BACKGROUND:CKM syndrome involves obesity, type 2 diabetes (T2D), chronic kidney disease (CKD) and cardiovascular disease (CVD). However, most preclinical models fail to reproduce the progressive renal and cardiac dysfunction characteristic of advanced CKM syndrome, limiting their ability to accurately reflect human disease. METHODS:Male uninephrectomized (UNx) KK-Ay mice received a high-fat diet (HFD) with or without the vasoconstrictor L-NNA for 13-16 weeks. RESULTS:UNx + HFD + L-NNA resulted in obesity, hyperglycemia and progressive kidney failure, indicated by a rapid increase in albuminuria and transient hyperfiltration followed by progressive glomerular filtration rate (GFR) decline over three months. Histopathological analysis revealed severe glomerular damage, fibrosis, inflammation and basement membrane thickening, most pronounced in UNx + HFD + L-NNA mice. Renal transcriptomics analysis revealed robust activation of inflammatory and fibrotic pathways, again most pronounced in UNx + HFD + L-NNA mice. In the heart, UNx + HFD + L-NNA resulted in increased ejection fraction and fractional shortening, reduced end-systolic volume and increased left ventricular posterior wall thickness. Alongside pronounced right ventricular fibrosis, this phenotype points toward a phenotype of heart failure with preserved ejection fraction (HFpEF). CONCLUSIONS:The UNx + HFD + L-NNA KK-Ay model reproduces key metabolic, renal and cardiac components of CKM syndrome. While obesity and hyperglycemia contribute substantially to disease burden, L-NNA-induced hypertension further exacerbates both renal decline and cardiac remodeling. Therefore, this model enables mechanistic investigation and evaluation of therapeutic strategies for CKM syndrome.

Abstract Background and Aims We developed a diet-induced hypertension-accelerated mouse model of diabetic kidney disease characterized by progressive loss of GFR resulting in chronic kidney disease. Since cardiovascular disease is the major cause of death in CKD, we characterized the functional and structural cardiac damage in this model. Additionally, we studied the efficacy of combination therapy with an ACE-inhibitor (Lisinopril) and SGLT2-inhibitor (Dapagliflozin) on cardiac histopathology. Method Male KKAy mice underwent uninephrectomy. After recovery mice received high fat diet (45% LARD) and drinking water with or without 50 mg/L LNNA (wk0). At 12 weeks, imaging was performed to study cardiac function and mice were terminated at week 13. In the intervention study, at week 4, lisinopril (2.5 mg/kg/day; drinking water) and at week 8 dapagliflozin (5 and 20 mg/kg/day; foodadmix) treatment were started. At week 16 mice were terminated and lung and heart weight and cardiac histology were determined. Results Upon termination, macroscopic evaluation of the hearts showed extensive scar tissue formation on the outside of the left ventricle. Histological evaluation showed the presence left ventricular hypertrophy, coronary calcification and myocardial fibrosis in male KKAy mice with UNX, HFD and LNNA. KKAy mice with UNX and HFD but without LNNA also showed myocardial fibrosis, monocyte infiltration and focal mineralization. Imaging showed preserved ejection fraction, a significant reduction, increased left ventricle posterior wall thickness and decrease left ventricle inner diameter. Treatment with combination therapy reduced lung wet weight, significantly reduced heart weight and significantly decreased cardiac fibrosis. Macroscopically, less scar tissue was observed after treatment. Conclusion This multifactorial mouse model shows cardiac damage on a background of hypertension, diabetes, renal dysfunction and obesity. Functional measurement including preserved ejection fraction, left ventricle hypertrophy, diastolic dysfunction and increased fibrosis resembling the clinical HFpEF phenotype. Combination therapy with Lisinopril and Dapagliflozin reduced cardiac weight and cardiac fibrosis. This indicates cardiac involvement in the DKD mouse model which confirms that this multifactorial model is clinically relevant.

