GLP-1 Treatment for Diabetes and Beyond

Abstracts

Novel Insights into GLP-1R Agonist Enhancement of Glucose-Stimulated Insulin Secretion
George G. Holz, PhD, State University of New York

Recent advances in Cre-mediated conditional gene targeting and cyclic nucleotide research reveal novel aspects of GLP-1 receptor (GLP-1R) agonist action that are of major importance to our understanding of how these drugs restore pancreatic insulin secretion in patients diagnosed with type 2 diabetes mellitus (T2DM). This lecture will first review recent evidence that GLP-1 released from intestinal L-cells activates enteric and autonomic reflexes to stimulate pancreatic insulin secretion. New findings demonstrating intra-islet secretion of GLP-1 will also be discussed, with special emphasis on the role of islet alpha cells as a source of GLP-1. Actions of dipeptidylpeptidase-4 (DPP-4) resistant GLP-1 analogs will be considered in light of their direct effect to activate the beta-cell GLP-1R and to potentiate glucose-stimulated insulin secretion (GSIS). Molecular determinants of GSIS under the control of the GLP-1R will be summarized, with a primary focus on cAMP-dependent protein kinase (PKA) and Epac2. Finally, I will discuss the potential for drug development strategies in which signal transduction bias is achieved so that GLP-1R agonists exert insulin secretagogue or growth factor-like effects.
 

Is GLP-1 a Hormone: Whether and when?
David D'Alessio, MD, Duke University

Glucagon-like peptide 1 (GLP-1) is a product of proglucagon cleavage synthesized in L-cells in the intestinal mucosa, a-cells in the pancreatic islet, and neurons in the nucleus of the solitary tract. GLP-1 is essential for normal glucose tolerance and acts through a specific GLP-1 receptor that is expressed by islet b-cells as well as other cell types. Because plasma concentrations of GLP-1 increase following meal ingestion it has been generally presumed that GLP-1 acts as a hormone, communicating information from the intestine to the endocrine pancreas through the circulation. However, there are a number of problems with this model including low circulating concentrations of GLP-1 in plasma, limited changes after meal ingestion and rapid metabolism in the plasma. Moreover, antagonism of systemic GLP-1 action impairs insulin secretion in the fasting state, suggesting that the GLP-1r is active even when plasma GLP-1 levels are low and unchanging. Consistent with this deletion of the GLP-1r from islet b-cells causes intolerance after IP or IV glucose, challenges that do not induce GLP-1 secretion. Taken together, these data support a model whereby GLP-1 acts through neural or paracrine mechanisms to regulate physiologic insulin secretion. In contrast, bariatric surgery seems to be a condition in which circulating GLP-1 has an endocrine effect. Both gastric bypass and sleeve gastrectomy are associated with substantial increases in postprandial GLP-1 release and in these conditions interference with GLP-1r signaling has a significant impact on glucose regulation after eating. Thus, with either bariatric surgery or treatment with long-acting GLP-1r agonists, circulating peptide mediates insulinotropic activity. Overall, a case can be made that physiologic actions of GLP-1 are not hormonal, but that an endocrine mechanism of GLP-1r activation can be co-opted for therapeutics.
 

Next Generation GLP-1 Agonists – Examples of Dual Strategies
Keld Fosgerau, PhD, Zealand Pharma A/S

