Bombesin receptor subtype-3-expressing neurons regulate energy homeostasis through a novel neuronal pathway in the hypothalamus.

Objectives: Bombesin receptor subtype-3 (BRS-3) has been suggested to play a potential role in energy homeostasis. However, the physiological mechanism of BRS-3 on energy homeostasis remains unknown. Thus, we investigated the BRS-3-mediated neuronal pathway involved in food intake and energy expenditure. Materials and Methods: Expression of BRS-3 in the rat brain was histologically examined. The BRS-3 neurons activated by refeeding-induced satiety or a BRS-3 agonist were identified by c-Fos immunostaining. We also analyzed expression changes in feeding-relating peptides in the brain of fasted rats administered with the BRS-3 agonist. Results: In the paraventricular hypothalamic nucleus (PVH), dorsomedial hypothalamic nucleus (DMH), and medial preoptic area (MPA), strong c-Fos induction was observed in the BRS-3 neurons especially in PVH after refeeding. However, the BRS-3 neurons in the PVH did not express feeding-regulating peptides, while the BRS-3 agonist administration induced c-Fos expression in the DMH and MPA, which were not refeeding-sensitive, as well as in the PVH. The BRS-3 agonist administration changed the Pomc and Cart mRNA level in several brain regions of fasted rats. Conclusion: These results suggest that BRS-3 neurons in the PVH are a novel functional subdivision in the PVH that regulates feeding behavior. As the MPA and DMH are reportedly involved in thermoregulation and energy metabolism, the BRS-3 neurons in the MPA/DMH might mediate the energy expenditure control. POMC and CART may contribute to BRS-3 neuron-mediated energy homeostasis regulation. In summary, BRS-3-expressing neurons could regulate energy homeostasis through a novel neuronal pathway.

A Selective Bombesin Receptor Subtype 3 Agonist Promotes Weight Loss in Male Diet-Induced-Obese Rats With Circadian Rhythm Change.

Bombesin receptor subtype 3 (BRS-3) is an orphan G protein-coupled receptor. Based on the obese phenotype of male BRS-3-deficient mice, BRS-3 has been considered an attractive target for obesity treatment. Here, we developed a selective BRS-3 agonist (compound-A) and evaluated its antiobesity effects. Compound-A showed anorectic effects and enhanced energy expenditure in diet-induced-obese (DIO)-F344 rats. Moreover, repeated oral administration of compound-A for 7 days resulted in a significant body weight reduction in DIO-F344 rats. We also evaluated compound-A for cardiovascular side effects using telemeterized Sprague-Dawley (SD) rats. Oral administration of compound-A resulted in transient blood pressure increases in SD rats. To investigate the underlying mechanisms of BRS-3 agonist effects, we focused on the suprachiasmatic nucleus (SCN), the main control center of circadian rhythms in the hypothalamus, also regulating sympathetic nervous system. Compound-A significantly increased the messenger RNA expression of Brs-3, c-fos, and circadian rhythm genes in SCN of DIO-F344 rats. Because SCN also controls the hypothalamic-pituitary-adrenal (HPA) axis, we evaluated the relationship between BRS-3 and the HPA axis. Oral administration of compound-A caused a significant increase of plasma corticosterone levels in DIO-F344 rats. On this basis, energy expenditure enhancement by compound-A may be due to a circadian rhythm change in central and peripheral tissues, enhancement of peripheral lipid metabolism, and stimulation of the sympathetic nervous system. Furthermore, the blood pressure increase by compound-A could be associated with sympathetic nervous system stimulation via SCN and elevation of plasma corticosterone levels through activation of the HPA axis.

A novel and selective melanin-concentrating hormone receptor 1 antagonist ameliorates obesity and hepatic steatosis in diet-induced obese rodent models.

