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Aim To evaluate the safety and pharmacokinetics of cotadutide, a dual glucagon-like peptide-1 (GLP-1) and glucagon receptor agonist, in overweight Asian participants with or without type 2 diabetes (T2D). Materials and methods In the phase 1, randomized, blinded, single-ascending dose study, 24 Japanese and eight Chinese healthy adults (body mass index [BMI] 23-40 kg/m2 ) received one subcutaneous dose of cotadutide (50-150 or 100 μg, respectively) or placebo. The primary endpoint was safety. In the phase 2a, randomized, double-blinded, parallel dose-ranging study with forced uptitration, 61 Japanese adults with T2D (BMI 24-40 kg/m2 ; HbA1c 7.0%-10.5%) received cotadutide (100, 200, 300 μg) or placebo for 48 days. Co-primary endpoints were safety/tolerability, change in glucose AUC0-4h and body weight. Results Significant reductions from baseline to day 48 were observed with cotadutide for glucose AUC0-4h (33.6%-42.1% reduction vs. +2.5% with placebo; 95% CIs: 100 μg -45.7%, -33.7%; 200 μg -35.6%, -23.7%; 300 μg -45.0%, -30.8%; placebo 3.4%, 8.3%) and body weight (1.3%-2.5% decrease vs. +0.8% with placebo; 95% CIs: 100 μg -3.4%, -0.8%; 200 μg -4.7%, -2.0%; 300 μg -4.6%, -2.1%; placebo -2.1%, 0.4%). The most common adverse events with cotadutide were mild gastrointestinal symptoms with no serious adverse events. Increased pulse rate with cotadutide versus placebo is consistent with GLP-1 monoagonists. Conclusions Once-daily cotadutide was effective and well tolerated up to 300 μg in overweight Japanese patients with T2D. Further evaluation in Asian populations is warranted.

Aim: To assess the efficacy, safety and tolerability of cotadutide in patients with type 2 diabetes mellitus and chronic kidney disease.Materials and Methods: In this phase 2a study (NCT03550378), patients with body mass index 25-45 kg/m2, estimated glomerular filtration rate 30-59 ml/min/1.73 m2 and type 2 diabetes [glycated haemoglobin 6.5-10.5% (48-91 mmol/mol)] controlled with insulin and/or oral therapy combination, were randomized 1:1 to once-daily subcutaneous cotadutide (50-300 μg) or placebo for 32 days. The primary endpoint was plasma glucose concentration assessed using a mixed-meal tolerance test.Results: Participants receiving cotadutide (n = 21) had significant reductions in the mixed-meal tolerance test area under the glucose concentration-time curve (–26.71% vs. +3.68%, p Conclusions: We established the efficacy of cotadutide in this patient population, with significantly improved postprandial glucose control and reduced bodyweight versus placebo. Reductions in urinary albumin-to-creatinine ratios suggest potential benefits of cotadutide on kidney function, supporting further evaluation in larger, longer-term clinical trials.

To characterize the impact of metabolic disease on the peptidome of human and mouse pancreatic islets, LC-MS was used to analyze extracts of human and mouse islets, purified mouse alpha, beta, and delta cells, supernatants from mouse islet incubations, and plasma from patients with type 2 diabetes. Islets were obtained from healthy and type 2 diabetic human donors, and mice on chow or high fat diet. All major islet hormones were detected in lysed islets as well as numerous peptides from vesicular proteins including granins and processing enzymes. Glucose-dependent insulinotropic peptide (GIP) was not detectable. High fat diet modestly increased islet content of proinsulin-derived peptides in mice. Human diabetic islets contained increased content of proglucagon-derived peptides at the expense of insulin, but no evident prohormone processing defects. Diabetic plasma, however, contained increased ratios of proinsulin and des-31,32-proinsulin to insulin. Active GLP-1 was detectable in human and mouse islets but 100-1000-fold less abundant than glucagon. LC-MS offers advantages over antibody-based approaches for identifying exact peptide sequences, and revealed a shift toward islet insulin production in high fat fed mice, and toward proglucagon production in type 2 diabetes, with no evidence of systematic defective prohormone processing.

Background: Cotadutide is a GLP1/glucagon receptor dual agonist under development for the treatment of nonalcoholic steatohepatitis and type 2 diabetes (T2D) with diabetic kidney disease. Early-phase clinical studies show that cotadutide achieves clinically significant weight loss. We aimed to establish mechanistically to what extent this effect is mediated via changes in energy intake (EI) or energy expenditure (EE). Methods: We conducted a phase 2a single-center randomized double-blind placebo-controlled trial in overweight/obese adults (BMI 27-40 kg/m2) with T2D (HbA1c 6.5-8%). Participants underwent a 16-day single-blind placebo run-in and were randomized to double-blind 42-day cotadutide or placebo. Cotadutide was given subcutaneously, up to 300 µg daily. Primary outcome: % weight change from day 17 to day 59. Secondary outcomes: % and absolute changes in EI (ad libitum lunch), and EE (indirect calorimetry). Results: A total of 19 (63%) cotadutide and 9 (78%) placebo participants completed the study. Least-squares (LS) mean weight change was -3.98% (90% CI -4.85%, -3.10%) and -1.40% (90% CI -2.66%, -0.13%) for cotadutide and placebo, respectively, p= 0.011 between groups. The cotadutide group had a striking 35% lower EI vs. placebo: LS mean -35.0% (90% CI -61.8%, -8.2%), p=0.037. Total EE (kJ/kg lean mass) after cotadutide treatment decreased vs. placebo: -6.0% vs. 0.3%, p Conclusions: Weight loss caused by cotadutide is predominantly due to reduced EI rather than increased EE. Transient preservation of EE in the early phases of cotadutide treatment may reflect glucagon receptor engagement. Disclosure R. Golubic: None. M. Evans: Advisory Panel; Self; Pila Pharma, Zucara Therapeutics Inc., Other Relationship; Self; Abbott Diabetes, Medtronic, Novo Nordisk, Research Support; Self; AstraZeneca, Imcyse. J. Kennet: None. V. E. Parker: Employee; Self; AstraZeneca, Stock/Shareholder; Self; AstraZeneca. D. Robertson: Employee; Self; AstraZeneca, Employee; Spouse/Partner; GlaxoSmithKline plc., Stock/Shareholder; Self; AstraZeneca. D. Luo: None. L. Hansen: Employee; Self; AstraZeneca. L. Jermutus: Employee; Self; AstraZeneca, Stock/Shareholder; Self; AstraZeneca. P. Ambery: Employee; Self; AstraZeneca. A. Park: Other Relationship; Self; Novo Nordisk.

