Antiobesity and emetic effects of a short-length peptide YY analog and its PEGylated and alkylated derivatives.

Neuropeptide Y2 receptor (Y2R) agonism is an important anorectic signal and a target of antiobesity drug discovery. Recently, we synthesized a short-length Y2R agonist, PYY-1119 (4-imidazolecarbonyl-[d-Hyp24,Iva25,Pya(4)26,Cha27,36,γMeLeu28,Lys30,Aib31]PYY(23-36), 1) as an antiobesity drug candidate. Compound 1 induced marked body weight loss in diet-induced obese (DIO) mice; however, 1 also induced severe vomiting in dogs at a lower dose than the minimum effective dose administered to DIO mice. The rapid absorption of 1 after subcutaneous administration caused the severe vomiting. Polyethylene glycol (PEG)- and alkyl-modified derivatives of 1 were synthesized to develop Y2R agonists with improved pharmacokinetic profiles, i.e., lower maximum plasma concentration (Cmax) and longer time at maximum concentration (Tmax). Compounds 5 and 10, modified with 20 kDa PEG at the N-terminus and eicosanedioic acid at the Lys30 side chain of 1, respectively, showed high Y2R binding affinity and induced significant body weight reduction upon once-daily administration to DIO mice. Compounds 5 and 10, with their relatively low Cmax and long Tmax, partially attenuated emesis in dogs compared with 1. These results indicate that optimization of pharmacokinetic properties of Y2R agonists is an effective strategy to alleviate emesis induced by Y2R agonism.

Differential effects of selective agonists of neuromedin U1 and U2 receptors in obese and diabetic mice.

BACKGROUND AND PURPOSE: Neuromedin U (NmU) may be a novel target for obesity treatment owing to its anorectic and energy expenditure enhancing effects. Although two receptors, NMU1 and NMU2, are both responsible for the NmU-mediated anti-obesity effects, the receptor agonist with the most appropriate profiles for treating obesity and diabetes in terms of efficacy and safety is as yet unknown. Thus, we developed and evaluated novel NMU1/2 receptor-selective agonists. EXPERIMENTAL APPROACH: Efficacy and safety were assessed in mice with diet-induced obesity (DIO) and those with leptin-deficient diabetes (ob/ob) through repeated peripheral administration of selective agonists to NMU1 (NMU-6102) and NMU2 (NMU-2084), along with non-selective NMU1/2 agonists (NMU-0002 and NMU-6014). We also performed immunohistochemistry for c-Fos protein expression in the brain to probe their mechanisms of action. KEY RESULTS: Although both non-selective NMU1/2 agonists and the NMU2-selective agonist had high efficacy compared with the NMU1-selective agonist, only the NMU2-selective agonist led to relatively low adverse effects, such as diarrhoea, in DIO mice. However, the non-selective NMU1/2 agonist and the NMU1-selective agonist, but not the NMU2-selective agonist, were effective in diabetic ob/ob mice. Mechanistically, NMU2-selective agonists preferentially activate pro-opiomelanocortin neurons in the hypothalamic arcuate nucleus but not in the paraventricular nucleus. CONCLUSIONS AND IMPLICATIONS: These results suggest that an NMU2 receptor-selective agonist may be a well-balanced drug for the treatment of obesity and that an NMU1 receptor-selective agonist may also be beneficial for treating obesity and diabetes once its side effects are minimized.

Highly potent antiobesity effect of a short-length peptide YY analog in mice.

Continuous administration of a 14-amino acid peptide YY (PYY) analog, Ac-[d-Pro24,Pya(4)26,Cha27,36,Aib28,31,Lys30]PYY(23-36) (4), which has a high binding affinity and agonist activity for the neuropeptide Y2 receptor (Y2R), has previously shown an antiobesity effect in a 2-week diet-induced obesity (DIO) study in mice. However, there remained a possibility to obtain more potent analogs by further improving its pharmacokinetic profile. A combination of the N-terminal 4-imidazolecarbonyl moiety and three amino acid substitutions, trans-4-hydroxy-d-proline (d-Hyp)24, isovaline (Iva)25, and γ-methylleucine (γMeLeu)28, not only improved the binding affinity of the peptide for Y2R but also increased its anorectic activity in lean mice. In a 2-week DIO study in mice, continuous administration of 4-imidazolecarbonyl-[d-Hyp24,Iva25,Pya(4)26,Cha27,36,γMeLeu28,Lys30,Aib31]PYY(23-36) (31, PYY-1119) at a dose of 0.03mg/kg/day showed a highly potent antiobesity effect, with more than 10% body weight reduction.