Several GLP-1 agonists have been developed into medicines for the treatment of Type 2 diabetes and obesity. However, despite the success for this drug-class there are still unmet medical needs for patients that should be addressed. These unmet needs include restoration of beta-cell function in diabetes and a substantial long-lasting body weight loss in Type 2 diabetes as well as obesity. At Zealand Pharma we believe that this unmet need could possibly be reached by development of second generation GLP-1 agonists, where the action of GLP-1 agonist is combined with a second activity in so-called multifunctional peptides. Specifically we at Zealand Pharma have developed a series of dual agonists combing GLP-1 agonism with agonistic properties of other gastrointestinal hormones exemplified by glucose-dependent insulinotropic peptide (GIP), glucagon (GCG), and gastrin. In a series of pre-clinical experiments in diabetic animal models we have shown that treatment with GLP-1-gastrin dual agonist causes an improvement glycemic control and protect beta cells, factors that can slow or prevent the T2D progression. Also, treatment of obese rodent models with either GLP-1-GCG or GLP-1-GIP dual agonists have demonstrated an increased efficacy in terms of body weight lowering effect, as compared to GLP-1 agonists alone. The concept and the chemical basis for developing dual GLP-1 agonists and the generated data will be presented and discussed.
 

GLP-1R Mechanisms for the Treatment of Obesity
Lotte Bjerre Knudsen, DMSc, Novo Nordisk

Liraglutide is a GLP-1R agonist marketed for the treatment of type 2 diabetes. Besides lowering blood glucose, liraglutide reduces body weight, and has recently been approved for chronic weight management. Acutely, GLP-1 markedly reduces gastric emptying, and this effect was previously believed to at least partly explain the effect on body weight loss. However, recent studies in both humans and animals have shown that GLP-1R agonists like liraglutide that lead to pharmacological concentrations for 24h/day only have minor effect on gastric emptying; such effects unlikely to have lasting effects on appetite reduction. Liraglutide has been shown to have direct effects to the arcuate nucleus of the rodent brain, activating POMC neurons and increase levels of the CART neuropeptide mRNA, which correlate nicely to clinical studies where liraglutide was shown to increase feelings of satiety. However, despite the lack of a GLP-1R on AGRP/NPY neurons liraglutide also was able to prevent a hunger associated increase in AGRP and NPY neuropeptide mRNA, again with a nice correlation to clinical studies that document reduced hunger feelings in patients while on liraglutide. Studies using fluorescent labelled liraglutide as well as other GLP-1R agonists and analysis using single plane illumination microscopy (SPIM) show that such medium size peptide based compounds can directly access not only the circumventricular organs of the brain but also directly access discrete regions in the hypothalamus. The direct effects of long-acting GLP-1R agonists in the hypothalamus are likely to be an important new pathway in understanding GLP-1R agonist mediated weight loss.
 

Pharmacological Analysis of Novel Biased Peptide Agonists of the Human Glucagon-like Peptide 1 Receptor
Jean-Philippe Fortin¹

Glucagon-like peptide-1 (GLP-1) is a polypeptide hormone secreted from enteroendocrine cells that potentiates glucose-dependent insulin secretion in pancreatic beta cells. The clinical success of injectable GLP-1 receptor (GLP-1R) peptide agonists for the treatment of type 2 diabetes mellitus has triggered industry-wide efforts aimed at developing orally bioavailable drugs against this target. More recently, the suggestion that differential engagement of intracellular signaling partners (Gs, β-arrestins) may impact the therapeutic actions of GLP-1 mimetics uncovered a new layer of complexity for lead optimization. We hypothesized that deeper understanding of the pharmacology of marketed injectables and novel biased compounds may help defining optimal signaling profiles for therapeutic efficacy. All agonists tested showed maximal activity on the cAMP pathway. Clinical agents liraglutide, lixisenatide were full agonists for β-arrestin recruitment, while exenatide displayed a slight reduction in β-arrestin efficacy relative to GLP-1. In contrast, we identified biased 11-mer peptides displaying a markedly reduced ability to engage β-arrestins, as compared to GLP-1. Bias factor quantification facilitated understanding of the structure-activity relationships underlying bias. Diminished β-arrestin recruitment was associated with altered GLP-1R endocytosis. To explore the biological relevance of β-arrestin recruitment, we assessed the acute insulinotropic effects of full and biased agonists using a human b-cell line, isolated human islets and a primate hyperglycemic clamp model. Importantly, these studies support that a certain degree of bias toward cAMP signaling is tolerated for these acute responses. The recent identification of novel tool compounds with more extreme biased profiles warrants additional work to define how β-arrestins impact the actions of GLP-1R activators.
 
