Novel TLR2xTLR4 Bispecific Antibody Inhibits Bacterial Sepsis.

Toll-like receptors (TLRs) sense microbial infection through recognition of pathogen-associated molecular patterns. For example, TLR4 responds to the lipopolysaccharide of gram-negative bacteria, whereas TLR2 recognizes a broad range of microbial ligands. Both receptors are, therefore, compelling targets for treating sepsis. Here, we developed a TLR2xTLR4 tetravalent bispecific antibody designated ICU-1, which inhibits both receptors. The inhibitory activity of ICU-1 was comparable to that of the parental antibodies in neutralization assays using a human monocyte cell line. Moreover, ICU-1 completely blocked stimulation of human peripheral blood mononuclear cells by clinically relevant bacterial species. These findings provide convincing evidence that ICU-1 offers a novel approach for treating bacterial sepsis.

AGS16F Is a Novel Antibody Drug Conjugate Directed against ENPP3 for the Treatment of Renal Cell Carcinoma.

PURPOSE: New cancer-specific antigens are required for the design of novel antibody-drug conjugates (ADC) that deliver tumor-specific and highly potent cytotoxic therapy. EXPERIMENTAL DESIGN: Suppression subtractive hybridization identified ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3 or CD203c) as a potential human cancer-specific antigen. Antibodies targeting the extracellular domain of human ENPP3 were produced and selected for specific binding to ENPP3. Expression of ENPP3 in normal and cancer tissue specimens was evaluated by immunohistochemistry (IHC). ADCs comprising anti-ENPP3 Ab conjugated with maleimidocaproyl monomethyl auristatin F via a noncleavable linker (mcMMAF) were selected for therapeutic potential using binding and internalization assays, cytotoxicity assays, and tumor growth inhibition in mouse xenograft models. Pharmacodynamic markers were evaluated by IHC in tissues and ELISA in blood. RESULTS: ENPP3 was highly expressed in clear cell renal cell carcinoma: 92.3% of samples were positive and 83.9% showed high expression. By contrast, expression was negligible in normal tissues examined, with the exception of the kidney. High expression was less frequent in papillary renal cell carcinoma and hepatocellular carcinoma samples. AGS16F, an anti-ENPP3 antibody-mcMMAF conjugate, inhibited tumor growth in three different renal cell carcinoma (RCC) xenograft models. AGS16F localized to tumors, formed the active metabolite Cys-mcMMAF, induced cell-cycle arrest and apoptosis, and increased blood levels of caspase-cleaved cytokeratin-18, a marker of epithelial cell death. CONCLUSIONS: AGS16F is a promising new therapeutic option for patients with RCC and is currently being evaluated in a phase I clinical trial.

NPY Y2 receptor agonist PYY(3-36) inhibits diarrhea by reducing intestinal fluid secretion and slowing colonic transit in mice.

Peptide YY (PYY)(3-36), a neuropeptide Y (NPY) Y2 receptor agonist, is a powerful inhibitor of intestinal secretion. Based on this anti-secretory effect, NPY Y2 receptor agonists may be useful as novel anti-diarrheal agents, but anti-diarrheal efficacy has yet to be determined. We therefore examined the anti-diarrheal efficacy of PYY(3-36) and a selective Y2 receptor agonist, N-acetyl-[Leu28, Leu31]-NPY(24-36), in experimental mouse models of diarrhea. Intraperitoneal administration of PYY(3-36) (0.01-1mg/kg) and N-acetyl-[Leu28, Leu31]-NPY(24-36) (10mg/kg) significantly inhibited diarrhea (increase in wet fecal weight and diarrhea score) induced by dimethyl-prostaglandin E2, 5-hydroxytryptamine, and castor oil. Anti-diarrheal activities of PYY(3-36) and N-acetyl-[Leu28, Leu31]-NPY(24-36) were comparable to the effects of loperamide (1mg/kg), a widely used anti-diarrheal drug. To clarify the anti-diarrheal mechanisms of NPY Y2 receptor agonists, we investigated the effects of PYY(3-36) and N-acetyl-[Leu28, Leu31]-NPY(24-36) on intestinal fluid secretion and colonic transit. PYY(3-36) (1mg/kg) and N-acetyl-[Leu28, Leu31]-NPY(24-36) (10mg/kg) significantly reduced dimethyl-prostaglandin E2-induced intestinal fluid accumulation in conscious mice, suggesting that NPY Y2 receptor agonists inhibit diarrhea, at least in part, by reducing intestinal secretion. In addition, PYY(3-36) (0.01-1mg/kg) and N-acetyl-[Leu28, Leu31]-NPY(24-36) (10mg/kg) potently inhibited normal fecal output, suggesting that NPY Y2 receptor activation inhibits colonic motor function and NPY Y2 receptor agonists inhibit diarrhea partly by slowing colonic transit. These results indicate that NPY Y2 receptor agonists inhibit diarrhea in mice by not only reducing intestinal fluid secretion, but also slowing colonic transit, and illustrate the therapeutic potential of NPY Y2 receptor agonists as effective treatments for diarrhea.

