The aim of this study was to evaluate amino acids as glucagon receptor (GCGR)‐specific biomarkers in rodents and cynomolgus monkeys in the presence of agonism of both glucagon‐like peptide‐1 receptor (GLP1R) and GCGR with a variety of dual agonist compounds.

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..

Glucagon-like peptide 1 receptor/glucagon receptor (GLP–1R/GCGR) dual agonists are being pursued for obesity and type 2 diabetes mellitus (T2DM). A biomarker for GCGR would aid in optimizing receptor balance and in modeling dose selection to avoid potential adverse events from excess GCGR agonism. It has been reported that glucagon treatment decreases CYP7A1 transcription in primary rat and human hepatocytes, whereas GCGR antagonists increase CYP7A1 mRNA in mice and its product, 7–HCO (the precursor of C4), in diabetic patients. We tested glucagon, insulin, dulaglutide, and a GLP–1R/GCGR dual agonist, Cpd. A, in primary human and cynomolgus monkey hepatocytes. All treatments decreased CYP7A1 expression in monkey hepatocytes. C4 was undetectable in the culture media. Lean mice or rats treated with Cpd. A showed no changes in non–fasting C4. Diet–induced obese mice treated with Cpd. A or a GCGR–null analog had decreased non–fasting C4. To test whether GCGR (and/or GLP–1R) agonism affects plasma C4 in higher species, we infused saline or glucagon (at two different rates) on a background of somatostatin infusion in cynomolgus monkeys dosed with dulaglutide the day before. C4 levels did not differ between treatment groups. To determine whether this lack of an effect was specific to pre-clinical species, T2DM patients were dosed once a week for 4 weeks with a placebo or 2 doses of a second GLP-1R/GCGR dual agonist. There were no statistically significant differences in C4 levels between groups. Hence, in contrast to GCGR antagonists, GCGR and/or GLP–1R agonists did not modulate C4 levels in pre–clinical species or in T2DM patients. Disclosure R. Camacho: Employee; Self; Janssen Research & Development. W. Li: None. T. Kirchner: None. R. Zhang: Employee; Self; Janssen Research & Development. F. Bonilla: None. W. Jian: None. B. Gao: None. R. SinhaRoy: Employee; Self; Janssen Research & Development. Employee; Spouse/Partner; Bristol–Myers Squibb Company. Stock/'Shareholder; Self; Merck & Co., Inc.. Stock/Shareholder; Spouse/Partner; Merck & Co., Inc. J. Leonard: Employee; Self; Janssen Research & Development. T. Connolly: None.

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.

A novel series of 2-thio-5-thiomethyl substituted imidazoles was discovered to be potent TGR5 agonists that possessed glucose-lowering effects while inhibiting gall bladder emptying in mice.

We have discovered a novel series of tetrahydrobenzimidazoles 3 as TGR5 agonists. Initial structure-activity relationship studies with an assay that measured cAMP levels in murine enteroendocrine cells (STC-1 cells) led to the discovery of potent agonists with submicromolar EC50 values for mTGR5. Subsequent optimization through methylation of the 7-position of the core tetrahydrobenzimidazole ring resulted in the identification of potent agonists for both mTGR5 and hTGR5 (human enteroendocrine NCI-H716 cells). While the lead compounds displayed low to moderate exposure after oral dosing, they significantly reduced blood glucose levels in C57 BL/6 mice at 30 mg/kg and induced a 13-22% reduction in the area under the blood glucose curve (AUC)0-120 min in oral glucose tolerance tests (OGTT).

Design and optimization of a novel series of imidazo[1,2-b]pyridazine PDE10a inhibitors are described. Compound 31 displays excellent pharmacokinetic properties and was also evaluated as an insulin secretagogue in vitro and in vivo.

A hallmark of type 2 diabetes is impaired insulin receptor (IR) signaling that results in dysregulation of glucose homeostasis. Understanding the molecular origins and progression of diabetes and developing therapeutics depend on experimental models of hyperglycemia, hyperinsulinemia, and insulin resistance. We present a novel monoclonal antibody, IRAB-B, that is a specific, potent IR antagonist that creates rapid and long-lasting insulin resistance. IRAB-B binds to the IR with nanomolar affinity and in the presence of insulin efficiently blocks receptor phosphorylation within minutes and is sustained for at least 3 days in vitro. We further confirm that IRAB-B antagonizes downstream signaling and metabolic function. In mice, a single dose of IRAB-B induces rapid onset of hyperglycemia within 6 h, and severe hyperglycemia persists for 2 weeks. IRAB-B hyperglycemia is normalized in mice treated with exendin-4, suggesting that this model can be effectively treated with a GLP-1 receptor agonist. Finally, a comparison of IRAB-B with the IR antagonist S961 shows distinct antagonism in vitro and in vivo. IRAB-B appears to be a powerful tool to generate both acute and chronic insulin resistance in mammalian models to elucidate diabetic pathogenesis and evaluate therapeutics.

The discovery of a novel series of cyclopenta[b]furans as CCR2 inhibitors is discussed. This series has excellent CCR2 potency and PK characteristics, and good cardiovascular safety.