Coauthors: Joseph-Brent Kuzmiski, Barbara Bernardo, Richard Derksen, Jocelyn E. Manning Fox, Patrick MacDonald, David Edmonds, David Tess, Cindy Li, Paula Loria, Amit Kalgutkar, Meg Landis, Lucy Rogers, Chris Limberakis, Alan Mathiowetz, Dan Lettiere, Heather Eng, David Price, Margaret Jackson and David Griffit
 
1 Pfizer Worldwide Research and Development, Cardiovascular and Metabolic Diseases Research Unit, Pfizer Inc. Cambridge, MA, USA
 

Sustained Release Formulation of Exenatide for the Treatment of Traumatic Brain Injury
Dong Seok Kim, PhD^1,2

Exenatide, a GLP-1 analogue, has been identified as a novel treatment for neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson’s disease (PD) by neuroprotective and neurotrophic mechanisms. Traumatic brain injury (TBI) occurs by an external force, which commonly results in demetia and/or parkinsonism decades later. Previously, Exenatide, delivered by a micro-osmotic pump, showed a neuroprotective effects in mild TBI (mTBI) animal model. In this study, the pharmaceutically acceptable sustained release (SR) formulation of exenatide, PT340, was examined in the mTBI mice model. mTBI mice displayed deficits in novel object recognition, while PT340-treated mTBI mice  performed similar to sham. These data suggest a strong beneficial action of PT-340 in treating mTBI. Convinient dosing regimen of SR-Exenatide will provide additional benefits for the treatment of mTBI as well as other neurodegenerative diseases such as AD and PD.
 
Coauthors: Yazhou Li PhD¹, Ian Tamargo¹, Hee Kyoung Kim², David Tweedie PhD¹, Chaim G. Pick PhD³, Lital Rachmany³, Heeyong Lee PhD² , Barry Hoffer MD/PhD⁴ and Nigel H Greig PhD¹
 
1 National Institute on Aging, Baltimore, MD
2 Peptron Inc., Daejeon, Korea
3 Tel Aviv university, Israel
4 Case Western Reserve University, Cleveland, OH
 

Neuroanatomical Mechanisms Mediating Central GLP-1 Effects on Energy Balance
Matthew R. Hayes, PhD, Perelman School of Medicine, University of Pennsylvania

Glucagon-like peptide-1 receptors (GLP-1R) expressed in exra-hypothalamic nuclei, such as the ventral tegmental area (VTA), nucleus accumbens (NAc) core, nucleus tractus solitarius (NTS), and parabrachial nucleus (PBN) are pharmacologically and physiologically relevant for the regulation of palatable food intake. In separate sets of experiments we are demonstrating similar behavioral, molecular and neurophysiological mechanisms mediating the food intake suppressive effects following GLP-1R activation in these nuclei. Within the NAc core as an example, we investigated whether GLP-1R signaling modulates GABAergic medium spiny neurons (MSNs) through presynaptic-glutamatergic and/or -dopaminergic signaling to control feeding. Ex vivo fast-scan cyclic voltammetry showed that the GLP-1R agonist exendin-4 does not alter dopamine release in the NAc core. Rather support for a glutamatergic mechanism was provided by ex vivo electrophysiological analyses showing that exendin-4 activates presynaptic GLP-1Rs in the NAc core to increase the activity of MSNs via a glutamatergic, AMPA/kainate receptor-mediated mechanism, indicated by increased mEPSC frequency and decreased paired pulse ratio. Only a small direct excitatory effect on MSNs by Ex-4 was observed, suggesting that the contribution of postsynaptic GLP-1R in the NAc core to MSN activity is minimal. The behavioral relevance of the electrophysiological data was confirmed by the finding that blockade of NAc AMPA/kainate receptors attenuated the ability of NAc core GLP-1R activation to suppress food intake and body weight gain; in contrast, NAc NMDA receptor blockade did not inhibit the energy balance effects of NAc GLP-1R activation. Collectively, these experiments demonstrate that GLP-1R signaling in the mesolimbic reward system modulates glutamatergic signaling to affect palatable food intake. DK096139 (MRH).
 

