Glucagon Solution Research Articles

New Fast Acting Glucagon for Recovery from Hypoglycemia, a Life-Threatening Situation: Nasal Powder and Injected Stable Solutions.

The goal of diabetes care is to achieve and maintain good glycemic control over time, so as to prevent or delay the development of micro- and macrovascular complications in type 1 (T1D) and type 2 diabetes (T2D). However, numerous barriers hinder the achievement of this goal, first of all the frequent episodes of hypoglycemia typical in patients treated with insulin as T1D patients, or sulphonylureas as T2D patients. The prevention strategy and treatment of hypoglycemia are important for the well-being of patients with diabetes. Hypoglycemia is strongly associated with an increased risk of cardiovascular disease in diabetic patients, due probably to the release of inflammatory markers and prothrombotic effects triggered by hypoglycemia. Treatment of hypoglycemia is traditionally based on administration of carbohydrates or of glucagon via intramuscular (IM) or subcutaneous injection (SC). The injection of traditional glucagon is cumbersome, such that glucagon is an under-utilized drug. In 1983, it was shown for the first time that intranasal (IN) glucagon increases blood glucose levels in healthy volunteers, and in 1989–1992 that IN glucagon is similar to IM glucagon in resolving hypoglycemia in normal volunteers and in patients with diabetes, both adults and children. IN glucagon was developed in 2010 and continued in 2015; in 2019 IN glucagon obtained approval in the US, Canada, and Europe for severe hypoglycemia in children and adults. In the 2010s, two ready-to-use injectable formulations, a stable non-aqueous glucagon solution and the glucagon analog dasiglucagon, were developed, showing an efficacy similar to traditional glucagon, and approved in the US in 2020 and in 2021, respectively, for severe hypoglycemia in adults and in children. Fast-acting glucagon (nasal administration and injected solutions) appears to represent a major breakthrough in the treatment of severe hypoglycemia in insulin-treated patients with diabetes, both adults and children. It is anticipated that the availability of fast-acting glucagon will expand the use of glucagon, improve overall metabolic control, and prevent hypoglycemia-related complications, in particular cardiovascular complications and cognitive impairment.

A microfluidic system for monitoring glucagon secretion from human pancreatic islets of Langerhans.

Glucagon is a 29-amino acid peptide released from α-cells within pancreatic islets of Langerhans to help raise blood glucose levels. While a plethora of methodologies have been developed for quantitative measurement of insulin released from islets, such methods are not well developed for glucagon despite its importance in blood sugar regulation. In this work, a simple yet robust microfluidic device was developed for holding human pancreatic islets and perfuse them with glucose. The perfusate was collected into 2 min fractions and glucagon quantified using a homogeneous time-resolved Förster resonance energy transfer (TR-FRET) sandwich immunoassay. Simulation of fluid flow within the microfluidic device indicated the device produced low amounts of shear stress on islets, and characterization of the flow with standard glucagon solutions revealed response times within 2 fractions (<4 min). Results with human islets from multiple donors demonstrated either a “burst” of glucagon or a “sustained” glucagon release across the entire period of stimulation. The simplicity, yet robustness, of the device and method is expected to appeal to a number of researchers examining pancreatic islet physiology.

Inhalable Sustained-Release Formulation of Glucagon: In Vitro Amyloidogenic and Inhalation Properties, and In Vivo Absorption and Bioactivity

The present study aimed to develop novel glucagon-loaded PLGA nanospheres without cytotoxic fibril formation for chronic glucagon replacement therapy. Glucagon-loaded nanospheres (GLG/NS) were prepared by an emulsion solvent diffusion method in oil, and a respirable powder formulation (GLG/NS-RP) was prepared with a jet mill. Physicochemical and inhalation properties of GLG/NS-RP were characterized, and pharmacokinetic behavior and hyperglycemic effect of intratracheally instilled GLG/NS-RP were evaluated in rats. Although preparation of GLG/NS using glucagon solution at concentrations over 10 mg/mL led to significant formation of cytotoxic glucagon aggregates, glucagon solution at less than 5 mg/mL did not cause structural changes. Drug release behavior of GLG/NS showed a biphasic pattern with an initial burst and slow diffusion. Laser diffraction and cascade impactor analyses of GLG/NS-RP suggested high dispersion and deposition in the respiratory organs with a fine particle fraction of 20.5%. After the intratracheal administration of the GLG/NS-RP (200 μg glucagon/kg) in rats, glucagon was released in a sustained manner, leading to sustained hyperglycemic effects compared with those of normal glucagon powder. These data would suggest a therapeutic benefit of the newly developed GLG/NS-RP as an alternative to the injection form of glucagon currently used.