Abstract Background and Aims Male KKAy mice develop diabetic kidney disease (DKD) with progressive decline of GFR upon high fat diet feeding which is accelerated by the vasoconstrictor LNNA. In this study, efficacy of combination therapy with clinical relevant therapeutics in the form of an ACE-inhibitor (Lisinopril) and SGLT2-inhibitor (Dapagliflozin) on renal function and histopathology was investigated. Method Male KKAy mice underwent uninephrectomy (UNX). After recovery mice received high fat diet (45% LARD) and 50 mg/L LNNA in drinking water (wk0). At week 4, Lisinopril (2.5 mg/kg/day in drinking water) and at week 8 Dapagliflozin (5 and 20 mg/kg/day in diet) were started. Body weight, blood glucose, food and water intake and albuminuria were determined regularly. GFR was measured transdermally by FITC-sinistrin clearance. Mice were terminated at week 16 and renal histology was scored by a team of renal histopathologists. Non-induced and induced non-treated mice were used as controls. Results Treatment of KKAy mice on HFD+LNNA with Dapagliflozin reduced blood glucose immediately. Combination therapy with Lisinopril and Dapagliflozin (5 mg/kg/day) rescued the progressive GFR decline to levels seen in non-induced chow-controls (p < 0.05 vs UNX+HFD+LNNA). Pathology showed that the percentage of healthy glomeruli increased from 14% to 26% after combination therapy (Dapa 5). Interstitial fibrosis and tubular atrophy were significantly reduced by combination therapy (p < 0.05 vs UNX+HFD+LNNA). Conclusion Male KKAy mice on a HFD and LNNA developed DKD resulting in CKD. Combination therapy with Lisinopril and Dapagliflozin rescued GFR decline and reduced glomerular damage and interstitial fibrosis and tubular atrophy This indicates the clinical relevance of the model which can be used to study compound efficacy in both early and more advanced stages of DKD.

GDF15 is a circulating polypeptide associated with cellular stress, and recently linked to metabolic adaptation. GDF15 has a half-life of approximately 3 hours in and acts at the GFRAL receptor selectively expressed in the area postrema. To characterize the effects of sustained GFRAL agonism on food intake (FI) and body weight (BW), we developed a half-life extended analog of GDF15 (Compound H; CpdH) suitable for reduced dosing frequency and tested its effects in obese cynomolgus monkeys. Animals were treated once weekly for 12 weeks with 0.048, 0.16, or 1.6 mg/Kg of CpdH or with 0.02 mg/Kg of the long-acting GLP-1 analog dulaglutide as a positive control. FI was measured daily and BW was measured biweekly. Mechanism-based longitudinal exposure-response (E-R) modeling was performed to characterize the effects of CpdH and dulaglutide on FI and BW. The integrated novel model accounts for both acute, exposure-dependent effects of treatments to reduce FI and the compensatory changes in energy expenditure (EE) and FI that occur over time in response to weight loss. CpdH had approximately linear, dose-proportional pharmacokinetics with a half-life of ≈8 days and treatment with CpdH led to dose- and exposure-dependent reductions in FI and BW. The 1.6 mg/Kg CpdH dose reduced mean FI by 57.5% at 1 week and provided sustained FI reductions of 31.5% from weeks 9-12, leading to a peak reduction in BW of 16±5%. Dulaglutide had more modest effects on FI (reductions between 15-40%) and peak BW loss was 3.8±4.0%. Longitudinal modeling of both the FI and BW profiles suggested reductions in BW observed with both CpdH and dulaglutide were fully explained by the exposure-dependent reductions in FI without any increase in EE. Upon verification of pharmacokinetic/pharmacodynamic relationship established in monkeys and human for dulaglutide, we predicted that CpdH could reach double digit BW loss in human. In summary, treatment with a long-acting GDF15 analog led to sustained dose- and exposure-dependent reductions in food intake in a monkey model of obesity and holds potential for effective clinical obesity pharmacotherapy. Significance Statement GDF15 activation of GFRAL receptors in the hindbrain controls food intake and body weight. Here we describe the effect and durability of a circulating half-life extended analog of GDF15 (Compound H) on food intake and body weight loss in a spontaneously obese cynomolgus monkey model. Inclusion of a translational treatment arm with a weight loss agent, dulaglutide, permitted pharmacokinetic/pharmacodynamic modeling and comparison of both GDF15 and GLP-1 based weight loss mechanisms, and development of an allometric scaling based mathematical model to estimate the efficacy of Compound H in human obese subjects.

