A novel nasal powder formulation of glucagon: toxicology studies in animal models.

BACKGROUND: Glucagon nasal powder (GNP), a novel intranasal formulation of glucagon being developed to treat insulin-induced severe hypoglycemia, contains synthetic glucagon (10% w/w), beta-cyclodextrin, and dodecylphosphocholine. The safety of this formulation was evaluated in four studies in animal models. METHODS: The first study evaluated 28-day sub-chronic toxicology in rats treated intranasally with 1 and 2 mg of GNP/day (0.1 and 0.2 mg glucagon/rat/day). The second study evaluated 28-day sub-chronic toxicology in dogs administered 20 and 40 mg of formulation/dog/day (2 and 4 mg glucagon/dog/day) intranasally. A pulmonary insufflation study assessed acute toxicology following intra-tracheal administration of 0.5 mg of GNP (0.05 mg glucagon) to rats. Local tolerance to 30 mg of GNP (equivalent to 3 mg glucagon, the final dose for humans) was tested through direct administration into the eyes of rabbits. RESULTS: There were no test article-related adverse effects on body weight and/or food consumption, ophthalmology, electrocardiography, hematology, coagulation parameters, clinical chemistry, urinalysis, or organ weights, and no macroscopic findings at necropsy in any study. In rats, direct intra-tracheal insufflation at a dose of 0.5 mg of GNP/rat (0.05 mg glucagon/rat) did not result in adverse clinical, macroscopic, or microscopic effects. In dogs, the only adverse findings following sub-chronic use were transient (<30 s) salivation and sneezing immediately post-treatment and mild to moderate reversible histological changes to the nasal mucosa. Daily dosing over 28 days in rats resulted in mild to moderate, unilateral or bilateral erosion/ulceration of the olfactory epithelium, frequently with minimal to mild, acute to sub-acute inflammation of the lamina propria at the dorsal turbinates of the nasal cavity in 2/10 males and 3/10 females in the high-dose group (0.2 mg glucagon/day). These lesions resolved completely over 14 days. Histological examination of tissues from both sub-chronic studies in dogs and rats revealed no microscopic findings. In rabbits, clinical observations noted in the GNP-treated eye and/or surrounding areas included ≥1 of the following: clear discharge, red conjunctiva, partial closure, and swelling of the peri-orbital area, which correlated with erythema and edema noted during ocular observations and grading. DISCUSSION: The studies reported here revealed no safety concerns associated with GNP in animal models. Studies published earlier have highlighted the local safety profile of intranasally administered cyclodextrins (a component of GNP). The choline group, the phosphate group, and the saturated 12-carbon aliphatic chain that are present in the dodecylphosphocholine excipient used in GNP are all present in the phospholipids and lecithins seen ubiquitously in mammalian cell membranes and are unlikely to pose safety concerns; this notion is supported by several studies conducted by the authors that revealed no safety concerns. Taken together, these results suggest that intranasal delivery of GNP holds promise as a future rescue medication for use by caregivers to treat insulin-induced hypoglycemic episodes in patients with type 1 or type 2 diabetes. CONCLUSION: This novel drug product is well tolerated in animal models.

Evaluation of genotoxicity testing of FDA approved large molecule therapeutics.

Large molecule therapeutics (MW>1000daltons) are not expected to enter the cell and thus have reduced potential to interact directly with DNA or related physiological processes. Genotoxicity studies are therefore not relevant and typically not required for large molecule therapeutic candidates. Regulatory guidance supports this approach; however there are examples of marketed large molecule therapeutics where sponsors have conducted genotoxicity studies. A retrospective analysis was performed on genotoxicity studies of United States FDA approved large molecule therapeutics since 1998 identified through the Drugs@FDA website. This information was used to provide a data-driven rationale for genotoxicity evaluations of large molecule therapeutics. Fifty-three of the 99 therapeutics identified were tested for genotoxic potential. None of the therapeutics tested showed a positive outcome in any study except the peptide glucagon (GlucaGen®) showing equivocal in vitro results, as stated in the product labeling. Scientific rationale and data from this review indicate that testing of a majority of large molecule modalities do not add value to risk assessment and support current regulatory guidance. Similarly, the data do not support testing of peptides containing only natural amino acids. Peptides containing non-natural amino acids and small molecules in conjugated products may need to be tested.

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

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.

Mishandling of the therapeutic peptide glucagon generates cytotoxic amyloidogenic fibrils.