Background: Cotadutide, a dual glucagon-like peptide (GLP-1) and glucagon receptor agonist, is in development for NASH and T2DM with DKD. GLP-1 receptor agonists have been shown to delay the onset of macroalbuminuria in DKD. The glycemic and renal effects of cotadutide were assessed in a phase 2a, randomized, controlled trial (NCT03550378). Methods: The trial enrolled 41 patients with T2DM (HbA1c 6.5-10.5%) and CKD stage 3 (eGFR 30-59 mL/min/1.73 m2), on insulin and/or oral glucose-lowering therapy and BMI 25-45 kg/m2. Patients were randomized to once-daily subcutaneous cotadutide (n = 21) titrated up to 300 µg or placebo (PBO; n = 20) for 32 days. Endpoints included glucose response during a mixed meal tolerance test (MMTT), time spent within a target glucose range over a 7-day period on continuous glucose monitoring (CGM) and body weight. Exploratory measures included eGFR, UACR, C-peptide and NT-proBNP. Results: After 32 days, cotadutide compared with PBO reduced MMTT glucose AUC (-26.7%; 90% CI -34.6% to -18.8%; 7-day mean glucose -47.9 mg/dL vs. PBO) and increased CGM time spent in target range over 32 days by 36.0% vs. PBO to 78.7% (both p ≤ 0.003). Cotadutide was associated with 35.2% reduction in insulin dose, 0.88 µg/L increase in C-peptide and 3.7% reduction in body weight (all p ≤ 0.012). There was no change in eGFR. In patients with baseline micro- or macroalbuminuria (n = 18), UACR was reduced by 50.6% (p = 0.0504 vs. PBO). In addition, NT-proBNP level was reduced with cotadutide vs. PBO (-79.7 ng/L vs. -9.42 ng/L, p = 0.040). Safety and tolerability findings were consistent with the known mechanism of action of GLP-1 receptor agonists. There were no episodes of severe hypoglycemia, and CGM time < 54 mg/dL was 1.65% with cotadutide vs. 0.82% with PBO. Conclusion: In patients with T2DM and DKD, short term treatment with cotadutide had robust effects on glucose lowering. The reduction in albuminuria suggests that cotadutide has renoprotective potential in DKD, but further evaluation is warranted. Disclosure V. E. Parker: Employee; Self; AstraZeneca, Stock/Shareholder; Self; AstraZeneca. P. Ambery: Employee; Self; AstraZeneca. H. L. Heerspink: Consultant; Self; AbbVie Inc., Astellas Pharma Inc., AstraZeneca, Bayer AG, Boehringer Ingelheim International GmbH, Chinook, CSL Behring, Fresenius Medical Care, Gilead Sciences, Inc., Janssen Research & Development, LLC, Merck & Co., Inc., Mitsubishi Corporation Life Sciences Limited, Mundipharma International, Novo Nordisk, Retrophin, Inc., Research Support; Self; AbbVie Inc., AstraZeneca, Boehringer Ingelheim International GmbH, Janssen Research & Development, LLC. R. J. Mccrimmon: Advisory Panel; Self; Novo Nordisk Inc., Sanofi-Aventis, Board Member; Self; Novo Nordisk Foundation, Research Support; Self; AstraZeneca. L. Jermutus: Employee; Self; AstraZeneca, Stock/Shareholder; Self; AstraZeneca. T. Hoang: None. H. Schlichthaar: Advisory Panel; Self; AstraZeneca, Eli Lilly and Company, Novo Nordisk. F. W. Gibb: Speaker’s Bureau; Self; Abbott Diabetes. B. Wenzel: None. M. G. Posch: None. Y. Chang: None. M. Petrone: None. L. Hansen: Employee; Self; AstraZeneca.

OBJECTIVE Cotadutide, a dual GLP-1 and glucagon receptor agonist, is under development for nonalcoholic steatohepatitis (NASH) and chronic kidney disease with type 2 diabetes. The effects of cotadutide on hepatic and metabolic parameters were evaluated in participants with overweight/obesity and type 2 diabetes. RESEARCH DESIGN AND METHODS In this phase 2b study, 834 adults with BMI ≥25 kg/m2 and type 2 diabetes inadequately controlled with metformin (glycated hemoglobin A1c [HbA1c] of 7.0%–10.5% [53–91 mmol/mol]) were randomized to double-blind cotadutide 100 μg (n = 100), 200 μg (n = 256), or 300 μg (n = 256); placebo (n = 110); or open-label liraglutide 1.8 mg (n = 110)—all administered subcutaneously. Coprimary end points were changes in HbA1c and body weight at week 14. The originally randomized interventions were continued to week 54. Liver damage biomarkers and liver fibrosis algorithms were assessed. RESULTS Cotadutide significantly decreased HbA1c and body weight at weeks 14 and 54 versus placebo (all P < 0.001). Improvements in lipid profile, AST and ALT levels, propeptide of type III collagen level, fibrosis-4 index, and nonalcoholic fatty liver disease fibrosis score were observed with cotadutide 300 μg versus placebo, but not with liraglutide. Weight loss with cotadutide 200 μg was similar to that with liraglutide 1.8 mg and greater with cotadutide 300 μg versus liraglutide 1.8 mg. The most common adverse events with cotadutide (nausea, 35%; vomiting, 17%) decreased over time. CONCLUSIONS Cotadutide treatment for 54 weeks improved glycemic control and weight loss in participants with overweight/obesity and type 2 diabetes. Ad hoc analyses demonstrated improvements in hepatic parameters and support further evaluation of cotadutide in NASH.

Non-alcoholic fatty liver disease and steatohepatitis are highly associated with obesity and type 2 diabetes mellitus. Cotadutide, a glucagon-like protein-1 receptor (GLP-1R) and glucagon receptor (GCGR) agonist, was shown to reduce blood glycaemia, body weight and hepatic steatosis in people with type 2 diabetes mellitus. Here, we demonstrate that the effects of cotadutide in reducing body weight and food intake and improving glucose control are predominantly mediated through Glp-1 signalling, whereas its action on the liver to reduce lipid content, drive glycogen flux and improve mitochondrial turnover and function are directly mediated through Gcg signalling. This was confirmed by the identification of phosphorylation sites on key lipogenic and glucose metabolism enzymes in liver of mice treated with cotadutide. Complementary metabolomic and transcriptomic analyses implicated lipogenic, fibrotic and inflammatory pathways, consistent with a unique therapeutic contribution of GCGR agonism by cotadutide in vivo. Notably, cotadutide also alleviated fibrosis to a greater extent than liraglutide or obeticholic acid, despite dose adjustment to achieve similar weight loss in two preclinical mouse models of NASH. Thus, cotadutide, via direct hepatic (GcgR) and extrahepatic (Glp-1R) effects, exerts multifactorial improvement in liver function and is a promising therapeutic option for the treatment of steatohepatitis.

