NN1213 - A Potent, Long-Acting, and Selective Analog of Human Amylin.

Amylin, a member of the calcitonin family, acts via amylin receptors in the hindbrain and hypothalamus to suppress appetite. Native ligands of these receptors are peptides with short half-lives. Conjugating fatty acids to these peptides can increase their half-lives. The long-acting human amylin analog, NN1213, was generated from structure-activity efforts optimizing solubility, stability, receptor affinity, and selectivity, as well as in vivo potency and clearance. In both rats and dogs, a single dose of NN1213 reduced appetite in a dose-dependent manner and with a long duration of action. Consistent with the effect on appetite, studies in obese rats demonstrated that daily NN1213 dosing resulted in a dose-dependent reduction in body weight over a 21-day period. Magnetic resonance imaging indicated that this was primarily driven by loss of fat mass. Based on these data, NN1213 could be considered an attractive option for weight management in the clinical setting.

Development of Zalfermin, a Long-Acting Proteolytically Stabilized FGF21 Analog.

Here, we describe the development of the FGF21 analog zalfermin (NNC0194-0499, 15), intended for once-weekly sc dosing. Protein engineering was needed to address inherent druggability issues of the natural FGF21 hormone. Thus, deamidation of Asp121 was solved by mutation to glutamine, and oxidation of Met168 was solved by mutation to leucine. N-terminal region degradation by dipeptidyl peptidase IV was prevented by alanine residue elongation. To prevent inactivating metabolism by fibroblast activation protein and carboxypeptidase-like activity in the C-terminal region, and to achieve t1/2 extension (53 h in cynomolgus monkeys), we introduced a C18 fatty diacid at the penultimate position 180. The fatty diacid binds albumin in a reversible manner, such that the free fraction of zalfermin potently activates the FGF-receptor complex and retains receptor selectivity compared with FGF21, providing strong efficacy on body weight loss in diet-induced obese mice. Zalfermin is currently being clinically evaluated for the treatment of metabolic dysfunction-associated steatohepatitis.

NPFF Decreases Activity of Human Arcuate NPY Neurons: A Study in Embryonic-Stem-Cell-Derived Model.

Restoring the control of food intake is the key to obesity management and prevention. The arcuate nucleus (ARC) of the hypothalamus is extensively being studied as a potential anti-obesity target. Animal studies showed that neuropeptide FF (NPFF) reduces food intake by its action in neuropeptide Y (NPY) neurons of the hypothalamic ARC, but the detailed mode of action observed in human neurons is missing, due to the lack of a human-neuron-based model for pharmacology testing. Here, we validated and utilized a human-neural-stem-cell-based (hNSC) model of ARC to test the effects of NPFF on cellular pathways and neuronal activity. We found that in the human neurons, decreased cAMP levels by NPFF resulted in a reduced rate of cytoplasmic calcium oscillations, indicating an inhibition of ARC NPY neurons. This suggests the therapeutic potential of NPFFR2 in obesity. In addition, we demonstrate the use of human-stem-cell-derived neurons in pharmacological applications and the potential of this model to address functional aspects of human hypothalamic neurons.

Development of Cagrilintide, a Long-Acting Amylin Analogue.

A hallmark of the pancreatic hormone amylin is its high propensity toward the formation of amyloid fibrils, which makes it a challenging drug design effort. The amylin analogue pramlintide is commercially available for diabetes treatment as an adjunct to insulin therapy but requires three daily injections due to its short half-life. We report here the development of the stable, lipidated long-acting amylin analogue cagrilintide (23) and some of the structure-activity efforts that led to the selection of this analogue for clinical development with obesity as an indication. Cagrilintide is currently in clinical trial and has induced significant weight loss when dosed alone or in combination with the GLP-1 analogue semaglutide.

Type II Turn of Receptor-bound Salmon Calcitonin Revealed by X-ray Crystallography.

Calcitonin is a peptide hormone consisting of 32 amino acid residues and the calcitonin receptor is a Class B G protein-coupled receptor (GPCR). The crystal structure of the human calcitonin receptor ectodomain (CTR ECD) in complex with a truncated analogue of salmon calcitonin ([BrPhe(22)]sCT(8-32)) has been determined to 2.1-Å resolution. Parallel analysis of a series of peptide ligands showed that the rank order of binding of the CTR ECD is identical to the rank order of binding of the full-length CTR, confirming the structural integrity and relevance of the isolated CTR ECD. The structure of the CTR ECD is similar to other Class B GPCRs and the ligand binding site is similar to the binding site of the homologous receptors for the calcitonin gene-related peptide (CGRP) and adrenomedulin (AM) recently published (Booe, J. M., Walker, C. S., Barwell, J., Kuteyi, G., Simms, J., Jamaluddin, M. A., Warner, M. L., Bill, R. M., Harris, P. W., Brimble, M. A., Poyner, D. R., Hay, D. L., and Pioszak, A. A. (2015) Mol. Cell 58, 1040-1052). Interestingly the receptor-bound structure of the ligand [BrPhe(22)]sCT(8-32) differs from the receptor-bound structure of the homologous ligands CGRP and AM. They all adopt an extended conformation followed by a C-terminal ß turn, however, [BrPhe(22)]sCT(8-32) adopts a type II turn (Gly(28)-Thr(31)), whereas CGRP and AM adopt type I turns. Our results suggest that a type II turn is the preferred conformation of calcitonin, whereas a type I turn is the preferred conformation of peptides that require RAMPs; CGRP, AM, and amylin. In addition the structure provides a detailed molecular explanation and hypothesis regarding ligand binding properties of CTR and the amylin receptors.