PURPOSE: Some therapeutic peptides exhibit amyloidogenic properties that cause insolubility and cytotoxicity against neuronal cells in vitro. Here, we characterize the conformational change in monomeric therapeutic peptide to its fibrillar aggregate in order to prevent amyloidogenic formation during clinical application. METHODS: Therapeutic peptides including glucagon, porcine secretin, and salmon calcitonin were dissolved in acidic solution at concentrations ranging from 1 mg/ml to 80 mg/ml and then aged at 37 degrees C. Amyloidogenic properties were assessed by circular dichroism (CD), electron microscopy (EM), staining with beta-sheet-specific dyes, and size-exclusion chromatography (SEC). Cytotoxic characteristics were determined concomitantly. RESULTS: By aging at 2.5 mg/ml or higher for 24 h, monomeric glucagon was converted to fibrillar aggregates consisting of a beta-sheet-rich structure with multimeric states of glucagon. Although no aggregation was observed by aging at the clinical concentration of 1 mg/ml for 1 day, 30-day aging resulted in the generation of fibrillar aggregates. The addition of anti-glucagon serum significantly inhibited fibrillar conversion of monomeric glucagon. Glucagon fibrils induced significant cell death and activated an apoptotic enzyme, caspase-3, in PC12 cells and NIH-3T3 cells. Caspase inhibitors attenuated this toxicity in a dose-dependent manner, indicating the involvement of apoptotic signaling pathways in the fibrillar formation of glucagon. On the contrary to glucagon, salmon calcitonin exhibited aggregation at a much higher concentration of 40 mg/ml and secretin showed no aggregation at the concentration as high as 75 mg/ml. CONCLUSIONS: These results indicated that glucagon was self-associated by its beta-sheet-rich intermolecular structure during the aging process under concentrated conditions to induce fibrillar aggregates. Glucagon has the same amyloidogenic propensities as pathologically related peptides such as beta-amyloid (Abeta)1-42 and prion protein fragment (PrP)106-126 including conformational change to a beta-sheet-rich structure and cytotoxic effects by activating caspases. These findings suggest that inappropriate preparation and application of therapeutic glucagon may cause undesirable insoluble products and side effects such as amyloidosis in clinical application.

Evaluación del riesgo carcinogénico de las sustancias: parte 2: ejemplos de valoraciones de resultados experimentales / Evaluation of the carcinogenic risk of substances: part II

Se presenta una recopilación de análisis de resultados experimentales de evaluación de potencial genotóxico y carcinogénico y la influencia del análisis mecanístico y análisis caso por caso en la valoración del riesgo carcinogénico para el humano(AU)

Experimental study of the detrimental effect of dopamine/glucagon combination in d,l-propranolol intoxication.

1. Respiratory and cardiovascular failure are the principle toxic effects of beta-blocker overdose. Respiratory arrest is the primary cause of death in beta-blocker intoxicated rats. 2. The effect of glucagon, dopamine and the combination of glucagon/dopamine on respiratory and cardiovascular function and survival time in beta-blocker overdose was investigated in a model of acute d,l-propranolol (resp. 30 and 15 mg kg-1 h-1 in rat and rabbit) intoxication in spontaneously breathing rats and artificially ventilated rats and rabbits. 3. Glucagon (initial dose of 100 micrograms kg-1 (bolus), followed by 1 microgram kg-1 min-1), dopamine (25 micrograms kg-1 min-1) or the combination of glucagon/dopamine did not improve survival time (ST) in d,l-propranolol intoxicated spontaneously breathing rats and artificially ventilated rats and rabbits, although some haemodynamic variables i.e. heart rate (HR), mean arterial blood pressure (MAP), left ventricular pressure (LVPmax) and the differentiated left ventricular pressure (LVdp/dtmax) temporarily improved. 4. Survival time was considerably reduced in d,l-propranolol intoxicated spontaneously breathing and artificially ventilated rats treated with a combination of glucagon/dopamine, which induced a decrease in PaO2 and pH and an increase in PaCO2 partly due to ventilation/perfusion mismatch. 5. The combination of glucagon/dopamine should be used carefully in the treatment of beta-blocker overdose in man.

Antídotos de uso reciente

Los antídotos son sustancias cuya función es contrarrestar el efecto farmacológico y tóxico de otras sustancias, teniendo en cuenta la importancia de las medidas generales en el manejo del intoxicado (baño general, emesis, lavado gástrico, carbón activado, catárticos). Cada día aparecen sustancias nuevas con dichas características. En el presente artículo se pretende dar información breve y detallada sobre las propiedades farmacológicas, indicaciones, dosificación, efectos secundarios y contraindicaciones de algunos de uso general (carbón activado, soluciones electrolíticas con polietilenglycol) y principalmente de algunos específicos de uso reciente: flumazenil, fragmentos Fab-antidigoxina, glucagón, naloxona, clonidina, N-acetil-cisteína, azul de metileno, nitrito y tiosulfato de sodio, ácido-2-3-dimercaptosuccínico, penicilina benzatínica, glicopirrolato y S-adenosil-metionina.