Pancreatic polypeptide enhances colonic muscle contraction and fecal output through neuropeptide Y Y4 receptor in mice.

Pancreatic polypeptide is released mainly from the pancreas, and is thought to be one of the major endogenous agonists of the neuropeptide Y Y(4) receptor. Pancreatic polypeptide has been shown to stimulate colonic muscle contraction, but whether pancreatic polypeptide has in vivo functional activity with respect to colonic transit is unclear. The present report investigated the effects of pancreatic polypeptide on fecal output as an index of colonic transit as well as intestinal motor activity, using wild-type (WT) and neuropeptide Y Y(4) receptor-deficient (KO) mice. Peripheral administration of pancreatic polypeptide increased fecal weight and caused diarrhea in WT mice in a dose-dependent manner (0.01-3mg/kg s.c.). Pancreatic polypeptide-induced increases in fecal weight and diarrhea completely disappeared in KO mice, while basal fecal weights did not differ between WT and KO mice. In longitudinal and circular muscles of mouse isolated colon, pancreatic polypeptide (0.01-1 microM) increased basal tone and frequency of spontaneous contraction in WT mice, but not in KO mice. Atropine did not affect pancreatic polypeptide-induced fecal output or increase in colonic muscle tone, indicating that the actions of pancreatic polypeptide are not mediated through cholinergic mechanisms. The present findings demonstrate that pancreatic polypeptide enhances colonic contractile activity and fecal output through neuropeptide Y Y(4) receptor, and a neuropeptide Y Y(4) receptor agonist might offer a novel therapeutic approach to ameliorate constipation.

Activation of sodium-glucose cotransporter 1 ameliorates hyperglycemia by mediating incretin secretion in mice.

Glucose ingestion stimulates the secretion of the incretin hormones, glucose-dependent insulinotropic peptide (GIP) and glucagon-like peptide-1 (GLP-1). Despite the critical role of incretins in glucose homeostasis, the mechanism of glucose-induced incretin secretion has not been established. We investigated the underlying mechanism of glucose-induced incretin secretion in vivo in mice. Injection of glucose at 1 g/kg in the upper intestine significantly increased plasma GIP and GLP-1 levels, whereas injection of glucose in the colon did not increase GIP or GLP-1 levels. This finding indicates that the glucose sensor for glucose-induced incretin secretion is in the upper intestine. Coadministration of a sodium-glucose cotransporter-1 (SGLT1) inhibitor, phloridzin, with glucose in the upper intestine blocked glucose absorption and glucose-induced incretin secretion. alpha-methyl-d-glucopyranoside (MDG), an SGLT1 substrate that is a nonmetabolizable sugar, significantly increased plasma GIP and GLP-1 levels, whereas phloridzin blocked these increases, indicating that concomitant transport of sodium ions and glucose (substrate) via SGLT1 itself triggers incretin secretion without the need for subsequent glucose metabolism. Interestingly, oral administration of MDG significantly increased plasma GIP, GLP-1, and insulin levels and reduced blood glucose levels during an intraperitoneal glucose tolerance test. Furthermore, chronic MDG treatment in drinking water (3%) for 13 days reduced blood glucose levels after a 2-h fast and in an oral glucose tolerance test in diabetic db/db mice. Our findings indicate that SGLT1 serves as the intestinal glucose sensor for glucose-induced incretin secretion and that a noncalorigenic SGLT1 substrate ameliorates hyperglycemia by stimulating incretin secretion.

Discovery of imidazo[1,2-a]pyridines as potent MCH1R antagonists.

A series of imidazo[1,2-a]pyridine derivatives was identified and evaluated for MCH1R binding and antagonistic activity. Introduction of a methyl substituent at the 3-position of imidazo[1,2-a]pyridine provided compounds with a significant improvement in MCH1R affinity. Representative compounds in this series exhibited good potency and brain exposure in rats.

Synergistic interaction between neuropeptide Y1 and Y5 receptor pathways in regulation of energy homeostasis.