Background and Aims: Cotadutide, a dual receptor agonist with glucagon-like peptide-1 (GLP-1) and glucagon activity, is being researched for the potential treatment of nonalcoholic steatohepatitis. Given the known effects of glucagon on hepatic glycogen and lipid metabolism, the aim of this phase 2a study was to investigate the effect of cotadutide on hepatic glycogen. Methods: In this double-blind, placebo-controlled study, 21 overweight or obese adults with type 2 diabetes mellitus (HbA1c ≤ 8%) were randomized to receive once-daily subcutaneous cotadutide (n = 12) or placebo (n = 9) for 28 days. Following 48 hours of standardized meals, serial magnetic resonance spectroscopy scans relying on signals from naturally abundant 13C were performed to measure glycogen, and liver fat was also estimated. Fasting amino acid and ketone levels were also assessed. Results: Significant reductions from baseline in postprandial hepatic glycogen content were observed with cotadutide (-100.2 mmol/L; 90% CI: -150.2, -50.1) vs. placebo (+5.5 mmol/L; 90% CI: -47.2, 58.3; P = 0.023). Statistically significant reductions in hepatic glycogen content were observed across serial measures and after a 14-hour fast (P < 0.05), with an overall change in glycogen AUC24h of -27.3% (P = 0.003) vs. placebo. In addition, a relative reduction in liver fat of 33.2% vs. placebo was reported (P = 0.006), and significant reductions in fasting alanine, glutamate, glycine, lysine, and threonine levels were also observed with cotadutide vs. placebo (all P ≤ 0.05). Beta-hydroxybutyrate and acetoacetate levels were not significantly different between the two arms. Conclusions: Cotadutide promoted a generalized reduction in hepatic glycogen and selected amino acid levels. Substantial reductions in liver fat were also observed with cotadutide, suggesting glucagon receptor engagement. Disclosure D. Robertson: Employee; Self; AstraZeneca. Stock/Shareholder; Self; AstraZeneca. L. Hansen: Employee; Self; AstraZeneca. P. Ambery: Employee; Self; AstraZeneca. R.L. Esterline: Employee; Self; AstraZeneca. L. Jermutus: Employee; Self; AstraZeneca. Stock/Shareholder; Self; AstraZeneca. Y. Chang: None. M. Petrone: Employee; Self; AstraZeneca. E. Johansson: None. L. Johansson: Employee; Self; Antaros Medical AB. Stock/Shareholder; Self; Antaros Medical AB. F.B. Sjöberg: None. V. Parker: None. Funding AstraZeneca

Cotadutide (MEDI0382) is a peptide with targeted GLP-1/glucagon (Gcg) receptor activity that has beneficial effects on T2D and obesity in humans and mice, and shows marked improvements in NAFLD/NASH in mice. We performed hyperinsulinemic clamps in diet induced obese (DIO) mice to better understand whether improved insulin sensitivity and β-cell function are responsible for the improved glycemia in our clinical studies. Following 28 days of daily dosing with Cotadutide (10 nmol/kg), Liraglutide (5 nmol/kg; GLP-1 agonist) or g1437 (5 nmol/kg; Gcg analog) mice underwent continuous infusion of 4mU/kg/min insulin and [3H]-glucose to assess glucose turnover. Tissue-specific glucose uptake was assessed using 14C-2-deoxyglucose. We observed lower body weight in all groups compared with vehicle control, with g1437 eliciting the largest response and Liraglutide the least. Fasting glucose was significantly higher in g1437 treated mice. Fasting insulin was dramatically lower in Cotadutide and g1437 groups compared with vehicle. Despite an equal rate of human insulin infusion between groups, the total insulin levels during the clamp remained lower in Cotadutide and g1437 treated mice. The glucose infusion rate, to maintain euglycemia ∼130 mg/dL, was significantly higher in Cotadutide treated mice in line with increased glucose disposal rate. Hepatic glucose production was suppressed in all groups, despite significantly lower levels of insulin in Cotadutide and g1437 treated mice. Finally, glucose uptake was elevated in brown adipose tissue in Cotadutide treated mice, which was confirmed in a separate study of [18F]FDG uptake by PET imaging. These experiments demonstrate that Cotadutide improved insulin sensitivity in concert with dramatic reductions for insulin demand, which may result in recovery of endogenous β-cell function in T2D. Disclosure R.C. Laker: Employee; Self; AstraZeneca. L. Lantier: None. O. McGuinness: None. S. Will: Employee; Self; AstraZeneca. Stock/Shareholder; Self; AstraZeneca. K. Kuszpit: None. A.R. Alfaro: None. L. Jermutus: Employee; Self; AstraZeneca. Stock/Shareholder; Self; AstraZeneca. C.J. Rhodes: Employee; Self; AstraZeneca.

Background and Aims: Previously, cotadutide demonstrated significant glucose- and body weight-lowering effects in doses up to 300 µg. We examined the safety and efficacy of cotadutide in doses up to 600 µg in patients with T2DM. Methods: In this double-blind, phase 2a study, 20 adults with T2DM (HbA1c ≤8.5%) were randomized to receive QD SC cotadutide (n=15) or placebo (n=5) for 77 days. A dose-escalation committee approved titrations >400 µg and ≤600 µg following a safety data review. Patients underwent continuous glucose monitoring. Results: The adverse event profile suggests that the highest clinically tolerated dose has not been identified. Nausea was reported by 3 (20%) patients treated with cotadutide, with one episode of vomiting. There was a significant reduction from baseline in the 7-day average glucose levels (Figure). By day 77, 76.9% of patients in the cotadutide group achieved a >5% body weight loss. A significant reduction in least-squares mean from baseline in ALT level with cotadutide versus placebo was reported (-7.17 U/L, 90% CI: -12.21, -2.14). Conclusions: Cotadutide was well-tolerated at doses up to 600 µg and reduced hyperglycemia, with clinically significant weight loss and beneficial effects on liver enzymes. Disclosure D. Robertson: Employee; Self; AstraZeneca. Stock/Shareholder; Self; AstraZeneca. L. Hansen: Employee; Self; AstraZeneca. M. Jain: Employee; Self; CSL Behring. Stock/Shareholder; Self; AstraZeneca. M. Petrone: Employee; Self; AstraZeneca. T. Wang: Employee; Self; AstraZeneca. T. Heise: Advisory Panel; Self; Mylan, Novo Nordisk A/S. Research Support; Self; ADOCIA, Aerami, Becton, Dickinson and Company, Biocon, Boehringer Ingelheim International GmbH, Eli Lilly and Company, Gan & Lee Pharmaceuticals, MedImmune, Merck KGaA, Mylan, Nordic Bioscience, Novo Nordisk A/S, Poxel SA, Sanofi, Xeris Pharmaceuticals, Inc., Zealand Pharma A/S. Speaker’s Bureau; Self; Eli Lilly and Company, Novo Nordisk A/S. L. Jermutus: Employee; Self; AstraZeneca. Stock/Shareholder; Self; AstraZeneca. Funding AstraZeneca

Aims MEDI0382 is a balanced glucagon-like peptide-1/glucagon receptor dual agonist under development for the treatment of type 2 diabetes mellitus and non-alcoholic steatohepatitis. The primary objective was to assess the safety of MEDI0382 in healthy subjects. Methods In this placebo-controlled, double-blind, Phase 1 study, healthy subjects (aged 18-45 years) were randomized (3:1) to receive a single subcutaneous dose of MEDI0382 or placebo after ≥8 h of fasting. The study consisted of six cohorts that received study drug at 5 μg, 10 μg, 30 μg, 100 μg, 150 μg or 300 μg. The primary objective was safety and tolerability. Secondary endpoints included assessments of pharmacokinetics and immunogenicity. All subjects were followed for up to 28 days. Results A total of 36 subjects received MEDI0382 (n = 6 per cohort) and 12 subjects received placebo (n = 2 per cohort). Treatment-emergent adverse events (TEAEs) occurred more frequently with MEDI0382 vs. placebo, which was mostly due to an increased occurrence at MEDI0382 doses ≥150 μg. All TEAEs were mild or moderate in severity. The most common TEAEs were vomiting, nausea and dizziness. There appeared to be a dose-dependent increase in heart rate with MEDI0382 treatment. MEDI0382 showed linear pharmacokinetic profile (time to maximum plasma concentration: 4.50-9.00 h; elimination half-life: 9.54-12.07 h). No immunogenicity was observed in the study. Conclusions In this single-dose, Phase 1 study in healthy subjects, the safety and pharmacokinetic profiles of MEDI0382 support once-daily dosing and further clinical development of MEDI0382.