Circular Dichroism Reveals Sensitivity of Glucagon Solution Structure to Fluoroalcohols, pH and Ionic Strength

Circular dichroism reports that glucagon in solution becomes increasingly helical with the addition of fluoroalcohol (which also decreases solution pH), and with changes in pH and ionic strength. Given the variability of structure observed, these data indicate that care must be taken when comparing results obtained under different solution conditions.

Characterization of seed nuclei in glucagon aggregation using light scattering methods and field-flow fractionation

BackgroundGlucagon is a peptide hormone with many uses as a therapeutic agent, including the emergency treatment of hypoglycemia. Physical instability of glucagon in solution leads to problems with the manufacture, formulation, and delivery of this pharmaceutical product. Glucagon has been shown to aggregate and form fibrils and gels in vitro. Small oligomeric precursors serve to initiate and nucleate the aggregation process. In this study, these initial aggregates, or seed nuclei, are characterized in bulk solution using light scattering methods and field-flow fractionation.ResultsHigh molecular weight aggregates of glucagon were detected in otherwise monomeric solutions using light scattering techniques. These aggregates were detected upon initial mixing of glucagon powder in dilute HCl and NaOH. In the pharmaceutically relevant case of acidic glucagon, the removal of aggregates by filtration significantly slowed the aggregation process. Field-flow fractionation was used to separate aggregates from monomeric glucagon and determine relative mass. The molar mass of the large aggregates was shown to grow appreciably over time as the glucagon solutions gelled.ConclusionThe results of this study indicate that initial glucagon solutions are predominantly monomeric, but contain small quantities of large aggregates. These results suggest that the initial aggregates are seed nuclei, or intermediates which catalyze the aggregation process, even at low concentrations.

Amyloid Fibrils of Glucagon Characterized by High-Resolution Atomic Force Microscopy

Glucagon solutions at pH 2.0 were subjected to mechanical agitation at 37°C in the presence of a hydrophobic surface to explore the details of aggregation and fiber formation. High-resolution intermittent-contact atomic force microscopy performed in solution revealed the presence of aggregates after 0.5h; however, longer agitation times resulted in the formation of fibrillated structures with varying levels of higher-order assembly. Height, periodicity, and amplitude measurements of these structures allowed the identification of four distinct fiber types. The most elementary fiber form, designated a filament, self-associates in a specific wound fashion to produce protofibrils composed of two filaments. Subsequent self-assembly of these filaments and protofibrils leads to two well-defined fibrillar motifs, termed Type I and Type II. Atomic force microscopy imaging of pH 2.8 glucagon solutions not agitated or exposed to elevated temperature revealed the presence of amorphous aggregates before the formation of fibrillar structures similar to those seen at pH 2.0. Time-course solution Fourier transform infrared spectroscopy and thioflavin T binding studies suggested that glucagon aggregation and fibril formation were associated with the development of β-sheet structure. The results of these studies are used to describe a possible mechanism for glucagon aggregation and fibrillation that is consistent with a hierarchical assembly model proposed for amyloid fibril formation.

From Physical Pharmacy to Clinical Pharmacology

AbstractFor Abstract see ChemInform Abstract in Full Text.

From Physical Pharmacy to Clinical Pharmacology

Research works on molecular interactions in solutions were carried out at School of Pharmacy, the University of Wisconsin under the direction of Prof. T. Higuchi and at Faculty of Pharmaceutical Sciences, Kyoto University under the direction of Prof. H. Sezaki. Studies on permeation of drugs through polymer membranes were carried out at Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Canada and at Pharmaceutical Chemistry Research Laboratories at Food and Drug Directorate, Department of Health and Welfare, Canada. Studies on modification of delivery patterns by means of pharmaceutical approaches were carried out at Faculty of Pharmaceutical Sciences, Hokkaido University. Topics related to modification of drug delivery patterns include employment of amorphous forms such as ground mixture with micro-crystalline cellulose and coprecipitate with polyvinylpyrrolidone, use of biodegradable polymers such as polylactic acid and polycarbonates, gel-forming materials such as konjac, agar and hydroxypropylcellulose, and physicochemical systems such as complexation. Works related to drug delivery and disposition of drugs in humans were carried out at Department of Pharmacy, Kumamoto University Hospital. Topics related to drug delivery in humans include injections containing anticancer drugs for intra-arterial administration, lidocaine gels for dermal anesthesia, glucagon solution for nasal administration. Topics related to disposition of drugs in humans include clinical pharmacokinetic studies in infants and elderly and medical uses of adsorbents.