Potent antiobesity effect of a short-length peptide YY-analogue continuously administered in mice.

The gastrointestinal peptide, peptide YY3-36 (PYY3-36) and its shorter peptide analogues have been reported to reduce appetite by activating the neuropeptide Y2 receptor (Y2R), which is associated with obesity and other metabolic diseases. A 14-amino acid PYY analogue, Ac-[d-Pro24,Cha27,28,36,Aib31]PYY(23-36) (3), showed high binding affinity and agonist activity for the Y2R, similar to that of PYY3-36, but had weak anorectic activity upon continuous administration in lean mice. Three amino acid substitutions [Pya(4)26, Aib28, Lys30], which contributed to the decreased hydrophobicity of 3, efficiently increased its anorectic activity. The compound containing these three amino acids, Ac-[d-Pro24,Pya(4)26,Cha27,36,Aib28,31,Lys30]PYY(23-36) (22), exerted more potent and durable food intake suppression than that by PYY3-36 in lean mice, as well as excellent Y2R agonist activity (EC50: 0.20nM) and good subcutaneous bioavailability (66.6%). The 11-day continuous administration of 22 at 1mg/kg/day successfully produced antiobese and antidiabetic effects, with more than 20% body weight loss in obese and Type 2 diabetes ob/ob model mice.

Design of potent dipeptidyl peptidase IV (DPP-4) inhibitors by employing a strategy to form a salt bridge with Lys554.

We report a design strategy to obtain potent DPP-4 inhibitors by incorporating salt bridge formation with Lys554 in the S1' pocket. By applying the strategy to the previously identified templates, quinoline 4 and pyridines 16a, 16b, and 17 have been identified as subnanomolar or nanomolar inhibitors of human DPP-4. Docking studies suggested that a hydrophobic interaction with Tyr547 as well as the salt bridge interaction is important for the extremely high potency. The design strategy would be useful to explore a novel design for DPP-4 inhibitors having a distinct structure with a unique binding mode.

Potent Body Weight-Lowering Effect of a Neuromedin U Receptor 2-selective PEGylated Peptide.

Neuromedin U (NMU) is a neuropeptide that mediates a variety of physiological functions via its receptors, NMUR1 and NMUR2. Recently, there has been an increased focus on NMU as a promising treatment option for diabetes and obesity. A short form of NMU (NMU-8) has potent agonist activity for both receptors but is metabolically unstable. Therefore, we designed and synthesized NMU-8 analogues modified by polyethylene glycol (PEG; molecular weight, 20 kDa; PEG20k) via a linker. 3-(2-Naphthyl)alanine substitution at position 19 increased NMUR2 selectivity of NMU-8 analogues with retention of high agonist activity. Compound 37, an NMUR2-selective PEG20k analogue containing piperazin-1-ylacetyl linker, exhibited a potent body weight-lowering effect with concomitant inhibition of food intake in a dose-dependent manner (body weight loss of 12.4% at 30 nmol/kg) by once-daily repeated dosing for 2 weeks in mice with diet-induced obesity.

Discovery of potent, selective, and orally bioavailable quinoline-based dipeptidyl peptidase IV inhibitors targeting Lys554.

Dipeptidyl peptidase IV (DPP-4) inhibition is a validated therapeutic option for type 2 diabetes, exhibiting multiple antidiabetic effects with little or no risk of hypoglycemia. In our studies involving non-covalent DPP-4 inhibitors, a novel series of quinoline-based inhibitors were designed based on the co-crystal structure of isoquinolone 2 in complex with DPP-4 to target the side chain of Lys554. Synthesis and evaluation of designed compounds revealed 1-[3-(aminomethyl)-4-(4-methylphenyl)-2-(2-methylpropyl)quinolin-6-yl]piperazine-2,5-dione (1) as a potent, selective, and orally active DPP-4 inhibitor (IC50=1.3 nM) with long-lasting ex vivo activity in dogs and excellent antihyperglycemic effects in rats. A docking study of compound 1 revealed a hydrogen-bonding interaction with the side chain of Lys554, suggesting this residue as a potential target site useful for enhancing DPP-4 inhibition.