Pramlintide is an equipotent amylin analogue that reduces food intake and body weight in obese subjects and has been clinically approved as an adjunctive therapy for the treatment of adult diabetic patients. However, due to its extremely short half-life in vivo, a regimen of multiple daily administrations is required for achieving clinical effectiveness. Herein is described the development of prototypical long acting pramlintide bioconjugates, in which pramlintide's disulfide-linked macrocycle was replaced by a cyclic thioether motif. This modification enabled stable chemical conjugation to a half-life extending antibody. In contrast to pramlintide (t(1/2) < 0.75 h), bioconjugates 35 and 38 have terminal half-lives of similar to 2 days in mice and attain significant exposure levels that are maintained up to 7 days. Single dose subcutaneous administration of 35 in lean mice, given 18-20 h prior to oral acetaminophen (AAP) administration, significantly reduced gastric emptying (as determined by plasma AAP levels). In a separate study, similar administration of 35 in fasted lean mice effected a reduction in food intake for up to 48 h. These data are consistent with durable amylinomimetic responses and provide the basis for further development of such long-acting amylinomimetic conjugates for the potential treatment of obesity and associated pathologies. (c) 2022 Elsevier Masson SAS. All rights reserved.

Nonalcoholic fatty liver disease (NAFLD) and its progression to nonalcoholic steatohepatitis (NASH) is highly prevalent in modern societies frequently consuming diets rich in fats and carbohydrates. NASH is characterized by liver steatosis, inflammation and increased risk for liver fibrosis in human. There is no single animal model available that encompasses the defining features of NASH; therefore, it is of importance to thoroughly characterize available rodent models for NASH research and new drug discovery. Foz/foz mice have a mutation in the Alms1 gene, a ubiquitous protein essential for proper primary cilium function. They have similar defects such as metabolic syndrome, NASH and liver fibrosis to human with the same gene mutation. To better characterize the model, foz/foz mice were fed with western diet (WD, D12079B) for various duration. Compared with WT mice, foz/foz mice on WD had (1) increased BW gain driven by hyperphagia; (2) insulin/leptin resistance with decreased adiponectin levels and hyperlipidemia; (3) increased fat mass and hepatomegaly; (4) increased liver enzymes and elevated TIMP1 in plasma; (5) Liver gene expressions profiles of foz/foz mice showed up-regulation of fibrogenic genes and Lipogenesis, inflammation genes. Histopathology assessment showed foz/foz mice developed severe hepatic steatosis and increased marker of macrophage populations (F4/80 IHC staining). liver collagen content was significantly increased at 16 and 24 wks post WD feeding (PSR staining) compared to wild type mice fed the same diet. Our results suggest that the foz/foz mouse model has robust hallmark features of NASH such as metabolic syndrome, steatohepatitis, and fibrosis. In addition, our data suggest the presence of metabolic complications in other organs as well, as foz/foz mice develop proteinuria over time. The WD-induced foz/foz model exhibits several clinically relevant features and serves as a translational model of NASH. Disclosure J. Liu: None. S. Wang: None. K.E. D’Aquino: Employee; Self; Janssen Pharmaceuticals, Inc. Employee; Spouse/Partner; Janssen Pharmaceuticals, Inc. N.H. Wallace: None. M.M. Rankin: Employee; Self; Janssen Pharmaceuticals, Inc. Stock/Shareholder; Self; Johnson & Johnson. F. Du: None. S.A. Hinke: Employee; Self; Janssen Pharmaceuticals, Inc. Stock/Shareholder; Self; Johnson & Johnson. K.A. Albarazanji: None. A.R. Nawrocki: Employee; Self; Janssen Pharmaceuticals, Inc.

Obesity is associated with increased risk of diabetes and cardiovascular disease. Evidence is accumulating that significant WL (>10%) may reduce this risk. GDF15 is a secreted circulating polypeptide associated with energy balance. The receptor for GDF15, Gfral, is expressed in the area postrema and mediates the inhibitory effects of GDF15 on food intake. Compound H (CpdH) is a GDF15 agonist on a half-life extension platform, suitable for once-weekly dosing. We sought to test the durability of the food intake suppression and body weight loss with once weekly dosing of CpdH in the spontaneously obese cynomolgus non-human primate (NHP) model. As a translational control we included dulaglutide (dula), a long-acting GLP-1 agonist, to enable efficacy modeling and validation of the animal model. PK/PD simulations derived from single-dose lean and obese NHP studies were used for dose selection; CpdH or dula was administered SC once weekly in a biologic naïve spontaneously obese cynomolgus (population BMI 46.0±1.0Kg/m2 with 15.1±1.5% total body fat (n=32) at baseline). Chronic treatment of animals with CpdH at 1 and 10nmol/kg once weekly reduced food intake in a dose related manner relative to vehicle. A commensurate, sustained reduction in body weight (up to 16.2±4.9%) was observed at steady state exposures. The injection of dulaglutide control caused rapid, transient reduction in food intake and a 3.8% reduction in vehicle corrected body weight over 12 weeks. A dose-response relationship for CpdH was formally demonstrated. In conclusion, CpdH is a novel agent for the treatment of human obesity and type 2 diabetes. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. Abstracts presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

