Diabetes Publish Ahead of Print, published online July 14, 2009 Dual GLP-1/GCGR agonist for the treatment of obesity GLP-1/GCGR dual agonism reverses obesity in mice Short-running title: Dual GLP-1/GCGR agonist for the treatment of obesity Alessandro Pocai, Paul E. Carrington, Jennifer R. Adams, Wright Michael, George Eiermann, Lan Zhu, Xiaobing Du, Aleksandr Petrov, Michael E. Lassman, Guoqiang Jiang, Franklin Liu, Corey Miller, Laurie M. Tota, Gaochao Zhou, Xiaoping Zhang, Michael M. Sountis, Alessia Santoprete*, Elena Capito'*, Gary G. Chicchi, Nancy Thornberry, Elisabetta Bianchi*, Antonello Pessi*, Donald J. Marsh, and Ranabir SinhaRoy Merck Research Laboratories, Rahway, NJ 07065, USA and *Istituto di Ricerche di Biologia Molecolare P. Angeletti, Pomezia 00040, Rome, Italy. Corresponding authors: Ranabir SinhaRoy, e-mail: [email protected] or Donald J. Marsh, e-mail: [email protected] Additional information for this article can be found in an online appendix at http://diabetes.diabetesjournals.org Submitted 24 February 2009 and accepted 6 July 2009. This is an uncopyedited electronic version of an article accepted for publication in Diabetes. The American Diabetes Association, publisher of Diabetes, is not responsible for any errors or omissions in this version of the manuscript or any version derived from it by third parties. The definitive publisher-authenticated version will be available in a future issue of Diabetes in print and online at http://diabetes.diabetesjournals.org. Copyright American Diabetes Association, Inc., 2009 Dual GLP-1/GCGR agonist for the treatment of obesity Objective— Oxyntomodulin (OXM) is a GLP-1 receptor (GLP1R)/glucagon receptor (GCGR) dual agonist peptide that reduces body weight in obese subjects through increased energy expenditure and decreased energy intake. The metabolic effects of OXM have been attributed primarily to GLP1R agonism. We examined whether a long acting GLP1R/GCGR dual agonist peptide exerts metabolic effects in diet-induced obese mice that are distinct from that obtained with a GLP1R-selective agonist. Research design and methods— We developed a protease-resistant dual GLP1R/GCGR agonist, DualAG, and a corresponding GLP1R-selective agonist, GLPAG, matched for GLP1R agonist potency and pharmacokinetics. The metabolic effects of these two peptides with respect to weight loss, caloric reduction, glucose control, and lipid lowering, were compared upon chronic dosing in DIO mice. Acute studies in DIO mice revealed metabolic pathways that were modulated independent of weight loss. Studies in Glp1r-/- and Gcgr-/- mice enabled delineation of the contribution of GLP1R versus GCGR activation to the pharmacology of DualAG. Results— Peptide DualAG exhibits superior weight loss, lipid lowering activity, and antihyperglycemic efficacy comparable to GLPAG. Improvements in plasma metabolic parameters including insulin, leptin, and adiponectin were more pronounced upon chronic treatment with DualAG than with GLPAG. Dual receptor agonism also increased fatty acid oxidation and reduced hepatic steatosis in DIO mice. The anti-obesity effects of DualAG require activation of both GLP1R and GCGR. Conclusions— Sustained GLP1R/GCGR dual agonism reverses obesity in DIO mice and is a novel therapeutic approach to the treatment of obesity. 2 Dual GLP-1/GCGR agonist for the treatment of obesity besity is an important risk effects on murine islets and inhibition of factor for T2DM and gastric acid secretion appear to be O approximately 90% of patients independent of GLP1R signaling (14). In with T2DM are overweight or obese (1). addition, pharmacological activation of Among new therapies for T2DM, peptidyl GCGR by glucagon, a master regulator of mimetics of the gut-derived incretin hormone fasting metabolism (16), decreases food glucagon-like peptide 1 (GLP-1) stimulate intake in rodents and humans (17-19), insulin biosynthesis and secretion in a suggesting a potential role for GCGR glucose-dependent manner (2; 3) and cause signaling in the pharmacology of OXM. modest weight loss in T2DM patients. The Since both OXM and GLP-1 are labile in vivo glucose lowering and anti-obesity effects of (T1/2 ~12 min and 2-3 min, respectively (20; incretin-based therapies for T2DM has 21), and are substrates for the cell surface prompted evaluation of the therapeutic protease dipeptidyl peptidase 4 (DPP-4) (22), potential of other glucagon-family peptides, we developed two long acting DPP-4-resistant in particular oxyntomodulin (OXM). The OXM analogs as pharmacological agents to OXM peptide is generated by post- better investigate the differential translational processing of preproglucagon pharmacology and therapeutic potential of (PPG) in the gut and is secreted post- dual GLP1R/GCGR agonism versus