Identification of 3-aminomethyl-1,2-dihydro-4-phenyl-1-isoquinolones: a new class of potent, selective, and orally active non-peptide dipeptidyl peptidase IV inhibitors that form a unique interaction with Lys554.

The design, synthesis, and structure-activity relationships of a new class of potent and orally active non-peptide dipeptidyl peptidase IV (DPP-4) inhibitors, 3-aminomethyl-1,2-dihydro-4-phenyl-1-isoquinolones, are described. We hypothesized that the 4-phenyl group of the isoquinolone occupies the S1 pocket of the enzyme, the 3-aminomethyl group forms an electrostatic interaction with the S2 pocket, and the introduction of a hydrogen bond donor onto the 6- or 7-substituent provides interaction with the hydrophilic region of the enzyme. Based on this hypothesis, intensive research focused on developing new non-peptide DPP-4 inhibitors has been carried out. Among the compounds designed in this study, we identified 2-[(3-aminomethyl-2-(2-methylpropyl)-1-oxo-4-phenyl-1,2-dihydro-6-isoquinolinyl)oxy]acetamide (35a) as a potent, selective, and orally bioavailable DPP-4 inhibitor, which exhibited in vivo efficacy in diabetic model rats. Finally, X-ray crystallography of 35a in a complex with the enzyme validated our hypothesized binding mode and identified Lys554 as a new target-binding site available for DPP-4 inhibitors.

Discovery of a 3-pyridylacetic acid derivative (TAK-100) as a potent, selective and orally active dipeptidyl peptidase IV (DPP-4) inhibitor.

Inhibition of dipeptidyl peptidase IV (DPP-4) is an exciting new approach for the treatment of diabetes. To date there has been no DPP-4 chemotype possessing a carboxy group that has progressed into clinical trials. Originating from the discovery of the structurally novel quinoline derivative 1, we designed novel pyridine derivatives containing a carboxy group. In our design, the carboxy group interacted with the targeted amino acid residues around the catalytic region and thereby increased the inhibitory activity. After further optimization, we identified a hydrate of [5-(aminomethyl)-6-(2,2-dimethylpropyl)-2-ethyl-4-(4-methylphenyl)pyridin-3-yl]acetic acid (30c) as a potent and selective DPP-4 inhibitor. The desired interactions with the critical active-site residues, such as a salt-bridge interaction with Arg125, were confirmed by X-ray cocrystal structure analysis. In addition, compound 30c showed a desired preclinical safety profile, and it was encoded as TAK-100.

Design and synthesis of 3-pyridylacetamide derivatives as dipeptidyl peptidase IV (DPP-4) inhibitors targeting a bidentate interaction with Arg125.

We have previously discovered nicotinic acid derivative 1 as a structurally novel dipeptidyl peptidase IV (DPP-4) inhibitor. In this study, we obtained the X-ray co-crystal structure between nicotinic acid derivative 1 and DPP-4. From these X-ray co-crystallography results, to achieve more potent inhibitory activity, we targeted Arg125 as a potential amino acid residue because it was located near the pyridine core, and some known DPP-4 inhibitors were reported to interact with this residue. We hypothesized that the guanidino group of Arg125 could interact with two hydrogen-bond acceptors in a bidentate manner. Therefore, we designed a series of 3-pyridylacetamide derivatives possessing an additional hydrogen-bond acceptor that could have the desired bidentate interaction with Arg125. We discovered the dihydrochloride of 1-{[5-(aminomethyl)-2-methyl-4-(4-methylphenyl)-6-(2-methylpropyl)pyridin-3-yl]acetyl}-l-prolinamide (13j) to be a potent and selective DPP-4 inhibitor that could interact with the guanidino group of Arg125 in a unique bidentate manner.