GDF-15 is a secreted circulating polypeptide that regulates systemic energy balance. GDF-15 agonists may have therapeutic potential as anorectic agents in obesity and type 2 diabetes. The receptor for GDF-15, Gfral, is expressed on specific neurons in the area postrema (AP) of the hindbrain, and is necessary for the effect of GDF-15 on food intake. Given the role of the AP in vagal control of gastric motility, we sought to investigate the potential effects of GDF-15 on gastric emptying. Food intake reduction by GDF-15 was confirmed in C57Bl/6N mice using BioDAQ continuous food consumption monitoring. Animals were treated sc with recombinant His-tagged human GDF-15 prior to initiation of the dark cycle; GDF-15 treated mice showed significant reduction in food intake relative to vehicle treated controls (12 hour cumulative food intake: 3nmol/kg, -19.9±10.5%; 10nmol/kg, -58.0±10.0%; P<0.0001, n=8). Gastric emptying was assessed using an oral acetaminophen (AAP) absorption test over 90 minute using LC/MS detection. This method was validated using known inhibitor of gastric emptying, Exendin-4 as a positive control; 7.2nmol/kg Exendin-4 significantly reduced integrated AAP absorption by -44.7±5.1% (P<0.01, n=8). In this assay, GDF-15 caused a significant dose-dependent inhibition of gastric emptying, reducing acetaminophen AUC levels by -18.1±10.6% at 1nmol/kg, and by -36.0±9.9% at 10nmol/kg, relative to vehicle treated control mice (P<0.01, n=8). Comparable results were obtained in an independent repetition of the study. We extended the results obtained in mice to SD rats, where we similarly observed a significant reduction in gastric emptying following GDF-15 treatment. Hence, GDF-15 appears to reduce gastric emptying rate in both mouse and rat, potentially contributing to the food intake suppression mechanism of action. Disclosure S.A. Hinke: Employee; Self; Janssen Research & Development. C.R. Cavanaugh: Employee; Self; Janssen Research & Development. T. Kirchner: None. W. Lang: None. R. Meng: None. N.H. Wallace: None. J. Liu: None. K.E. D'Aquino: Employee; Self; Janssen Research & Development. Employee; Spouse/Partner; Janssen Research & Development. G. Ho: None. M.M. Rankin: Employee; Self; Janssen Research & Development. S. Wang: None. J.A. Chavez: None. S.M. Nelson: None. J. Furman: Employee; Self; Janssen Research & Development. S. Mullican: Employee; Self; Janssen Research & Development. S.M. Rangwala: Employee; Self; Janssen Research & Development. A.R. Nawrocki: Employee; Self; Janssen Pharmaceuticals, Inc..

A new series of (2S, 3R, 4R, 5S, 6R)-5-fluoro-6-(hydroxymethyl)-2-aryltetrahydro-2H-pyran-3,4-diols as dual inhibitors of sodium glucose co-transporter proteins (SGLTs) were disclosed. Two methods were developed to efficiently synthesize C-5-fluoro-lactones 3 and 4, which are key intermediates to the C-5-fluoro-hexose based C-aryl glucosides. Compound 2b demonstrated potent hSGLT1 and hSGLT2 inhibition (IC50 = 43 nM for SGLT1 and IC50 = 9 nM for SGLT2). It showed robust inhibition of blood glucose excursion in oral glucose tolerance test (OGTT) in Sprague Dawley (SD) rats and exerted pronounced antihyperglycemic effects in db/db mice and high-fat diet-fed ZDF rats when dosed orally at 10 mg/kg.