Diabetes plays an important role in the complex relationship between chronic kidney disease (CKD) and cardiovascular disease. This retrospective observational study compared the influence of estimated glomerular filtration rate (eGFR) and proteinuria on the risk of major adverse cardiovascular event (MACE; myocardial infarction or stroke) in CKD patients with and without diabetes. Data were from a linked database of UK electronic health records. Individuals with CKD and no prior MACE were classified as type 1 diabetes (T1DM; n = 164), type 2 diabetes (T2DM; n = 9,711), and non-diabetes (non-DM; n = 75,789). Monthly updated time-dependent Cox proportional hazard models were constructed to calculate adjusted hazard ratios (aHRs) for progression to MACE from first record of abnormal eGFR or proteinuria (index date). In non-DM, aHRs (95% CIs) by baseline eGFR category (referent G2) were G1: 0.70 (0.55–0.90), G3a: 1.28 (1.20–1.35), G3b: 1.64 (1.52–1.76), G4: 2.19 (1.98–2.43), and G5: 3.12 (2.44–3.99), and by proteinuria category (referent A1) were A2: 1.13 (1.00–1.28), A2/3 (severity indeterminable): 1.58 (1.28–1.95), and A3: 1.64 (1.38–1.95). In T2DM, aHRs were G1: 0.98 (0.72–1.32), G3a: 1.18 (1.03–1.34), G3b: 1.31 (1.12–1.54), G4: 1.87 (1.53–2.29), G5: 2.87 (1.82–4.52), A2: 1.22 (1.04–1.42), A2/3: 1.45 (1.17–1.79), and A3: 1.82 (1.53–2.16). Low numbers in T1DM precluded analysis. Modelling T2DM and non-DM together, aHRs were, respectively, G1: 3.23 (2.38–4.40) and 0.70 (0.55–0.89); G2: 3.18 (2.73–3.70) and 1.00 (referent); G3a: 3.65 (3.13–4.25) and 1.28 (1.21–1.36); G3b: 4.01 (3.40–4.74) and 1.65 (1.54–1.77); G4: 5.78 (4.70–7.10) and 2.21 (2.00–2.45); G5: 9.00 (5.71–14.18) and 3.14 (2.46–4.00). In conclusion, reduced eGFR and proteinuria were independently associated with increased risk of MACE regardless of diabetes status. However, the risk of MACE in the same eGFR state was 4.6–2.4 times higher in T2DM than in non-DM.

MEDI0382 is an oxyntomodulin-like peptide with targeted GLP-1/glucagon receptor activity that is in clinical development for type 2 diabetes (T2DM), NASH, and obesity and provides glycemic control and weight loss as well as marked improvements of liver outcomes in humans and mice. In mice, reductions in hepatic lipid were independent of weight loss and resulted from improved mitochondrial respiration and turnover and reduced de novo lipogenesis. As hepatocytes lack the GLP-1 receptor, this effect of MEDI0382 is predominantly modulated via glucagon receptors. After treatment with MEDI0382 or g1437, a glucagon analog, isolated hepatocytes showed increased basal and maximal oxygen consumption, increased mitophagosome number, and a ∼6-fold induction in expression of mitochondrial biogenesis regulator PGC-1α (P ≤ 0.0001). We used phosphoproteomics to identify potential mechanisms of action of MEDI0382 and g1437 on lipogenesis inhibition. After g1437 treatment, primary murine hepatocytes exhibited increased phosphorylation on AMPK, GSK3-α, PHK-β, ChREBP, and HSL—all known PKA substrates. We also identified new glucagon-induced phosphorylations on PHK-α2, PYGL, and ChREBP that were not necessarily PKA consensus motifs, implicating involvement of kinases downstream of PKA. In obese mice, MEDI0382 given for 7 days showed time-dependent phosphorylation of the same sites on AMPK, ChREBP, ACC2, ACLY, and DGAT1. These phosphorylation events are predicted to be inhibitory for lipogenesis. To understand translational potential, we treated primary human hepatocytes with g1437 and found 12 homologous phospho sites, including those on AMPK, ACLY, and DGAT1. Thus, MEDI0382 provides unique antisteatotic benefits for obesity/T2DM comorbidities of NASH/NAFLD via enhanced mitochondrial function and novel glucagon-mediated inhibition of lipogenesis. Disclosure R.C. Laker: Employee; Self; MedImmune. M. Boland: Employee; Self; Gubra, MedImmune. A. Nawrocki: None. K.M. Mather: None. L. Jermutus: Employee; Self; AstraZeneca. Stock/Shareholder; Self; AstraZeneca. M.R. Larsen: None. J. Trevaskis: Employee; Self; Gilead Sciences, Inc., MedImmune. Stock/Shareholder; Self; AstraZeneca. C.J. Rhodes: Employee; Self; AstraZeneca, MedImmune. Funding AstraZeneca

Background: MEDI0382 is an oxyntomodulin-like peptide with targeted GLP-1/glucagon receptor activity under development for type 2 diabetes mellitus (T2DM). GLP-1 and glucagon promote delayed gastric emptying, and GLP-1 tolerance has been described. Methods: This exploratory part of a double-blind phase 2a study measured gastric emptying time (GET) in T2DM patients randomized to daily SC MEDI0382 (n = 20) or placebo (n = 6) for 49 days. Doses were up-titrated fortnightly from 50 to 300 µg. GET was measured after 13C-octanoate ingestion via serial breath collection with plasma sampling for glucose, insulin, and MEDI0382 Ctrough levels. GET (t1/2 = time for 13C retention to decline 50%; tlag = time when percent excreted 13C dose peaks) was measured at baseline; 15, 29, 43, 50 days; and 28 days post-dose. Results: GET t1/2 was significantly prolonged from baseline, by 117.2 min (90% CI 56.2, 178.3) v placebo (-42.9 min; 90% CI -152.0, 66.2; P = 0.039), at 200 µg after 43 days of dosing. Also, at 43 days, tlag was significantly prolonged, by 46.5 min (90% CI, 16.9, 76) v placebo (-27.3 min; 90% CI, -80.0, 25.4; P = 0.048). Numerical increases in t1/2 and tlag were seen at all dose levels; tlag increased with exposure, as exposure increased with dose up to 200 µg. Lesser delay occurred in GET on day 50 at 300 µg with similar exposure, but alongside significant reduction in glucose AUC of -25.3% (90% CI, -28.2, -22.4; P < 0.0001). Postprandial insulin levels were unchanged on day 50 despite marked glucose reduction; delay in peak insulin levels was evident. Discussion: MEDI0382 promoted dose-dependent increase in GET. Although tolerance to this effect may be evident from day 50, reduced postprandial glucose was observed concurrently and may indicate an insulinotropic effect. This unique profile displays characteristics of both short- and long-acting GLP-1 agonism, suggesting that these effects may be mediated by both glucagon and GLP-1 receptor agonism. Disclosure D. Robertson: Employee; Self; AstraZeneca. Employee; Spouse/Partner; GlaxoSmithKline plc. Stock/Shareholder; Self; AstraZeneca. V.E. Parker: Employee; Self; MedImmune. Stock/Shareholder; Self; AstraZeneca. P. Ambery: Employee; Self; AstraZeneca. M. Petrone: Employee; Self; MedImmune. T. Wang: Employee; Self; MedImmune. T. Heise: Advisory Panel; Self; Mylan. Research Support; Self; ADOCIA, Boehringer Ingelheim International GmbH, Dance Biopharm Holdings Inc., Eli Lilly and Company, Gan & Lee Pharmaceuticals, Johnson & Johnson, MedImmune, Mylan, Nordic Bioscience, Novo Nordisk A/S, Pfizer Inc., Poxel, Saniona, Sanofi, Wockhardt, Zealand Pharma A/S. Speaker's Bureau; Self; Eli Lilly and Company, Novo Nordisk A/S. L. Plum-Moerschel: None. L. Jermutus: Employee; Self; AstraZeneca. Stock/Shareholder; Self; AstraZeneca. B. Hirshberg: Employee; Self; AstraZeneca. J.J. Meier: Advisory Panel; Self; AstraZeneca, Boehringer Ingelheim International GmbH, Eli Lilly and Company, Merck Sharp & Dohme Corp., Novo Nordisk A/S, Sanofi. Funding AstraZeneca