A novel dipeptidyl peptidase-4 inhibitor, alogliptin (SYR-322), is effective in diabetic rats with sulfonylurea-induced secondary failure.

AIMS: Loss of efficacy over time or secondary failure occurs somewhat often and remains a major concern of sulfonylurea (SU) therapy. In this study, we investigated the benefits of alogliptin, an oral, potent and highly selective dipeptidyl peptidase-4 (DPP-4) inhibitor, in a rat model exhibiting SU secondary failure. MAIN METHODS: Neonatally streptozotocin-induced diabetic rats (N-STZ-1.5 rats), a non-obese model of type 2 diabetes, were used in these studies. The effects of alogliptin on DPP-4 activity and glucagon-like peptide 1 (GLP-1) concentration were determined by measuring their levels in plasma. In addition, the effects of alogliptin on an oral glucose tolerance test were investigated by using an SU secondary failure model. KEY FINDINGS: Alogliptin dose dependently suppressed plasma DPP-4 activity leading to an increase in the plasma active form of GLP-1 and improved glucose excursion in N-STZ-1.5 rats. Repeated administration of glibenclamide resulted in unresponsiveness or loss of glucose tolerance typical of secondary failure. In these rats, alogliptin exhibited significant improvement of glucose excursion with significant increase in insulin secretion. By contrast, glibenclamide and nateglinide had no effect on the glucose tolerance of these rats. SIGNIFICANCE: The above findings suggest that alogliptin was effective at improving glucose tolerance and therefore overcoming SU induced secondary failure in N-STZ-1.5 rats.

The dipeptidyl peptidase-4 inhibitor alogliptin in combination with pioglitazone improves glycemic control, lipid profiles, and increases pancreatic insulin content in ob/ob mice.

The combination of two agents with different but complementary mechanisms of action is a logical approach for treating patients with type 2 diabetes. Thus, we evaluated chronic combination therapy with alogliptin, a highly selective dipeptidyl peptidase-4 inhibitor that enhances the action of incretins, and pioglitazone, a thiazolidinedione that improves peripheral and hepatic insulin sensitivity. Studies were designed to investigate the chronic metabolic and pancreatic effects of alogliptin (0.03%) plus pioglitazone (0.003%) combination treatment in obese ob/ob mice. After 4-5 weeks of treatment, alogliptin significantly increased plasma active glucagon-like peptide-1 levels up to 4.1-fold and decreased plasma glucagon up to 25%, whereas pioglitazone significantly increased plasma adiponectin up to 1.3-fold. Combination treatment exhibited a complementary effect, increasing plasma insulin levels by 3.2-fold (alogliptin alone, 1.6-fold; pioglitazone alone, 1.5-fold) and decreasing glycosylated hemoglobin by 2.3% (alogliptin alone, 1.0%; pioglitazone alone, 1.5%), and non-fasting and fasting plasma glucose by 37% and 62% (alogliptin alone, 17% and 24%; pioglitazone alone, 30% and 45%), respectively. Combination treatment also decreased plasma triglycerides by 67% and non-esterified fatty acids by 25% (alogliptin alone, 24% and 11%; pioglitazone alone, 54% and 8%). Moreover, combination treatment increased pancreatic insulin content by 2.2-fold (alogliptin alone, 1.3-fold; pioglitazone alone, 1.6-fold), with no significant changes in body weight. These results indicate that combination treatment with alogliptin and pioglitazone improved glycemic control, lipid profiles and increased pancreatic insulin content in ob/ob mice by preventing incretin inactivation and improving insulin resistance. These results provide a strong argument for using alogliptin in combination with pioglitazone.

Pharmacokinetic, pharmacodynamic, and efficacy profiles of alogliptin, a novel inhibitor of dipeptidyl peptidase-4, in rats, dogs, and monkeys.