Increased expression of toll-like receptor 4 and inflammatory cytokines, interleukin-6 in particular, in islets from a mouse model of obesity and type 2 diabetes.

Toll-like receptor 4 (TLR4) has received much attention in the recent years due to its role in development of insulin resistance in type 2 diabetes mellitus. Its expression is elevated in fat and muscle from insulin-resistant mice. Several cells of the pancreatic islets, including ß-cells and resident macrophages, express TLR4. Our hypothesis is that expression of TLR4 and downstream signalling molecules in islets increases during progression of type 2 diabetes, thereby contributing to ß-cell damage. We investigated the hypothesis in the db/db mouse. Islets from male db/db (4, 8 and 15 weeks old) and control db/+ (4 and 15 weeks old) mice were examined for mRNA expression of TLR4 and selected cytokines using qPCR. In addition, cytokine secretion from islets was quantified. TLR4 is expressed in islets from lean and obese mice, displaying a 7.4-fold higher level in 15 weeks old db/db relative to age-matched control (p < 0.01). During progression of clinical type 2 diabetes manifested by hyperglycaemia, TLR4 expression increases 5.6-fold in islets from 15 weeks compared with 4 weeks old db/db mice (p < 0.01). Furthermore, both protein and mRNA levels of all cytokines examined increased. In particular, expression of IL-6 increased with 37 fold. Expression of TLR4 in db/db mouse islets increased in parallel with hyperglycaemia. A similar increase in expression and secretion of TNFα, IL-1 and IL-6 was observed. Our results demonstrate that, in addition to its contribution to insulin resistance, TLR4 might also play a role in ß-cell dysfunction in type 2 diabetes.

Interleukin-6 enhances insulin secretion by increasing glucagon-like peptide-1 secretion from L cells and alpha cells.

Exercise, obesity and type 2 diabetes are associated with elevated plasma concentrations of interleukin-6 (IL-6). Glucagon-like peptide-1 (GLP-1) is a hormone that induces insulin secretion. Here we show that administration of IL-6 or elevated IL-6 concentrations in response to exercise stimulate GLP-1 secretion from intestinal L cells and pancreatic alpha cells, improving insulin secretion and glycemia. IL-6 increased GLP-1 production from alpha cells through increased proglucagon (which is encoded by GCG) and prohormone convertase 1/3 expression. In models of type 2 diabetes, the beneficial effects of IL-6 were maintained, and IL-6 neutralization resulted in further elevation of glycemia and reduced pancreatic GLP-1. Hence, IL-6 mediates crosstalk between insulin-sensitive tissues, intestinal L cells and pancreatic islets to adapt to changes in insulin demand. This previously unidentified endocrine loop implicates IL-6 in the regulation of insulin secretion and suggests that drugs modulating this loop may be useful in type 2 diabetes.

Neuronal responses to transient hypoglycaemia in the dorsal vagal complex of the rat brainstem.

Several regions of the mammalian brain contain glucosensing neurones. In vivo studies have suggested that those located in the hypothalamus and lower brainstem are involved in glucoprivic feeding and homeostatic control of blood glucose. We have identified and characterized hypoglycaemia-sensitive neurones in the dorsal vagal complex of the brainstem using in situ hybridization, single-cell RT-PCR and whole-cell patch-clamp recordings from rat brainstem slices. Approximately 80% of neurones did not respond to hypoglycaemia (changing artificial cerebrospinal fluid (ACSF) glucose from 10 mM to 0 mM) within 5 min (non-responsive: NR). Another 10% depolarized within 155+/-31 s (mean+/-s.e.m.) of glucose removal (glucose-inhibited: GI), and the remaining neurones hyperpolarized within 53+/-7 s (glucose-excited: GE). The hyperpolarization was reversed by the KATP channel blocker tolbutamide. Single-cell RT-PCR revealed that GI and GE, but not NR, cells expressed glucokinase (GLK). In contrast, SUR1, a KATP channel subunit, was expressed in GE and some NR cells. In situ hybridization with biotin-labelled riboprobes in the dorsal vagal complex revealed ubiquitous expression of SUR1, and widespread, but sparse, expression of GLK. Identification of astrocytes using a GFAP (glial fibrillary acidic protein) antibody showed that GLK and GFAP were not colocalized. In summary, we have demonstrated that GI and GE neurones exist in the brainstem and that GLK is essential for their function. It seems likely that GE neurones work in a way analogous to pancreatic beta-cells in that they require both GLK and KATP channels.