Effect of various glucagon/insulin molar ratios on blood ketone body levels in rats by use of osmotic minipumps

The bihormonal control by insulin and glucagon of blood ketone body level was studied. Mixed solutions with various molar ratios of glucagon and insulin ( G I ) were subcutaneously infused continuously for five days by use of the osmotic minipump in the normal rats. The concentrations of insulin and glucagon solution were set at the high G I molar ratio, the moderate G I molar ratio and the low G I molar ratio. In addition, the moderate G I molar ratio group was divided into three sub-groups: low glucagon and low insulin, moderate glucagon and moderate insulin, and high glucagon and high insulin. After five days, the rats were decapitated to measure plasma ketone body, free fatty acid (FFA), glucose, insulin and glucagon. The FFA level was not significantly different among three groups. The glucose level was not different between the high and moderate G I molar ratio groups, and decreased in the low G I molar ratio group. 3-β-hydroxybutyrate (3-OHBA) and acetoacetate (AcAc) levels in the high G I molar ratio group were elevated, and 3-OHBA level in the low G I molar ratio group was lowered compared to those in the moderate G I molar ratio group. Among three moderate G I molar ratio sub-groups, there was no difference in 3-OHBA and AcAc levels. These results demonstrate that plasma ketone body levels are controlled by the plasma G I molar ratio.

Effects of inorganic ions on autophagy in hepatocytes.

The effects of inorganic phosphate and divalent cations, Ca2+ and Mg2+, on autophagy were morphologically studied using perfused livers. High concentrations of phosphates in perfusate caused an increased number of macroautophagic vacuoles and translocation of lysosomes around a nucleus, like a glucagon solution. On the other hand, the depletion of phosphate in a glucagon solution showed no effect on the lysosomal elements or showed decreased numbers of microautophagic vacuoles. A divalent cation-free solution caused a similar decrease in the microautophagic vacuoles. There was a significant difference in the number of the macroor microautophagic vacuoles between either the high phosphate or the glucagon solution and either the phosphate-free or the divalent cation-free solution. The use of those inorganic ions in autopahgy was suggested. It has been proposed that inorganic phosphates along with Ca2+ are used for membrane fusion. The present study shows an increased number of autophagosomes in the high phosphate solution and high incidence of the autophagosomes in the phosphate-free or the divalent cation-free solution. Thus, these inoganic ions may be connected with the formation of autophagosomes and in the fusion of them with lysosomes.

Laser Raman spectra of glucagon

Laser Raman spectroscopy study indicates that in concentrated fresh acidic solution (30 mg/ml), glucagon remains predominantly α-helix and not random-coil. The splitting of the amide III band into three components in the crystal at 1262, 1275, and 1295 cm −1 is due to the α-conformation as expected. The presence of a small fraction of β-conformation is demonstrated by the appearance of the weak band at 1230 cm −1 in the fresh solution. This study also established the frequencies of amide III′ bands for the α- and β-conformations of glucagon: 957 and 988 cm −1 for α and β forms, respectively. The conformations of acidic and basic glucagon solutions are apparently different.

Thermodynamics of glucagon aggregation

Heats of dilution of concentrated glucagon solutions have been measured calorimetrically at 10 and 25 degrees C in 0.2 M potassium phosphate buffer of pH 10.6. Analysis of the data in terms of a monomer-trimer equilibrium gives the following thermodynamic parameters for the association reaction at 25 degrees C: delta G degrees = 7.34 kcal/mol of trimer, delta H degrees = -31.2 kcal/mol, deltaS degrees = -80 cal/(K mol), deltaCp = 430 cal/(K mol). The sensitivity of heat of dilution data to the association constant and stoichiometry of the reaction is discussed.

Self-association of glucagon as measured by the optical properties of rhodamine 6G.

The fluorescence of rhodamine 6G is completely quenched in glucagon solutions in 0.6 M K2HOP4 at pH 10.6. The absorption of rhodamine 6G is red-shifted by the same reaction. A single rhodamine 6G molecule appears to be bound to a hydrophobic patch in the center of the trimer of glucagon. Since the glucagon monomer has almost no organized structure this site exists only in the associated trimer form of glucagon. The self-association of glucagon to the trimer has been determined from the variation in rhodamine 6G fluorescence and absorption measured over a 60-fold range of dye concentration. The self-association constant agrees with values determined by other methods in the absence of dye. The binding isotherms of rhodamine 6G to glucagon shift with glucagon concentration and exhibit negative cooperativity.

