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Aims/hypothesis. Pulsatile secretion is important for insulin action and suitable animal models are important tools for examining the role of impaired pulsatile insulin secretion as a possible link between beta-cell mass, function and morphology and insulin resistance. This study examines the vascular sampling site, insulin kinetics, pulsatility and the response to glucose pulse entrainment to evaluate the Göttingen minipig as a model for studying pulsatile insulin secretion. Methods. Basal and glucose entrained insulin secretion was examined in normal minipigs and evaluated by autocorrelation, cross correlation and deconvolution. Results. Cross correlation showed a relation between oscillations in insulin concentrations in the portal and jugular vein in anaesthetised animals (p<0.001 in all animals), confirming the usefulness of jugular vein sampling for pulse detection. Jugular vein sampling in conscious animals showed obvious oscillations allowing estimates of burst shape and insulin kinetics. Glucose entrainment improved the pulsatile pattern (autocorrelation: 0.555±0.148 entrained vs 0. 350±0.197 basal, p=0.054). Deconvolution analysis resolved almost all insulin release as secretory bursts (69±20 basal vs 99.5±1.2% entrained, p<0.01) with a pulse interval (min) of 6.6±2.2 (basal) and 9.4±1.5 (entrained) (p<0.05) and a pulse mass (pmol/l per pulse) which was higher after entrainment (228±117 vs 41.2±18.6 basal, p<0.001). Conclusion/interpretation. The ability to fit kinetic parameters directly by deconvolution of peripheral endogenous insulin concentration time series in combination with the suitability of jugular vein sampling, rapid kinetics and entrainability makes the Göttingen minipig ideal for mechanistic studies of insulin pulsatility and its effects on insulin action. [Diabetologia (2002[Diabetologia ( ) 45:1389[Diabetologia ( -1396 Keywords Pulsatile insulin secretion, insulin kinetics, deconvolution, in vivo model, insulin action. dent) diabetes mellitus is characterized by defects in both insulin secretion and action, impaired pulsatile secretion could be an important link between beta-cell malfunction and impaired insulin action [8]. When addressing the cause of these impairments in humans, studies are often influenced by long-term impaired metabolism, and therefore demonstrate that defects could be secondary. This problem can, to some extent, be circumvented by studying first degree relatives of diabetic patients who are at risk of the disease but are still not affected by the effects of hyperglycaemia [9]. However, animal models are a valuable research tool for examining the relation between beta-cell mass, function and morphology on the one hand and insulin It is well documented that insulin concentrations show large amplitude oscillations in the peripheral circulation with a periodicity of 5 to 15 min [1,2,3], and it has been shown that these oscillations are of importance for insulin action in liver [4], muscle [5,6] and adipose tissues [7]. Since Type II (non-insulin-depen-

Several groups are developing fully integrated dual-hormone artificial pancreas systems, which deliver insulin or glucagon in response to changes in glucose to maintain euglycemia. These systems hold the potential to transform management of type 1 diabetes, but in order to be realized these systems require a stable-in-solution glucagon analog suitable for chronic use. Dasiglucagon is a novel glucagon analog stable in liquid formulation. The present studies evaluated safety and tolerability of chronically administered dasiglucagon in rats and dogs. Rats and dogs were dosed s.c. daily for 26 and 39 weeks, respectively, in 4 groups of 20 rats with 0 (vehicle), 0.5, 2, or 8 mg/kg/day; 4 groups of 4 beagle dogs with 0, 0.02, 0.1, or 0.3 mg/kg/day. Glucose and insulin levels increased in a dose-dependent manner in all treated animals. In dogs, heart rate increased at the 0.1 and 0.3 mg/kg/day dose level. Liver and kidney weights increased at all dose levels for in both species, and microscopic pathology demonstrated increase in hepatocyte glycogen vacuolation and increase in chronic progressive nephropathy (a normal finding in aging rats). Heart weights increased with no histopathological correlate. All findings showed full/partial recovery after 4-week treatment free periods. Some animals developed anti-drug-antibodies but with no apparent effect on pharmacodynamics. The NOAEL (no observed adverse effect level) dose of 2 mg/kg/day in rats and 0.1 mg/kg/day in dogs represents exposure multiples (AUC) of 22 and 2 compared to anticipated human doses of up to 1 mg/day. Chronic administration of dasiglucagon multiple times above relevant human doses was well tolerated. All findings were consistent with the known pharmacological effects of glucagon. The risk of glycogen accumulation is not considered relevant for humans treated with dasiglucagon to maintain euglycemia. These findings support long-term human testing of dasiglucagon in dual-hormonal artificial pancreas systems. Disclosure J.R. Castle: Consultant; Self; Zealand Pharma A/S. Advisory Panel; Self; Novo Nordisk Inc. M. Elander: Employee; Self; Zealand Pharma A/S. S.A. O'Halloran: None.

The selection of a vehicle for oral formulations of compounds to be used in non-clinical safety studies is a challenge for poorly soluble compounds. Typically a compromise between solubility and tolerability has to be reached. Vehicle tolerability data are not readily available for a number of vehicles, and a series of oral tolerability studies were, therefore, conducted with Gelucire and Gelucire:PEG400 formulations in rats, dogs and minipigs in order to determine tolerable daily dose volumes in these species. Gelucire and Gelucire:PEG400 formulations were assessed in studies for up to 5 days in minipigs, 7 days in rats and up to 39 weeks in dogs. Gastrointestinal side effects in terms of soft and/or liquid faeces were noted in all species, but the sensitivity to these effects differed between species with the dog being the most sensitive. It was concluded that Gelucire:PEG400 (90:10) was tolerated in Beagle dogs when administered at 1 ml kg(-1) once daily for 39 weeks, and 100% Gelucire was tolerated in the rat and the minipig when administered once daily at 5 ml kg(-1) for 5 days. Copyright © 2016 John Wiley & Sons, Ltd.

Preclinical ResearchPreclinical Development Toxicology, Formulation Drug Delivery, Pharmacokinetics The occurrence of convulsions in nonclinical safety studies during development of a new drug often complicates further development of the compound. Based on experiences with a previous development project at H. Lundbeck A/S, where convulsions were noted in animals, a structured approach to investigate possible mechanisms behind the convulsions is suggested. Because it is typically difficult to determine the exact reason for convulsions, it is important to focus on the assessment of possible human risk rather than finding the exact mechanism behind the convulsions. The suggested investigations include the following: calculation of safety margin and assessment of human risk, evaluation of data already captured in performed studies such as clinical chemistry data, description of clinical signs, batches of compound used, expert advice on activities that could enable further development of the compound, investigation of extended in vitro receptor profiles of parent compound and major metabolites, extended histopathology on brain sections, seizure threshold tests of parent compound and major metabolites (in vivo/in vitro), evaluation of electroencephalograms, and finally contact with authorities to discuss a plan for continued clinical development.