The aim of the present research was to characterize the pharmacokinetic, pharmacodynamic, and efficacy profiles of alogliptin, a novel quinazolinone-based dipeptidyl peptidase-4 (DPP-4) inhibitor. Alogliptin potently inhibited human DPP-4 in vitro (mean IC(50), ~ 6.9 nM) and exhibited > 10,000-fold selectivity for DPP-4 over the closely related serine proteases DPP-2, DPP-8, DPP-9, fibroblast activation protein/seprase, prolyl endopeptidase, and tryptase (IC(50) > 100,000 nM). Absolute oral bioavailability of alogliptin in rats, dogs, and monkeys was 45%, 86%, and 72% to 88%, respectively. After a single oral dose of alogliptin, plasma DPP-4 inhibition was observed within 15 min and maximum inhibition was > 90% in rats, dogs, and monkeys; inhibition was sustained for 12 h in rats (43%) and dogs (65%) and 24 h in monkeys (> 80%). From E(max) modeling, 50% inhibition of DPP-4 activity was observed at a mean alogliptin plasma concentration (EC(50)) of 3.4 to 5.6 ng/ml (10.0 to 16.5 nM) in rats, dogs, and monkeys. In Zucker fa/fa rats, a single dose of alogliptin (0.3, 1, 3, and 10 mg/kg) inhibited plasma DPP-4 (91% to 100% at 2 h and 20% to 66% at 24 h), increased plasma GLP-1 (2- to 3-fold increase in AUC(0-20 min)) and increased early-phase insulin secretion (1.5- to 2.6-fold increase in AUC(0-20 min)) and reduced blood glucose excursion (31%-67% decrease in AUC(0-90 min)) after oral glucose challenge. Alogliptin (30 and 100 mg/kg) had no effect on fasting plasma glucose in normoglycemic rats. In summary, these data suggest that alogliptin is a potent and highly selective DPP-4 inhibitor with demonstrated efficacy in Zucker fa/fa rats and potential for once-daily dosing in humans.

Chronic administration of alogliptin, a novel, potent, and highly selective dipeptidyl peptidase-4 inhibitor, improves glycemic control and beta-cell function in obese diabetic ob/ob mice.

Dipeptidyl peptidase-4 (DPP-4) inhibitors improve glycemic control in patients with type 2 diabetes by increasing plasma active glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide levels. However, the effects of chronic DPP-4 inhibition on in vivo beta-cell function are poorly characterized. We thus evaluated the chronic effects of the DPP-4 inhibitor alogliptin benzoate (formerly SYR-322) on metabolic control and beta-cell function in obese diabetic ob/ob mice. Alogliptin (0.002%, 0.01%, or 0.03%) was administered in the diet to ob/ob mice for 2 days to determine effects on plasma DPP-4 activity and active GLP-1 levels and for 4 weeks to determine chronic effects on metabolic control and beta-cell function. After 2 days, alogliptin dose-dependently inhibited DPP-4 activity by 28-82% and increased active GLP-1 by 3.2-6.4-fold. After 4 weeks, alogliptin dose-dependently decreased glycosylated hemoglobin by 0.4-0.9%, plasma glucose by 7-28% and plasma triglycerides by 24-51%, increased plasma insulin by 1.5-2.0-fold, and decreased plasma glucagon by 23-26%, with neutral effects on body weight and food consumption. In addition, after drug washout, alogliptin (0.03% dose) increased early-phase insulin secretion by 2.4-fold and improved oral meal tolerance (25% decrease in glucose area under the concentration-time curve), despite the lack of measurable plasma DPP-4 inhibition. Importantly, alogliptin also increased pancreatic insulin content up to 2.5-fold, and induced intense insulin staining of islets, suggestive of improved beta-cell function. In conclusion, chronic treatment with alogliptin improved glycemic control, decreased triglycerides, and improved beta-cell function in ob/ob mice, and may exhibit similar effects in patients with type 2 diabetes.

Discovery of alogliptin: a potent, selective, bioavailable, and efficacious inhibitor of dipeptidyl peptidase IV.

Alogliptin is a potent, selective inhibitor of the serine protease dipeptidyl peptidase IV (DPP-4). Herein, we describe the structure-based design and optimization of alogliptin and related quinazolinone-based DPP-4 inhibitors. Following an oral dose, these noncovalent inhibitors provide sustained reduction of plasma DPP-4 activity and a lowering of blood glucose in animal models of diabetes. Alogliptin is currently undergoing phase III trials in patients with type 2 diabetes.