Glucagon produced by recombinant DNA technology: repeated dose toxicity studies, intravenous administration to CD rats and beagle dogs for four weeks.

The toxicity of glucagon produced by recombinant DNA technology (Glucagon (ge)) was studied by daily intravenous administration to rats and dogs for 4 weeks. Pancreatic glucagon of bovine or porcine origin (Glucagon Novo) was used as a reference control in the dogs. Glucagon (ge) has the same sequence of the 29 amino acids as pancreatic glucagon of humans, cows, pigs, rats and dogs. The dosages were 0 (control), 0.2, 1.0 and 5.0 mg Glucagon (ge)/kg/day in the rats, and 0 (control), 1.0 and 5.0 mg Glucagon (ge) and 5.0 mg Glucagon (Novo)/kg/day in the dogs. The studies complied with current EEC, US and Japanese guidelines for 4 week toxicity studies of drugs. All dose levels were well tolerated. The plasma glucose and cardiovascular responses to dosing were monitored in the dogs and found to be in agreement with well-known effects of pancreatic glucagon. The most consistent finding in both species was an increase in liver weight. This change was without concomitant pathological deviations in the other parameters examined. There were no differences in the reaction of dogs following treatment with Glucagon (ge) or Glucagon (Novo). A dose of 1 mg Glucagon (ge)/kg/day was regarded as a clear no-toxic-effect-level in both species.

Glucagon control of carcinogenesis.

Long term administration of glucagon markedly enhanced carcinoma formation in mice simultaneously treated with a chemical carcinogen, 3-methylcholanthrene (3-MCA). The number and size of squamous cell carcinomas were 3-fold greater compared to those in mice treated with MCA alone. DNA labeling and the percentage of [3H]thymidine-labeled cells were also 2- to 3-fold higher in glucagon- and MCA-treated mice than in those treated with MCA only or 5- to 6-fold higher than those in control mice. Ultrastructural studies of glucagon- and MCA-treated tumors revealed the predominance of acinar-cystoid and secretory types of tumors, with the occurrence of large and dense secretory granules (Zymogen-like granules), markedly dilated endoplasmic reticulum cisternae, and lysosomes compared to only typical squamous neoplastic cells observed in MCA-treated mice. Scanning electron microscopic observations also indicated that neoplastic cells of MCA and glucagon-treated tumors exhibited characteristic secretory features on their cell surfaces (a marked increase in blebs and microvilli). These findings demonstrate that glucagon in pharmacological doses significantly enhanced the carcinogenic process and changed the cytodifferentiation of neoplastic cells and, thus, plays an important role in controlling tumor cell biology and neoplastic transformation.

[Treatment of acute pancreatitis by glucagon. Experimental studies in dogs]. / Tratamento da pancreatite aguda pelo glucagon. Estudo experimental em caes.

The authors made a comparative study of the use of glucagon (4 mg/day IV) in 30 dogs with acute pancreatitis. They found that the mortality and amylasemia were significantly lower in the glucagon-treated group than in the control groups (saline and glucose solution 5%). In the glucagon group, the areas of necrosis were smaller (1.5 cm) and rarely found; microscopically, the areas of necrosis and the inflammatory reaction were much smaller than in the other groups. These findings lead to the conclusion that the beneficial action of glucagon is due to another mechanism other than its hyperglycemic effect and that the administration of hypertonic solutions of glucose does not have a beneficial effect in acute pancreatitis.

Toxic effects of glucagon-induced acute lipid mobilization in geese.

The toxic effects associated with rapid lipid mobilization and a high plasma free fatty acid (FFA) concentration produced by glucagon were evaluated. Glucagon (0.5 mg/kg of body wt) was injected intravenously into nonfasting geese. The geese developed rapid respirations and high plasma FFA levels within 15 min after the glucagon injection; three of eleven died. Control geese, injected with saline, did not exhibit toxic signs. Peak FFA concentrations developed 15 min after glucagon and high levels persisted for over 90 min. Geese injected with glucagon frequently developed electrocardiographic abnormalities that included supraventricular tachycardia, premature ventricular contractions, and signs of myocardial ischemia. Light and electron microscopy revealed acute myocardial degeneration and fatty infiltration of the liver. The increase in plasma FFA concentrations and toxic effects were not prevented by pretreatment with nicotinic acid or propranolol.