Background: MEDI0382 is an oxyntomodulin-like peptide with targeted GLP-1/glucagon receptor activity under development for type 2 diabetes mellitus (T2DM). GLP-1 has been shown to promote glucose-dependent insulin release, delay gastric emptying, and suppress appetite. Glucagon has similar effects on appetite and gastric emptying and enhances energy expenditure by upregulating energy-expensive processes such as gluconeogenesis and fatty acid oxidation. Methods: This exploratory component of a phase 2a study measured fasting amino acid, β-hydroxybutyrate, and free fatty acid levels in overweight or obese T2DM patients randomized to once-daily SC MEDI0382 (n = 26) or placebo (n = 13) for 49 days. Doses were up-titrated weekly from 50 to 300 µg; measurements were taken at baseline and after 49 days of dosing. Results: A significant decrease from baseline to day 49 with MEDI0382 vs. placebo in alanine was observed (-0.5 vs. 0.4 mg/dL; P = 0.017). Numerical reductions in other amino acid levels, including glutamate, cystine, valine, lysine, and tyrosine, were also seen. There were no clinically or statistically significant changes from baseline to day 49 between treatment groups in β-hydroxybutyrate or free fatty acid levels. Conclusion: Despite the known effects of glucagon on fatty acid oxidation, MEDI0382 had no effect on ketogenesis or free fatty acids. Although the reason for this is unclear, enhanced insulin secretion may have counter-regulated this effect. MEDI0382 did promote reductions in key glucogenic amino acids, and this reduction could be related to increased gluconeogenesis and therefore glucagon receptor engagement. Similar patterns of amino acid reduction have been observed after bariatric surgery and have been attributed to excess circulating glucagon. We postulate that upregulation of an energy-consuming process such as gluconeogenesis could contribute to significant weight loss in subjects dosed with MEDI0382. Disclosure V.E. Parker: Employee; Self; MedImmune. Stock/Shareholder; Self; AstraZeneca. D. Robertson: Employee; Self; AstraZeneca. Employee; Spouse/Partner; GlaxoSmithKline plc. Stock/Shareholder; Self; AstraZeneca. T. Wang: Employee; Self; MedImmune. D.C. Hornigold: Employee; Self; AstraZeneca. Stock/Shareholder; Self; AstraZeneca. M.G. Posch: None. L. Plum-Moerschel: None. J.J. Meier: Advisory Panel; Self; AstraZeneca, Boehringer Ingelheim International GmbH, Eli Lilly and Company, Merck Sharp & Dohme Corp., Novo Nordisk A/S, Sanofi. H. Schlichthaar: None. B.M. Klaus: None. L. Jermutus: Employee; Self; AstraZeneca. Stock/Shareholder; Self; AstraZeneca. P. Ambery: Employee; Self; AstraZeneca. B. Hirshberg: Employee; Self; AstraZeneca. Funding AstraZeneca

Apelin-36 was discovered as the endogenous ligand for the previously orphan receptor APJ. Apelin-36 has been linked to two major types of biological activities: cardiovascular (stimulation of cardiac contractility and suppression of blood pressure) and metabolic (improving glucose homeostasis and lowering body weight). It has been assumed that both of these activities are modulated through APJ. Here, we demonstrate that the metabolic activity of apelin-36 can be separated from canonical APJ activation. We developed a series of apelin-36 variants in which evolutionarily conserved residues were mutated, and evaluated their ability to modulate glucose homeostasis and body weight in chronic mouse models. We found that apelin-36(L28A) retains full metabolic activity, but is 100-fold impaired in its ability to activate APJ. In contrast to its full metabolic activity, apelin-36(L28A) lost the ability to suppress blood pressure in spontaneously hypertensive rats (SHR). We took advantage of these findings to develop a longer-acting variant of apelin-36 that could modulate glucose homeostasis without impacting blood pressure (or activating APJ). Apelin-36-[L28C(30kDa-PEG)] is 10,000-fold less potent than apelin-36 at activating the APJ receptor but retains its ability to significantly lower blood glucose and improve glucose tolerance in diet-induced obese mice. Apelin-36-[L28C(30kDa-PEG)] provides a starting point for the development of diabetes therapeutics that are devoid of the blood pressure effects associated with canonical APJ activation.

Aims To characterize the pharmacology of MEDI0382, a peptide dual agonist of glucagon-like peptide-1 (GLP-1) and glucagon receptors. Materials and methods MEDI0382 was evaluated in vitro for its ability to stimulate cAMP accumulation in cell lines expressing transfected recombinant or endogenous GLP-1 or glucagon receptors, to potentiate glucose-stimulated insulin secretion (GSIS) in pancreatic β-cell lines and stimulate hepatic glucose output (HGO) by primary hepatocytes. The ability of MEDI0382 to reduce body weight and improve energy balance (i.e. food intake and energy expenditure), as well as control blood glucose, was evaluated in mouse models of obesity and healthy cynomolgus monkeys following single and repeated daily subcutaneous administration for up to 2 months. Results MEDI0382 potently activated rodent, cynomolgus and human GLP-1 and glucagon receptors and exhibited a fivefold bias for activation of GLP-1 receptor versus the glucagon receptor. MEDI0382 produced superior weight loss and comparable glucose lowering to the GLP-1 peptide analogue liraglutide when administered daily at comparable doses in DIO mice. The additional fat mass reduction elicited by MEDI0382 probably results from a glucagon receptor-mediated increase in energy expenditure, whereas food intake suppression results from activation of the GLP-1 receptor. Notably, the significant weight loss elicited by MEDI0382 in DIO mice was recapitulated in cynomolgus monkeys. Conclusions Repeated administration of MEDI0382 elicits profound weight loss in DIO mice and non-human primates, produces robust glucose control and reduces hepatic fat content and fasting insulin and glucose levels. The balance of activities at the GLP-1 and glucagon receptors is considered to be optimal for achieving weight and glucose control in overweight or obese Type 2 diabetic patients.