The sodium/glucose cotransporters (SGLT1 and SGLT2) transport glucose across the intestinal brush border and kidney tubule. Dual SGLT1/2 inhibition could reduce hyperglycemia more than SGLT2-selective inhibition in patients with type 2 diabetes. However, questions remain about altered gastrointestinal (Gl) luminal glucose and tolerability, and this was evaluated in slc5a1(-/-) mice or with a potent dual inhibitor (compound 8; SGLT1 K-i = 1.5 +/- 0.5 nM 100-fold greater potency than phlorizin; SGLT2 K-i = 0.4 +/- 0.2 nM). (13)C(6)glucose uptake was quantified in slc5a1(-/-) mice and in isolated rat jejunum. Urinary glucose excretion (UGE), blood glucose (Sprague-Dawley rats), glucagon-like peptide 1 (GLP-1), and hemoglobin A1c (HbA1c) levels (Zucker diabetic fatty rats) were measured. Intestinal adaptation and rRNA gene sequencing was analyzed in C57BI/6 mice. The blood C-13(6)-glucose area under the curve (AUC) was reduced in the absence of SGLT1 by 75% (245 +/- 6 vs. 64 +/- 6 mg/dl.h in wild-type vs. slc5a1(-/-) mice) and compound 8 inhibited its transport up to 50% in isolated rat jejunum. Compound 8 reduced glucose excursion more than SGLT2-selective inhibition (e.g., AUC = 129 +/- 3 vs. 249 +/- 5 mg/dl-h for 1 mg/kg compound 8 vs. dapagliflozin) with similar UGE but a lower renal glucose excretion threshold. In Zucker diabetic fatty rats, compound 8 decreased HbA1c and increased total GLP-1 without changes in jejunum SGLT1 expression, mucosal weight, or villus length. Overall, compound 8 (1 mg/kg for 6 days) did not increase cecal glucose concentrations or bacterial diversity in C57BL76 mice. In conclusion, potent dual SGLT1/2 inhibition lowers blood glucose by reducing intestinal glucose absorption and the renal glucose threshold but minimally impacts the intestinal mucosa or luminal microbiota in chow-fed rodents.

OBJECTIVE:Insulin resistance is a key feature of Type 2 Diabetes (T2D), and improving insulin sensitivity is important for disease management. Allosteric modulation of the insulin receptor (IR) with monoclonal antibodies (mAbs) can enhance insulin sensitivity and restore glycemic control in animal models of T2D. METHODS:A novel human mAb, IRAB-A, was identified by phage screening using competition binding and surface plasmon resonance assays with the IR extracellular domain. Cell based assays demonstrated agonist and sensitizer effects of IRAB-A on IR and Akt phosphorylation, as well as glucose uptake. Lean and diet-induced obese mice were used to characterize single-dose in vivo pharmacological effects of IRAB-A; multiple-dose IRAB-A effects were tested in obese mice. RESULTS:In vitro studies indicate that IRAB-A exhibits sensitizer and agonist properties distinct from insulin on the IR and is translated to downstream signaling and function; IRAB-A bound specifically and allosterically to the IR and stabilized insulin binding. A single dose of IRAB-A given to lean mice rapidly reduced fed blood glucose for approximately 2 weeks, with concomitant reduced insulin levels suggesting improved insulin sensitivity. Phosphorylated IR (pIR) from skeletal muscle and liver were increased by IRAB-A; however, phosphorylated Akt (pAkt) levels were only elevated in skeletal muscle and not liver vs. control; immunochemistry analysis (IHC) confirmed the long-lived persistence of IRAB-A in skeletal muscle and liver. Studies in diet-induced obese (DIO) mice with IRAB-A reduced fed blood glucose and insulinemia yet impaired glucose tolerance and led to protracted insulinemia during a meal challenge. CONCLUSION:Collectively, the data suggest IRAB-A acts allosterically on the insulin receptor acting non-competitively with insulin to both activate the receptor and enhance insulin signaling. While IRAB-A produced a decrease in blood glucose in lean mice, the data in DIO mice indicated an exacerbation of insulin resistance; these data were unexpected and suggested the interplay of complex unknown pharmacology. Taken together, this work suggests that IRAB-A may be an important tool to explore insulin receptor signaling and pharmacology.