Neuropeptide Y plays a key role in the physiological control of energy homeostasis. Five neuropeptide Y receptor subtypes have been cloned, and multiple neuropeptide Y receptor subtypes are thought to mediate neuropeptide Y activity. However, interactions among neuropeptide Y receptor subtypes have not been elucidated to date. Herein, we examined the interaction between neuropeptide Y(1) and Y(5) receptors in feeding regulation by employing selective neuropeptide Y(1) and Y(5) receptor antagonists in C57BL/6 and neuropeptide Y(1) receptor knockout mice fed a high-fat diet. A single-dose of a neuropeptide Y(1) receptor antagonist (10-30 mg/kg) suppressed spontaneous food intake and reduced body weight in high-fat diet-fed C57BL/6 mice, while treatment with a neuropeptide Y(5) receptor antagonist did not significantly reduce food intake or body weight. Coadministration of a neuropeptide Y(1) receptor antagonist with a neuropeptide Y(5) receptor antagonist further suppressed food intake and reduced body weight. Next, we evaluated the chronic efficacy of a neuropeptide Y(5) receptor antagonist in high-fat diet-fed neuropeptide Y(1) receptor knockout mice in order to mimic chronic combination treatment with neuropeptide Y(1) and Y(5) receptor antagonists. The neuropeptide Y(5) receptor antagonist produced greater body weight reductions in high-fat diet-fed neuropeptide Y(1) receptor knockout mice than in wild-type C57BL/6 mice. These findings confirm an interaction between neuropeptide Y(1) and Y(5) receptors in the regulation of energy homeostasis, as blockade of both the neuropeptide Y(1) and Y(5) receptors produced a greater anti-obesity effect than blocking either receptor alone.

Discovery of tetrasubstituted imidazolines as potent and selective neuropeptide Y Y5 receptor antagonists: reduced human ether-a-go-go related gene potassium channel binding affinity and potent antiobesity effect.

A series of novel imidazoline derivatives was synthesized and evaluated as neuropeptide Y (NPY) Y5 receptor antagonists. Optimization of previously reported imidazoline leads, 1a and 1b, was attempted by introduction of substituents at the 5-position on the imidazoline ring and modification of the bis(4-fluorphenyl) moiety. A number of potent derivatives without human ether-a-go-go related gene potassium channel (hERG) activity were identified. Selected compounds, including 2a, were shown to have excellent brain and CSF permeability. Compound 2a displayed a suitable pharmacokinetic profile for chronic in vivo studies and potently inhibited D-Trp(34)NPY-induced acute food intake in rats. Oral administration of 2a resulted in a potent reduction of body weight in a diet-induced obese mouse model.

Identification of 2-aminobenzimidazoles as potent melanin-concentrating hormone 1-receptor (MCH1R) antagonists.

A series of 2-aminobenzimidazole-based MCH1R antagonists was identified by core replacement of the aminoquinoline lead 1. Subsequent modification of the 2- and 5-positions led to improvement in potency and intrinsic clearance. Compound 25 exhibited good plasma and brain exposure, and attenuated MCH induced food intake at 30mg/kg PO in rats.

Comparison of independent and combined chronic anti-obese effects of NPY Y2 receptor agonist, PYY(3-36), and NPY Y5 receptor antagonist in diet-induced obese mice.

Neuropeptide Y (NPY) and its family of peptides are thought to have a major role in the physiological control of energy homeostasis. Among five NPY receptors described, stimulation of the Y2 receptor (Y2R) or inhibition of the Y5 receptor (Y5R) has recently been shown to produce weight-lowering effects in obese rodents. The present study examined and compared the effects of a Y2R agonist, PYY(3-36), and a Y5R antagonist, alone and in combination, on food intake and body weight in diet-induced obese (DIO) mice. Acute intraperitoneal injection of PYY(3-36) dose-dependently reduced spontaneous feeding in lean and DIO mice. In contrast, acute oral administration of the Y5R antagonist had no effect on spontaneous feeding or the anorexigenic effects of PYY(3-36). In a chronic study, subcutaneous infusion of PYY(3-36) (1 mg/kg/day for 14 days) significantly reduced food intake and body weight in DIO mice. The Y5R antagonist (10 mg/kg/day for 14 days, orally) reduced body weight to the same extent as PYY(3-36) without a significant feeding reduction. Combined administration of PYY(3-36) and the Y5R antagonist resulted in a greater body weight reduction than treatment with either agent alone. The combined effects on food intake, body weight, and adiposity are almost the same as a hypothetical sum of the effects of each drug alone. These results illustrate that the combination of a Y2R agonist, PYY(3-36), and a Y5R antagonist resulted in additive effects on body weight and adiposity in DIO mice, suggesting that Y2R stimulation signal and Y5R blockade signal act by distinct pathways.

Effects of a novel Y5 antagonist in obese mice: combination with food restriction or sibutramine.

