibril formation in atherosclerosis.
`Protein Cell. 2011; 2:116–127. [PubMed: 21400045]
`7. Maji SK, Perrin MH, Sawaya MR, Jessberger S, Vadodaria K, Rissman RA, Singru PS, Nilsson KP,
`Simon R, Schubert D, Eisenberg D, Rivier J, Sawchenko P, Vale W, Riek R. Functional amyloids
`as natural storage of peptide hormones in pituitary secretory granules. Science. 2009; 325:328–332.
`[PubMed: 19541956]
`8. El-Khatib FH, Jiang J, Damiano ER. A feasibility study of bihormonal closed-loop blood glucose
`control using dual subcutaneous infusion of insulin and glucagon in ambulatory diabetic swine. J
`Diabetes Sci Technol. 2009; 3:789–803. [PubMed: 20144330]
`9. Ward W, Engle J, Duman H, Bergstrom C, Kim S, Federiuk I. The Benefit of Subcutaneous
`Glucagon During Closed-Loop Glycemic Control in Rats With Type 1 Diabetes. IEEE, Sensors
`Journal. 2008; 8:88–96.
`10. El-Khatib FH, Russell SJ, Nathan DM, Sutherlin RG, Damiano ER. A bihormonal closed-loop
`artificial pancreas for type 1 diabetes. Sci Transl Med. 2010; 2:27ra27.
`11. Castle JR, Engle JM, El Youssef J, Massoud RG, Yuen KC, Kagan R, Ward WK. Novel use of
`glucagon in a closed-loop system for prevention of hypoglycemia in type 1 diabetes. Diabetes
`Care. 2010; 33:1282–1287. [PubMed: 20332355]
`12. Ward WK, Massoud RG, Szybala CJ, Engle JM, El Youssef J, Carroll JM, Roberts CT Jr,
`DiMarchi RD. 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. [PubMed: 21129325]
`
`Curr Diab Rep. Author manuscript; available in PMC 2014 March 31.
`
`NIH-PA Author Manuscript
`
`NIH-PA Author Manuscript
`
`NIH-PA Author Manuscript
`
`Page 7
`
`

`
`Jackson et al.
`
`Page 8
`
`13. Jackson, M.; Stonex, T.; Caputo, N.; El Youssef, J.; Branigan, D.; Castle, JR.; David, L.; Ward,
`WK. Development of a Stable Formulation of Liquid Glucagon for Use in a Bihormonal Pump;
`Presented at 2012 Scientific Session, American Diabetes Association, Philadelphia; 2012.
`14. Robinson NE, Robinson AB. Molecular clocks. Proc Natl Acad Sci U S A. 2001; 98:944–949.
`[PubMed: 11158575]
`15. Joshi AB, Sawai M, Kearney WR, Kirsch LE. Studies on the mechanism of aspartic acid cleavage
`and glutamine deamidation in the acidic degradation of glucagon. J Pharm Sci. 2005; 94:1912–
`1927. [PubMed: 16052557]
`16. Manning MC, Chou DK, Murphy BM, Payne RW, Katayama DS. Stability of protein
`pharmaceuticals: an update. Pharm Res. 2010; 27:544–575. [PubMed: 20143256]
`17. Steiner SS, Li M, Hauser R, Pohl R. Stabilized glucagon formulation for bihormonal pump use. J
`Diabetes Sci Technol. 4:1332–1337. [PubMed: 21129327]
`18. Steiner, SS.; Hauser, R.; Li, M.; Feldstein, R.; Pohl, R. Stabilized glucagon solutions. US Patent
`Application # 12/891,240.
`19. Chabenne JR, DiMarchi MA, Gelfanov VM, DiMarchi RD. Optimization of the native glucagon
`sequence for medicinal purposes. J Diabetes Sci Technol. 2010; 4:1322–1331. [PubMed:
`21129326]
`20. DiMarchi, MA.; Smiley, DL.; DiMarchi, M.; Chabenne, JR.; Day, J. Glucagon analogs exhibiting
`enhanced solubility and stability. US Patent Application # 12/999,284.