Presence ot trimers in glucagon solution.

Glucagon in solution at pH 10.2, at a concentration of 5 mg/ml, at which profiles of optical parameters against concentration show that it is substantially in its associated state, was covalently cross‐linked with dimethyl suberimidate. At a suberimidate concentration of 1 mg/ml the formation of high‐molecular‐weight aggregates, which is observed at higher reagent concentrations, is suppressed, and a largely homogeneous product is eluted from a gel‐filtration column in 6 M guanidine hydrochloride. This is also homogeneous as judged by polyacrylamide‐gel electrophoresis in the presence of sodium dodecylsulphate. Sedimentation equilibrium in 6 M guanidine hydrochloride led to a molecular weight of about 9600, showing that the unique cross‐linked form is the trimer; there is no evidence for dimers or hexamers, which have been inferred from sedimentation‐equilibrium distributions. It is suggested that the trimeric form of high α‐helix content may be equated with the rod‐like trimers making up the asymmetric unit in the crystal.

Studies on the conformation of glucagon.

Viscosity measurements of glucagon solutions at pH 11 demonstrate that the molecule has a compact shape in dilute aqueous solution. In more concentrated solutions gels are formed with time which have an antiparallel β-conformation, similar to that found in gels from acidic solution.

Evidence for the presence of inhibitor and some trials for its prevention in radioimmunoassay of plasma glucagon by two-antibody system.

The radioimmuno assay for glucagon using two-antibody system was described in detail. The value of sensitivity ranged 0.06 to 0.60mμg on the standard curves obtained with crystalline glucagon (0 to 14mμg per ml) in buffer solution. The precision of the standard curves was 0.8 to 1.5 in per cent precipitated radioactivity for 0 to 14mμg per ml range of glucagon. Glucagon added to human plasma was recovered at the rate of 74.6 to 116.3 per cent. The glucagon level in fasting human plasma measured by this method showed a wide range of variety and even high values (2.2 to 7.1mμg/ml) were obtained in non-diabetics than in diabetics who gave the values of 1.5 to 5.6 mμg/ml. Human plasma globulin reduced the percentage of precipitated radioactivity, whereas human plasma albumin did not appear to interfere with the immunological reaction. Since human plasma globulin did not exhibit any effect upon the rate of bound radioactivity in paper chromatography, it is suggested that the human plasma globulin may inhibit the immunological reaction at the second reaction. The cross reaction between antirabbit serum goat serum (ARGS) and human plasma or plasma globulin was observed on the immunoelectrophoresis and this was regarded as one of the inhibitors. EDTA failed to reduce the inhibition. The inhibition could be minimized by the extraction of plasma glucagon using acid-ethanol acetone method, by absorption of cross reactant in ARGS by a small amount of human globulin and by the use of anti-rabbit serum globulin goat serum. Even in the assay using anti-rabbit serum globulin goat serum as the second antibody, however, the rate of precipitated radioactivity in the assay of the standard glucagon added to the pooled human plasma with the minimun glucagon was lower than expected. This suggests the presence of another inhibitor (s) in human plasma and the necessity of the use of glucagon-free human plasma in the preparation of the standard glucagon solution.

The circular dichroism of glucagon solutions

Circular dichroic spectra of glucagon confirm that in dilute aqueous solutions (1 mg/ml) about 10–15% α-helix is present which increases to about 35% α-helix at concentrations greater than 10 mg/ml. Even in the helix-inducing solvent chloroethanol a concentration dependence of helicity is observed.

Blood glucose and liver glycogen levels in glucagon-treated chick embryos

The capabilities of chick embryos incubated 6.0–18.0 days to respond to glucagon and recover from its hyperglycemic effect have been examined. A single addition of glucagon solution (Lilly) at a dosage level of 150 or 300 μg was placed on the chorioallantois of 6.0-, 8.0-, 10.0-, 12.0-, 14.0-, and 16.0-day embryos. Blood glucose samples were collected from days 8.0 through 18.0 while livers were procured from embryos sacrificed from days 6.0 through 18.0 of incubation. Liver glycogen levels in glucagon-treated chick embryos indicate that the chick is capable of responding to the hormone as early as day 6 of incubation. Both blood glucose and liver glycogen levels in treated chick embryos indicate that the magnitude and duration of response varies with the age of the embryo. The recovery of blood glucose and liver glycogen levels in the 6.0–10.0-day incubation interval demonstrates that the early chick does have the capacity to regulate blood glucose as well as liver glycogen levels to normal after the addition of a hormonal “hyperglycemic agent.”