MEDI0382 is a GLP-1/glucagon receptor dual agonist under development for the treatment of type 2 diabetes mellitus (T2DM). This double-blind study (NCT02548585) evaluated MEDI0382 in T2DM patients with BMI 27-40 kg/m2. Subjects (n = 61) were randomized to once-daily subcutaneous MEDI0382 or placebo (PBO) at different dosing levels and uptitration schedules (100 μg [C1], 150 μg [C2], 200 μg [C3], and 300 μg (2 uptitration schedules, C4 and C5). Marked reduction in fasting and postprandial glucose was achieved with a ∼40% decrease in glucose AUC0-4h after mixed-meal testing across all cohorts (P ≤ 0.0102) (Figure 1A). Weight loss was observed at all dose levels and reached statistical significance (P = 0.024) at 300 μg (C4) (Figure 1B). Ambulatory systolic blood pressure decreased nonsignificantly from baseline in the 300-μg cohort (C4, -4.9 vs. -6.5 mmHg; C5, -9.1 vs. -5.0 mmHg). Similar to other marketed GLP-1 analogs, MEDI0382 increased pulse rate vs. PBO (C4, 8.4 vs. -2.4 BPM, P = 0.0039; C5, 5.8 vs. 0.2 BPM, P = 0.0796). Treatment-related adverse events occurred more often with MEDI0382 vs. PBO (86% [36/42] vs. 47% [9/19]); most events were mild or moderate in severity. In conclusion, MEDI0382 administered for up to 22 days showed a marked reduction in both fasting and postprandial glucose with weight reduction and an acceptable tolerability profile. Disclosure D. Robertson: Employee; Self; MedImmune. Employee; Spouse/Partner; GlaxoSmithKline plc. M.W. Stumvoll: Speaker's Bureau; Self; Novartis Pharmaceuticals Corporation. Advisory Panel; Self; AstraZeneca, Novo Nordisk A/S. Speaker's Bureau; Self; Merck Foundation. M.G. Posch: None. T. Heise: Research Support; Self; ADOCIA, Boehringer Ingelheim GmbH, Dance Biopharm, Eli Lilly and Company, Janssen Research & Development, MedImmune, Merck Sharp & Dohme Corp., Mylan, Nordic Bioscience, Novo Nordisk A/S, Poxel SA, Roche Diagnostics Corporation, Saniona, Sanofi, Senseonics, Zealand Pharma A/S. Advisory Panel; Self; Novo Nordisk A/S, Mylan. Speaker's Bureau; Self; Dexcom, Inc., Eli Lilly and Company, Novo Nordisk A/S, Sanofi. L. Plum-Moerschel: None. G. Klein: None. L. Tsai: Employee; Self; MedImmune. M. Petrone: Employee; Self; MedImmune. Stock/Shareholder; Self; MedImmune. B. Hirshberg: Employee; Self; AstraZeneca, MedImmune. C. Rondinone: Employee; Self; MedImmune. Stock/Shareholder; Self; AstraZeneca, F. Hoffmann-La Roche Ltd., Abbott, AbbVie Inc. V.E. Parker: Employee; Self; MedImmune. Stock/Shareholder; Self; Accenture. Employee; Self; AstraZeneca. L. Jermutus: Employee; Self; AstraZeneca. Stock/Shareholder; Self; AstraZeneca. P. Ambery: Employee; Self; MedImmune. Stock/Shareholder; Self; AstraZeneca.

MEDI0382 is under development for the treatment of type 2 diabetes mellitus (T2DM). In a double-blind study (NCT02548585), 51 T2DM patients with BMI 27-40 kg/m2 were randomized (1:1) to daily SC MEDI0382 200 μg or placebo (PBO). Based on ANCOVA with treatment and baseline (BL) values as covariates, MEDI0382 markedly reduced fasting glucose (change from BL at day 41, -49.9 vs. -19.2 mg/dL; P < 0.0001) and postprandial glucose in a mixed-meal tolerance test (percent change from BL in glucose AUC0-4h, -32.8 vs. -10.2; P < 0.0001) (Figure 1) with no increase in hypoglycemia. HbA1c levels decreased -0.9% with MEDI0382 and -0.6% with PBO (P = 0.0004). Weight loss was 3.8 kg vs. BL (1.7 kg vs. PBO; P = 0.0008); 92% of MEDI0382 patients lost >2 kg. Mean reduction in ambulatory systolic BP was -4.2 for MEDI0382 vs. -1.5 mmHg for PBO (P = ns). As with marketed GLP-1 analogs, a mean pulse increase of 6.8 BPM for MEDI0382 vs. a fall of 2.0 BPM for PBO was seen (P < 0.0001). Treatment-related AEs such as decreased appetite, vomiting, and headache were seen in more patients on MEDI0382 (20 vs. 15); 0 were grade >3 severity, 3 led to study discontinuation for MEDI0382 vs. 1 for PBO, and 1 SAE in the PBO arm. Overall, MEDI0382 normalized fasting and postprandial blood glucose, significantly reduced body weight, and had an acceptable safety profile over 41 days of dosing in obese/overweight T2DM patients. Disclosure P. Ambery: Employee; Self; MedImmune. Stock/Shareholder; Self; AstraZeneca. M.W. Stumvoll: Speaker's Bureau; Self; Novartis Pharmaceuticals Corporation. Advisory Panel; Self; AstraZeneca, Novo Nordisk A/S. Speaker's Bureau; Self; Merck Foundation. M.G. Posch: None. T. Heise: Research Support; Self; ADOCIA, Boehringer Ingelheim GmbH, Dance Biopharm, Eli Lilly and Company, Janssen Research & Development, MedImmune, Merck Sharp & Dohme Corp., Mylan, Nordic Bioscience, Novo Nordisk A/S, Poxel SA, Roche Diagnostics Corporation, Saniona, Sanofi, Senseonics, Zealand Pharma A/S. Advisory Panel; Self; Novo Nordisk A/S, Mylan. Speaker's Bureau; Self; Dexcom, Inc., Eli Lilly and Company, Novo Nordisk A/S, Sanofi. L. Plum-Moerschel: None. L. Tsai: Employee; Self; MedImmune. D. Robertson: Employee; Self; MedImmune. Employee; Spouse/Partner; GlaxoSmithKline plc. M. Petrone: Employee; Self; MedImmune. Stock/Shareholder; Self; MedImmune. C. Rondinone: Employee; Self; MedImmune. Stock/Shareholder; Self; AstraZeneca, F. Hoffmann-La Roche Ltd., Abbott, AbbVie Inc. V.E. Parker: Employee; Self; MedImmune. Stock/Shareholder; Self; Accenture. Employee; Self; AstraZeneca. B. Hirshberg: Employee; Self; AstraZeneca, MedImmune. L. Jermutus: Employee; Self; AstraZeneca. Stock/Shareholder; Self; AstraZeneca.