OBJECTIVE: To further address the function of the Y5 receptor in energy homeostasis, we investigated the effects of a novel spironolactone Y5 antagonist in diet-induced obese (DIO) mice. METHODS AND PROCEDURES: Male C57BL/6 or Npy5r(-/-) mice were adapted to high-fat (HF) diet for 6-10 months and were submitted to three experimental treatments. First, the Y5 antagonist at a dose of 10 or 30 mg/kg was administered for 1 month to DIO C57BL/6 or Npy5r(-/-) mice. Second, the Y5 antagonist at 30 mg/kg was administered for 1.5 months to DIO C57BL/6 mice, and insulin sensitivity was evaluated using an insulin tolerance test. After a recovery period, nuclear magnetic resonance measurement was performed to evaluate body composition. Third, DIO mice were treated with the Y5 antagonist alone, or in combination with 10% food restriction, or with another anorectic agent, sibutramine at 10 mg/kg, for 1.5 months. Plasma glucose, insulin, and leptin levels, and adipose tissue weights were quantified. RESULTS: The spironolactone Y5 antagonist significantly reduced body weight in C57BL DIO mice, but not in Npy5r(-/-) DIO mice. The Y5 antagonist produced a fat-selective loss of body weight, and ameliorated obesity-associated insulin resistance in DIO mice. In addition, the Y5 antagonist combined with either food restriction or sibutramine tended to produce greater body weight loss, as compared with single treatment. DISCUSSION: These findings demonstrate that the Y5 receptor is an important mediator of energy homeostasis in rodents.

A pair-feeding study reveals that a Y5 antagonist causes weight loss in diet-induced obese mice by modulating food intake and energy expenditure.

Neuropeptide Y (NPY) is thought to have a significant role in the physiological control of energy homeostasis. We recently reported that an NPY Y5 antagonist inhibits body weight gain in diet-induced obese (DIO) mice, with a moderate reduction in food intake. To clarify the mechanism of the antiobesity effects of the Y5 antagonist, we conducted a pair-feeding study in DIO mice. The Y5 antagonist at 100 mg/kg produced a moderate feeding suppression leading to an 18% decrease in body weight, without altering body temperature. In contrast, the pair-fed group showed only a transient weight reduction and a reduced body temperature, thus indicating that the Y5 antagonist stimulates thermogenesis. The Y5 antagonist-treated mice showed an up-regulation of uncoupling protein mRNA in brown adipose tissue (BAT) and white adipose tissue (WAT), suggesting that both BAT and WAT contribute to energy expenditure. Thus, the Y5 antagonist induces its antiobesity effects by acting on both energy intake and expenditure.

RFamide peptide QRFP43 causes obesity with hyperphagia and reduced thermogenesis in mice.

QRFP, an RFamide peptide, was recently identified as an endogenous ligand of an orphan G protein-coupled receptor, GPR103. Recent investigation revealed that acute intracerebroventricular (ICV) administration of QRFP26/P518/26RFa, a constitutive part of QRFP43 (43-amino acid-residue form of QRFP), increases appetite in mice, but its role in long-term energy homeostasis remains unknown. In the present study, we examined the effects of chronic administration of QRFP43 on feeding behavior, body weight regulation, and energy expenditure in mice. Intracerebroventricular infusion of QRFP43 for 13 d resulted in a significant increase in body weight and fat mass with hyperphagia. Weight gain and hyperphagia were more evident when mice were fed a moderately high-fat diet. Pair feeding of QRFP43-infused mice did not increase body weight but significantly increased fat mass and plasma concentrations of insulin, leptin, and cholesterol when compared with controls. Moreover, significant decreases in rectal temperature and expression of brown adipose tissue uncoupling protein-1 mRNA were observed in QRFP43-infused ad libitum- and pair-fed mice. The present results suggest that QRFP plays an important role in energy homeostasis by regulating appetite and energy expenditure.

Antiobesity effect of a melanin-concentrating hormone 1 receptor antagonist in diet-induced obese mice.

Melanin-concentrating hormone (MCH) is a cyclic orexigenic peptide expressed in the lateral hypothalamus, which plays an important role in regulating energy balance. To elucidate the physiological role of MCH in obesity development, the present study examined the effect of a selective MCH1 receptor (MCH1R) antagonist in the diet-induced obesity mouse model. The MCH1R antagonist has high affinity and selectivity for MCH-1R and potently inhibits intracerebroventricularly injected MCH-induced food intake in Sprague Dawley rats. Chronic intracerebroventricular infusion of the MCH1R antagonist (7.5 microg/d) completely suppressed body weight gain in diet-induced obese mice during the treatment periods and significantly decreased cumulative food intake, by 14%. Carcass analysis showed that the MCH1R antagonist resulted in a selective decrease of body fat in the diet-induced obese mice. In addition, the MCH1R antagonist ameliorated the obesity-related hypercholesterolemia, hyperinsulinemia, hyperglycemia, and hyperleptinemia. These results indicate that MCH has a major role in the development of diet-induced obesity in mice and that a MCH1R antagonist might be a useful candidate as an antiobesity agent.