`21. Prestrelski, S.; Fang, WJ.; Carpenter, JF.; Kinzell, J. Glucagon formulations for the treatment of
`hypoglycemia. US Patent Application # 13/186,275.
`22. Jezek, J. Stable aqueous systems comprising proteins. US Patent Application 11/994,408.
`23. Verma AK, da Silva JH, Kuhl DR. Diuretic effects of subcutaneous furosemide in human
`volunteers: a randomized pilot study. Ann Pharmacother. 2004; 38:544–549. [PubMed: 14982985]
`24. Ward WK, Castle JR, Branigan D, Massoud RG, El Youssef J. Discomfort from an Alkaline
`Formulation Delivered Subcutaneously in Humans: Albumin at pH 7 versus pH 10. Clinical Drug
`Investigation. 2012; 32:433–438. [PubMed: 22568666]
`25. Brubaker PL, Drucker DJ. Structure-function of the glucagon receptor family of G protein-coupled
`receptors: the glucagon, GIP, GLP-1, and GLP-2 receptors. Receptors Channels. 2002; 8:179–188.
`[PubMed: 12529935]
`26. Almholt K, Tullin S, Skyggebjerg O, Scudder K, Thastrup O, Terry R. Changes in intracellular
`cAMP reported by a Redistribution assay using a cAMP-dependent protein kinase-green
`fluorescent protein chimera. Cell Signal. 2004; 16:907–920. [PubMed: 15157670]
`27. Jiang Y, Cypess AM, Muse ED, Wu CR, Unson CG, Merrifield RB, Sakmar TP. Glucagon
`receptor activates extracellular signal-regulated protein kinase 1/2 via cAMP-dependent protein
`kinase. Proc Natl Acad Sci U S A. 2001; 98:10102–10107. [PubMed: 11517300]
`28. Pilar Lopez M, Gomez-Lechon MJ, Castell JV. Role of glucose, insulin, and glucagon in glycogen
`mobilization in human hepatocytes. Diabetes. 1991; 40:263–268. [PubMed: 1846828]
`29. Gomez-Lechon MJ, Ponsoda X, Castell JV. A microassay for measuring glycogen in 96-well-
`cultured cells. Anal Biochem. 1996; 236:296–301. [PubMed: 8660508]
`30. Cypess AM, Unson CG, Wu CR, Sakmar TP. Two cytoplasmic loops of the glucagon receptor are
`required to elevate cAMP or intracellular calcium. J Biol Chem. 1999; 274:19455–19464.
`[PubMed: 10383462]
`31. Cascieri MA, Koch GE, Ber E, Sadowski SJ, Louizides D, de Laszlo SE, Hacker C, Hagmann WK,
`MacCoss M, Chicchi GG, Vicario PP. Characterization of a novel, non-peptidyl antagonist of the
`human glucagon receptor. J Biol Chem. 1999; 274:8694–8697. [PubMed: 10085108]
`32. Parker JC, McPherson RK, Andrews KM, Levy CB, Dubins JS, Chin JE, Perry PV, Hulin B, Perry
`DA, Inagaki T, Dekker KA, Tachikawa K, Sugie Y, Treadway JL. Effects of skyrin, a receptor-
`selective glucagon antagonist, in rat and human hepatocytes. Diabetes. 2000; 49:2079–2086.
`[PubMed: 11118010]
`33. Ikegami T, Cypess AM, Bouscarel B. Modulation of glucagon receptor expression and response in
`transfected human embryonic kidney cells. Am J Physiol Cell Physiol. 2001; 281:C1396–C1402.
`[PubMed: 11546678]
`
`Curr Diab Rep. Author manuscript; available in PMC 2014 March 31.
`
`NIH-PA Author Manuscript
`
`NIH-PA Author Manuscript
`
`NIH-PA Author Manuscript
`
`Page 8
`
`

`
`Jackson et al.
`
`Page 9
`
`Figure 1.
`
`Curr Diab Rep. Author manuscript; available in PMC 2014 March 31.
`
`NIH-PA Author Manuscript
`
`NIH-PA Author Manuscript
`
`NIH-PA Author Manuscript
`
`Page 9