Nonalcoholic steatohepatitis (NASH), characterized by hepatic steatosis, inflammation and fibrosis is an unmet medical need. MEDI0382, a balanced GLP-1/glucagon dual receptor agonist, is under development for the treatment of T2DM. Here we examined the effects of MEDI0382 on NASH compared to liraglutide, a GLP-1 analog. Leptin-deficient ob/ob mice were maintained on high trans-fat, fructose and cholesterol diet for 8 weeks to induce NASH then randomized to four treatment groups: vehicle, MEDI0382 (30 nmol/kg), liraglutide (30 nmol/kg) or vehicle-treated and switched to low-fat diet (LFD). Treatment with MEDI0382 and liraglutide reduced body weight and improved glucose tolerance. Hepatic lipid was reduced by 40% with MEDI0382 treatment (p In conclusion, MEDI0382 exerted similar metabolic control relative to liraglutide, but exhibited superior effects on primary NASH endpoints. Disclosure M. Beaton: Employee; Self; MedImmune. Employee; Spouse/Partner; MedImmune. S. Guionaud: None. J.P. Conway: None. J. Grimsby: Employee; Self; AstraZeneca. C.J. Rhodes: Stock/Shareholder; Self; AstraZeneca. L. Jermutus: Employee; Self; AstraZeneca. Stock/Shareholder; Self; AstraZeneca. J. Trevaskis: Employee; Self; MedImmune. Stock/Shareholder; Self; AstraZeneca.

MEDI0382, a GLP-1/glucagon dual receptor agonist, is being developed for the treatment of T2DM. GLP-1 receptor agonists promote glucose-dependent insulin release, suppress glucagon, and increase or have minimal effect on glucose-dependent insulinotropic polypeptide (GIP); knowledge of the effect on GLP-1 is limited. We characterized fasting and postprandial hormone profiles associated with MEDI0382 therapy. In a phase 2a study (NCT02548585), 51 subjects with T2DM and BMI 27-40 kg/m2 received MEDI0382 (uptitrated to 200 µg) or placebo (PBO) daily for 41 days. Glucose, insulin, glucagon, GIP, and GLP-1 were measured at baseline (day -1) and day 41 during a liquid mixed-meal tolerance test. MEDI0382 significantly reduced fasting glucose (-49.9 vs. -19.2 mg/dL for PBO; P < 0.0001) and glucose AUC0-4h (-32.8 vs. -10.2% for PBO; P < 0.0001) in association with increased fasting insulin (2.2 mU/L vs. -3.9 mU/L for PBO; P = 0.016), but postprandial insulin AUC was unchanged. In contrast, fasting and postprandial endogenous glucagon, GIP (Figure), and active and inactive GLP-1 were suppressed, and a delay in their kinetics was observed after MEDI0382 therapy. The results suggest that MEDI0382 is insulinotropic and might also cause delayed gastric emptying. The effects of MEDI0382 on GIP have not been observed with GLP-1 receptor mono-agonists and may represent a footprint of dual GLP-1/glucagon receptor agonism in this complex hormone signaling network. Disclosure V.E.R. Parker: Employee; Self; MedImmune. Stock/Shareholder; Self; Accenture. Employee; Self; AstraZeneca. L. Tsai: Employee; Self; MedImmune. D. Robertson: Employee; Self; MedImmune. Employee; Spouse/Partner; GlaxoSmithKline plc. M. Petrone: Employee; Self; MedImmune. Stock/Shareholder; Self; MedImmune. C. Rondinone: Employee; Self; MedImmune. Stock/Shareholder; Self; AstraZeneca, F. Hoffmann-La Roche Ltd., Abbott, AbbVie Inc. B. Hirshberg: Employee; Self; AstraZeneca, MedImmune. L. Jermutus: Employee; Self; AstraZeneca. Stock/Shareholder; Self; AstraZeneca. P. Ambery: Employee; Self; MedImmune. Stock/Shareholder; Self; AstraZeneca.

Summary Ghrelin, an appetite-stimulatory hormone secreted by the stomach, was discovered as a ligand for the growth hormone secretagogue receptor (GHSR). Through GHSR, ghrelin stimulates growth hormone (GH) secretion, a function that evolved to protect against starvation-induced hypoglycemia. Though the biology mediated by ghrelin has been described in great detail, regulation of ghrelin action is poorly understood. Here, we report the discovery of liver-expressed antimicrobial peptide 2 (LEAP2) as an endogenous antagonist of GHSR. LEAP2 is produced in the liver and small intestine, and its secretion is suppressed by fasting. LEAP2 fully inhibits GHSR activation by ghrelin and blocks the major effects of ghrelin in vivo , including food intake, GH release, and maintenance of viable glucose levels during chronic caloric restriction. In contrast, neutralizing antibodies that block endogenous LEAP2 function enhance ghrelin action in vivo . Our findings reveal a mechanism for fine-tuning ghrelin action in response to changing environmental conditions.

Highlights • Apelin-36-[L28A] and apelin-36-[L28C(30kDa-PEG)] were initially reported to induce beneficial metabolic effects independent of the apelin receptor in vivo. • We have shown that these peptides activate the apelin receptor, competing for binding with nanomolar affinities in rat and human cardiac tissues. • Apelin-36-[L28A] and apelin-36-[L28C(30kDa-PEG)] were more potent at inhibiting cAMP release compared to β-arrestin activation. • Our study suggests that apelin-36-[L28A] and apelin-36-[L28C(30kDa-PEG)] are G protein biased ligands of the apelin receptor., Background Apelin signalling pathways have important cardiovascular and metabolic functions. Recently, apelin-36-[L28A] and apelin-36-[L28C(30kDa-PEG)], were reported to function independent of the apelin receptor in vivo to produce beneficial metabolic effects without modulating blood pressure. We aimed to show that these peptides bound to the apelin receptor and to further characterise their pharmacology in vitro at the human apelin receptor. Methods [Pyr1]apelin-13 saturation binding experiments and competition binding experiments were performed in rat and human heart homogenates using [125I]apelin-13 (0.1 nM), and/or increasing concentrations of apelin-36, apelin-36-[L28A] and apelin-36-[L28C(30kDa-PEG)] (50pM-100μM). Apelin-36 and its analogues apelin-36-[F36A], apelin-36-[L28A], apelin-36-[L28C(30kDa-PEG)], apelin-36-[A28 A13] and [40kDa-PEG]-apelin-36 were tested in forskolin-induced cAMP inhibition and β–arrestin assays in CHO-K1 cells heterologously expressing the human apelin receptor. Bias signaling was quantified using the operational model for bias. Results In both species, [Pyr1]apelin-13 had comparable subnanomolar affinity and the apelin receptor density was similar. Apelin-36, apelin-36-[L28A] and apelin-36-[L28C(30kDa-PEG)] competed for binding of [125I]apelin-13 with nanomolar affinities. Apelin-36-[L28A] and apelin-36-[L28C(30kDa-PEG)] inhibited forskolin-induced cAMP release, with nanomolar potencies but they were less potent compared to apelin-36 at recruiting β-arrestin. Bias analysis suggested that these peptides were G protein biased. Additionally, [40kDa-PEG]-apelin-36 and apelin-36-[F36A] retained nanomolar potencies in both cAMP and β-arrestin assays whilst apelin-36-[A13 A28] exhibited a similar profile to apelin-36-[L28C(30kDa-PEG)] in the β–arrestin assay but was more potent in the cAMP assay. Conclusions Apelin-36-[L28A] and apelin-36-[L28C(30kDa-PEG)] are G protein biased ligands of the apelin receptor, suggesting that the apelin receptor is an important therapeutic target in metabolic diseases.