Chemical Biotechnology Applied to Metabolic Diseases
Richard DiMarchi, PhD, Indiana University

The epidemic of obesity and its associated comorbidities represents a medicinal challenge that recruits broad molecular diversity. We have pioneered the application of endogenous hormones and physiological mechanisms optimized for pharmacological purposes as a means to address the broad heterogeneity constituted by the multiple diseases associated with the metabolic syndrome. From the earliest demonstration with lispro-insulin to the most recent discovery of incretin-based poly-pharmacophores we have pursued the discovery of therapeutics directed at the successful management of insulin-dependent diabetes, obesity and related diseases. We have coined the term “chemical biotechnology” to reflect the integration of classical small and large molecule-based chemistries. The integrated pharmacology of these peptides, proteins and nuclear hormones has provided a library of medicinal agents to be interrogated in cardio-metabolic diseases.

References

1. “A Rationally Designed Peptide of Triple Gut Hormone Action Cures Obesity and Diabetes” Nature Medicine (2015), 21:27-36
2. “Chemical Synthesis of Insulin Analogs Through a Novel Precursor” ACS Chem. Biol., (2014), 9:683
3. “Break on Through to the Other 1” Cell Metabolism (2014), 20:554
4. “Unimolecular Dual Incretins Maximize Metabolic Benefits in Rodents, Monkeys, and Humans” Sci. Trans. Med. (2013), 5:209
5. “Targeted Estrogen Delivery Reverses The Metabolic Syndrome” Nature Medicine Nature Medicine (2012), Volume:18:1847
6. FGF21 Analogs of Sustained Action Enabled by Orthogonal Biosynthesis Demonstrate Enhanced Antidiabetic Pharmacology in Rodents, Diabetes (2012), 61(2), 505-12.

 

GLP-1 and Dual GLP-1/GIP Receptor Agonists as Promising Therapeutics in Alzheimer’s Disease
Konrad Talbot, PhD, Cedars-Sinai Medical Center

Among the greatest medical challenges of the 21st century is finding an effective treatment for Alzheimer’s disease (AD), which robs those afflicted of their memories and the ability to think, use language, and relate to other people. Without effective treatment, the number of Americans suffering from AD will grow to 13.5 million people by 2050, a number that would overwhelm our healthcare system. Our research group established a very common abnormality in the brains of AD cases (even without diabetes) that could be a drug target for one of the first effective treatments of AD. That abnormality is brain insulin resistance, which normally protects the brain against the kind of tissue damage and memory losses seen in AD. We recently found that insulin resistance (measured by brain responsiveness to 1 nM insulin) is prominent in brain tissue from mild cognitive impairment (MCI), a condition often leading to AD dementia. One hour exposure of brain tissue to 100 nM of the GLP-1 receptor agonist liraglutide (Victoza) markedly enhances insulin responsiveness of such tissue from MCI cases but not from AD dementia cases. In the latter cases, however, brain insulin responsiveness is substantially raised after one hour exposure to 100 nM of a drug activating receptors for both major incretins: GLP-1 and glucose-dependent insulinotropic polypeptide (GIP). Administration of liraglutide or the dual incretin receptor agonist ip also reduces AD-related pathology and cognitive deficits in an AD mouse model. GLP-1 and dual GLP-1/GIP receptor agonists are thus emerging as likely AD therapeutics.
 