GLP1R- prandially from L-cells of the jejuno-ileum selective agonism. Peptide DualAG exhibits together with other PPG-derived peptides in vitro GLP1R and GCGR agonist potency including GLP-1 (4; 5). In rodents, OXM comparable to that of native OXM and is reduces food intake and body weight, conjugated to cholesterol via a Cys sidechain increases energy expenditure, and improves at the C-terminus for improved glucose metabolism (6-8). A 4-week clinical pharmacokinetics. Peptide GLPAG differs study in obese subjects demonstrated that from DualAG by only one residue repeated subcutaneous administration of (Gln3→Glu) and is an equipotent GLP1R OXM was well tolerated and caused agonist, but has no significant GCGR agonist significant weight loss with a concomitant or antagonist activity in vitro. The objective reduction in food intake (9). An increase in of this study was to leverage the matched activity-related energy expenditure was also GLP-1R agonist potencies and noted in a separate study involving short term pharmacokinetics of peptides DualAG and treatment with the peptide (10). GLPAG in comparing the metabolic effects OXM activates both, the GLP-1 and therapeutic potential of a dual receptor (GLP1R) and glucagon receptor GLP1R/GCGR agonist versus a GLP1R- (GCGR) in vitro, albeit with 10-100-fold selective agonist in a mouse model of obesity. reduced potency compared to the cognate ligands GLP-1 and glucagon, respectively RESEARCH DESIGN AND METHODS (11-13). It has been proposed that OXM Animals. Experiments were modulates glucose and energy homeostasis performed in lean and diet-induced obese solely by GLP1R agonism, since its acute (DIO) C57BL/6 mice and in weight- and sex- metabolic effects in rodents are abolished by matched Gcgr−/− (23), Glp1r−/− (24), or lean co-administration of the GLP1R antagonist C57BL/6 control mice. All mice were exendin(9-39) and are not observed in Glp1r-/- obtained from Taconic Farms (Germantown, mice (7; 8; 14; 15). Other aspects of OXM NY, USA) and were maintained on either pharmacology, however, such as protective standard chow (Teklad 7012; Harlan Teklad) 3 Dual GLP-1/GCGR agonist for the treatment of obesity or high fat diet (D12492: 60% kcal from fat; Measurement of plasma peptide Research Diets) in a 12 h light/12 h dark cycle exposures at the end of the chronic study. (light: 3:00 AM -3:00 PM). Animal protocols Diet-induced obese (DIO) mice, 23-weeks old used in these studies were approved by the and maintained for 16-weeks on a high fat Merck Research Laboratories Institutional diet, were anesthetized with isofluorane and Animal Care and Use Committee in Rahway, blood was collected by cardiocentesis into NJ. EDTA-coated microtainer tubes containing Peptides. The free thiol-containing DPP-4 inhibitor and aprotinin. The in vitro peptide precursors of DualAG and GLPAG cell-based cAMP bio-assay for determining were synthesized by standard solid-phase GLP1R agonist potency was used with CHO peptide synthesis using Fmoc/t-Bu chemistry. cells stably transfected with human GLP1R to Peptides were synthesized by reverse-phase determine peptide concentrations by HPLC using water/acetonitrile (0.1% comparing the degree of cAMP accumulation trifluoroacetic acid) gradients. Cholesterol- in plasma samples from treated animals peptide conjugates were synthesized by against a cAMP standard curve generated by reaction of the thiol-containing peptide spiking peptide standards into mouse plasma. precursors with cholest-5-en-3-yl 1-bromo-2- Single dose studies in DIO mice. oxo-6,9,12,15-tetraoxa-3-azaoctadecan-18- Weight- (~45 g) and age-matched DIO mice oate, which was previously assembled by (23-weeks old) were s.c. injected at 9:00 AM standard solution chemistry. The peptide with vehicle, DualAG, or GLPAG at a dose of conjugates were purified by reverse-phase 191 nmol/kg. Food was removed and 6 hours chromatography using water/acetonitrile later (3:00 PM, ~Tmax for GLPAG and (0.2% acetic acid) gradients. Purified peptides DualAG) the animals were sacrificed for were characterized by electrospray MS on a collection of plasma and tissue samples, Micromass LCZ platform spectrometer. which were immediately stored at -80 °C. Determination of murine GLP1R and RNA preparation and quantitative GCGR agonist potency. In vitro agonist RT-PCR. RNA was isolated from tissues potency of peptides was determined in CHO using UltraspecTM Total RNA Isolation cells stably expressing murine GLP1R or Reagent (Biotex Laboratories, Inc.). The murine GCGR using the Cisbio CAMP resulting total RNA was subjected to DNAse Dynamic 2 assay. Peptides were diluted in treatment using RNAse-freeDNAse (Qiagen). assay buffer and incubated with cells in the Following reverse transcription of the RNA to presence of 20%