Lowering levels of peripheral amyloid-β has been proposed as a strategy to reduce plaques in patients with Alzheimer’s disease. Henderson et al. test a modified version of the amyloid-degrading enzyme neprilysin in rats, monkeys and Tg2576 mice. Levels of amyloid-β were reduced in the bloodstream, but not in the CNS., Alzheimer’s disease is characterized by the accumulation of amyloid deposits in the brain and the progressive loss of cognitive functions. Although the precise role of amyloid-β in disease progression remains somewhat controversial, many efforts to halt or reverse disease progression have focussed on reducing its synthesis or enhancing its removal. It is believed that brain and peripheral soluble amyloid-β are in equilibrium and it has previously been hypothesized that a reduction in peripheral amyloid-β can lower brain amyloid-β, thereby reducing formation of plaques predominantly composed of insoluble amyloid-β; the so-called peripheral sink hypothesis. Here we describe the use of an amyloid-β degrading enzyme, the endogenous metallopeptidase neprilysin, which is fused to albumin to extend plasma half-life and has been engineered to confer increased amyloid-β degradation activity. We used this molecule to investigate the effect of degradation of peripheral amyloid-β on amyloid-β levels in the brain and cerebrospinal fluid after repeated intravenous dosing for up to 4 months in Tg2576 transgenic mice, and 1 month in rats and monkeys. This molecule proved highly effective at degradation of amyloid-β in the periphery but did not alter brain or cerebrospinal fluid amyloid-β levels, suggesting that the peripheral sink hypothesis is not valid and is the first time that this has been demonstrated in non-human primates.

Neurturin (NRTN), a member of the glial-derived neurotrophic factor family, was identified from an embryonic chicken pancreatic cDNA library in a screen for secreted factors. In this study, we assessed the potential antidiabetic activities of NRTN relative to liraglutide, a glucagon-like peptide 1 receptor agonist, in Zucker diabetic fatty (ZDF) rats. Subcutaneous administration of NRTN to 8-week-old male ZDF rats prevented the development of hyperglycemia and improved metabolic parameters similar to liraglutide. NRTN treatment increased pancreatic insulin content and β-cell mass and prevented deterioration of islet organization. However, unlike liraglutide-treated rats, NRTN-mediated improvements were not associated with reduced body weight or food intake. Acute NRTN treatment did not activate c-Fos expression in key feeding behavior and metabolic centers in ZDF rat brain or directly enhance glucose-stimulated insulin secretion from pancreatic β-cells. Treating 10-week-old ZDF rats with sustained hyperglycemia with liraglutide resulted in some alleviation of hyperglycemia, whereas NRTN was not as effective despite improving plasma lipids and fasting glucose levels. Interestingly, coadministration of NRTN and liraglutide normalized hyperglycemia and other metabolic parameters, demonstrating that combining therapies with distinct mechanism(s) can alleviate advanced diabetes. This emphasizes that therapeutic combinations can be more effective to manage diabetes in individuals with uncontrolled hyperglycemia.

Background: Immunosuppressive agents are being investigated for the treatment of\ud chronic kidney disease (CKD) but may increase risk of infection. This was a retrospective\ud observational study intended to evaluate the risk of hospitalized infection in\ud patients with CKD, by estimated glomerular filtration rate (eGFR) and proteinuria\ud status, aiming to identify the most appropriate disease stage for immunosuppressive\ud intervention.\ud Methods: Routine UK primary-care\ud and linked secondary-care\ud data were extracted\ud from the Clinical Practice Research Datalink. Patients with a record of CKD were\ud identified and grouped into type 2, type 1 and nondiabetes cohorts. Time-dependent,\ud Cox proportional hazard models were used to determine the likelihood of hospitalized\ud infection.\ud Results: We identified 97 839 patients with a record of CKD, of these 11 719 (12%)\ud had type 2 diabetes. In these latter patients, the adjusted hazard ratios (aHR) were\ud 1.00 (95% CI: 0.80-1.25),\ud 1.00, 1.03 (95% CI: 0.92-1.15),\ud 1.36 (95% CI: 0.20-1.54),\ud 1.82 (95% CI: 1.54-2.15)\ud and 2.41 (95% CI: 1.60-3.63)\ud at eGFR stages G1, G2 (reference),\ud G3a, G3b, G4 and G5, respectively; and 1.00, 1.45 (95% CI: 1.29-1.63)\ud and 1.91\ud (95% CI: 1.67-2.20)\ud at proteinuria stages A1 (reference), A2 and A3, respectively. All\ud aHRs (except G1 and G3a) were significant, with similar patterns observed within the\ud non-DM\ud and overall cohorts.\ud Conclusions: eGFR and degree of albuminuria were independent markers of hospitalized\ud infection in both patients with and without diabetes. The same patterns of\ud hazard ratios of eGFR and proteinuria were seen in CKD patients regardless of diabetes\ud status, with the risk of each outcome increasing with a decreasing eGFR and increasing\ud proteinuria. Infection risk increased significantly from eGFR stage G3b and\ud proteinuria stage A2 in type 2 diabetes. Treating type 2 DM patients with CKD at\ud eGFR stages G1-G3a\ud with immunosuppressive therapy may therefore provide a favourable\ud risk-benefit\ud ratio (G1-G3a\ud in type 2 diabetes; G1-G2\ud in nondiabetes and\ud overall cohorts) although the degree of proteinuria needs to be considered.

Bi-specific antibodies are suggested to possess unique advantages when compared with mono-specific antibodies as anti-cancer therapeutics. These advantages include greater efficacy, superior combination with standard of care agents, and evasion of resistance mechanisms. Data are presented supporting the hypothesis that the increased avidity of a tetravalent (non BiTE-like) bi-specific antibody mediates such advantages when compared with conventional bi-valent mono-specific antibodies. Experiments include functional comparisons of antibodies binding the Epidermal Growth Factor (EGFR) and the Insulin-like Growth Factor 1 Receptor (IGF1R) at the biochemical, cellular and tumor xenograft levels. These data support the ongoing use of multi-specific antibodies in oncology research and describe a mechanism that may contribute to superior activity compared to mono-specific antibodies and drug combinations. It is noted that advantages of bi-specific antibody formats remain to be seen clinically in terms of both efficacy and safety. Citation Format: Kris F. Sachsenmeier, Nazzareno Dimasi, Qihui Huang, Erin Sult, Binyam Bezabeh, Ryan Fleming, Carl Hay, Helen Zhong, Shannon Breen, Changshou Gao, Balaji Agoram, Lutz Jermutus, Robert Hollingsworth. The avidity hypothesis: comparing bispecific and monospecific antibodies in preclinical oncology models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4635. doi:10.1158/1538-7445.AM2013-4635