Preclinical Evidence for Glucagon-Like Peptide-1 Receptor Agonist Induced Cardioprotection: Evidence for Improving Cardiac Metabolic Efficiency
Beat M. Jucker, PhD^1,2

Because myocardial function and metabolism are inextricably linked it has been suggested that optimizing fatty acid and/or carbohydrate metabolism may be a novel therapeutic approach to the treatment of heart failure.  It is well known that myocardial ischemia and failure conditions can increase utilization of carbohydrate relative to fatty acids, a compensatory adjustment to increase myocardial efficiency. In this regard, prolonged infusion of glucagon-like peptide-1 (GLP-1) has been shown to increase myocardial glucose utilization and increase myocardial function in a model of dilated cardiomyopathy. Previous studies have shown that GLP-1 provides cardiovascular benefits independent of its role on peripheral glycemic control. However, the precise mechanism(s) by which GLP-1 treatment renders cardioprotection during myocardial ischemia remain unresolved. As such, we examined the role for GLP-1 treatment on glucose and fatty acid metabolism in normal and ischemic rat hearts following a 30 min ischemia and 24 h reperfusion injury, and in isolated cardiomyocytes.  In addition, cardioprotection studies using long acting GLP-1 mimetics including albiglutide and DPP-IV resistant GLP-1 bound to a domain antibody specific for albumin were performed. Our results show that the observed cardioprotection benefit may derive from distinct and complementary roles of GLP-1 treatment on metabolism in myocardial sub-regions in response to this injury. In addition, continuous activation may be needed for the observed cardioprotection. These findings suggest that in addition to providing peripheral glycemic control, GLP-1 mimetics may have direct therapeutic potential for improving cardiac energetics and function in the setting of myocardial ischemic injury.
 
Coauthors: Weike Bao, MD, PhD¹, Karpagam Aravindhan, PhD¹, Hasan Alsaid, PhD², Thimmaiah Chendrimada, PhD¹, Lucy J. Holt, PhD³, Elena DeAngelis, PhD³, Mathew Szapacs, PhD⁴, Mark R. Harpel, PhD¹, Larry Jolivette, PhD¹, Robert N. Willette, PhD¹, John J. Lepore, MD¹
 
1 Heart Failure Discovery Performance Unit, Metabolic Pathways and Cardiovascular Therapy Area Unit
2 Preclinical and Translational Imaging, Platform Technology
3 Innovation BDU, Biopharm Research
4 DMPK, Platform Technology and Science, GlaxoSmithKline, King of Prussia, Pennsylvania, United States
 

Signal Bias and Allosterism at the GLP-1R: Implications for Small Molecule GLP-1R Agonists
Patrick Sexton, PhD, Monash University

Glucagon-like peptide-1 (GLP-1) is a key incretin peptide that promotes insulin secretion in response to nutrient ingestion, but also has a range of other actions including preservation of b-cell mass, reduction in gastric emptying and reduction in appetite. GLP-1 exerts its effects by binding to the GLP-1 receptor (GLP-1R), which belongs to the Class B subfamily of G protein-coupled receptors (GPCR). In recent years, it has become clear that individual GPCRs can exist in multiple receptor conformations and can elicit numerous functional responses, both G protein- and non-G protein-mediated. This has led to the discovery that different ligands can stabilize distinct subsets of receptor conformations that can “traffic” stimulus to diverse functional outputs with varying prominence, a concept referred to as biased agonism or ligand-directed signaling. Regulation of GLP-1R function is complex, with multiple endogenous and exogenous peptides that interact with the receptor that result in the activation of numerous downstream signalling cascades. The current understanding of GLP-1R signalling and regulation is limited, with the desired spectrum of signalling required for the ideal therapeutic outcome still to be determined. We have recently demonstrated that both peptide and small molecule ligands of the GLP-1 receptor exhibit distinct bias for both activation of second messenger pathways and interaction with scaffolding and regulatory proteins. Furthermore, small molecule allosteric modulators can modify the signalling and regulatory bias of endogenous peptide ligands acting via the GLP-1 receptor. We are currently exploring the impact of differential signal bias on different aspects of β-cell function.