Melanin-concentrating hormone (MCH), a cyclic neuropeptide expressed predominantly in the lateral hypothalamus, plays an important role in the control of feeding behavior and energy homeostasis. Mice lacking MCH or MCH1 receptor are resistant to diet-induced obesity (DIO) and MCH1 receptor antagonists show potent anti-obesity effects in preclinical studies, indicating that MCH1 receptor is a promising target for anti-obesity drugs. Moreover, recent studies have suggested the potential of MCH1 receptor antagonists for treatment of non-alcoholic fatty liver disease (NAFLD). In the present study, we show the anti-obesity and anti-hepatosteatosis effect of our novel MCH1 receptor antagonist, Compound A. Repeated oral administration of Compound A resulted in dose-dependent body weight reduction and had an anorectic effect in DIO mice. The body weight lowering effect of Compound A was more potent than that of pair-feeding. Compound A also reduced lipid content and the expression level of lipogenesis-, inflammation-, and fibrosis-related genes in the liver of DIO mice. Conversely, intracerebroventricular infusion of MCH caused induction of hepatic steatosis as well as increase in body weight in high-fat diet-fed wild type mice, but not MCH1 receptor knockout mice. The pair-feeding study revealed the MCH-MCH1 receptor system affects hepatic steatosis through a mechanism that is independent of body weight change. Metabolome analysis demonstrated that Compound A upregulated lipid metabolism-related molecules, such as acylcarnitines and cardiolipins, in the liver. These findings suggest that our novel MCH1 receptor antagonist, Compound A, exerts its beneficial therapeutic effect on NAFLD and obesity through a central MCH-MCH1 receptor pathway.

Development of a Novel Carbon-11 Labeled PET Radioligand for Melanin- Concentrating Hormone Receptor 1.

BACKGROUND AND OBJECTIVE: Melanin-concentrating hormone (MCH) is an attractive target for antiobesity agents and many drug discovery programs have been dedicated to identify smallmolecule antagonists of melanin-concentrating hormone receptor 1 (MCHR1). The aim of this study was to develop a positron emission tomography (PET) tracer for MCHR1 for translation of preclinical pharmacology to clinic to enhance success rate of drug discovery programs. METHODS: We identified 4-(cyclopropylmethoxy)-N-[8-methyl-3-({[(1-methyl-1H-pyrrol-2-yl)methyl] amino}ethyl)quinolin-7-yl]benzamide (Compound II) from Takeda MCHR1 antagonist library by utilizing binding affinity, log D value, physicochemical parameters ideal for a central nerve system agent, and synthetic feasibility of corresponding carbon-11 labeled radioligands as selection parameters for tracer candidates. RESULTS: In the rat PET study, [11C] Compound II showed clear uptake in the caudate/putamen with the pretreatment of cyclosporine A and its uptake was higher than that in the cerebellum where expression of MCHR1 was reported to be low. CONCLUSION: In summary, [11C]Compound II is a promising lead compound for developing a suitable MCHR1 PET radioligand. [11C]Compound II, in combination with cyclosporine A, could be used as a research tool to visualize and quantify MCHR1 in rodents.

Amine-free melanin-concentrating hormone receptor 1 antagonists: Novel 1-(1H-benzimidazol-6-yl)pyridin-2(1H)-one derivatives and design to avoid CYP3A4 time-dependent inhibition.

Melanin-concentrating hormone (MCH) is an attractive target for antiobesity agents, and numerous drug discovery programs are dedicated to finding small-molecule MCH receptor 1 (MCHR1) antagonists. We recently reported novel pyridine-2(1H)-ones as aliphatic amine-free MCHR1 antagonists that structurally featured an imidazo[1,2-a]pyridine-based bicyclic motif. To investigate imidazopyridine variants with lower basicity and less potential to inhibit cytochrome P450 3A4 (CYP3A4), we designed pyridine-2(1H)-ones bearing various less basic bicyclic motifs. Among these, a lead compound 6a bearing a 1H-benzimidazole motif showed comparable binding affinity to MCHR1 to the corresponding imidazopyridine derivative 1. Optimization of 6a afforded a series of potent thiophene derivatives (6q-u); however, most of these were found to cause time-dependent inhibition (TDI) of CYP3A4. As bioactivation of thiophenes to form sulfoxide or epoxide species was considered to be a major cause of CYP3A4 TDI, we introduced electron withdrawing groups on the thiophene and found that a CF3 group on the ring or a Cl adjacent to the sulfur atom helped prevent CYP3A4 TDI. Consequently, 4-[(5-chlorothiophen-2-yl)methoxy]-1-(2-cyclopropyl-1-methyl-1H-benzimidazol-6-yl)pyridin-2(1H)-one (6s) was identified as a potent MCHR1 antagonist without the risk of CYP3A4 TDI, which exhibited a promising safety profile including low CYP3A4 inhibition and exerted significant antiobesity effects in diet-induced obese F344 rats.

Amine-free melanin-concentrating hormone receptor 1 antagonists: Novel non-basic 1-(2H-indazole-5-yl)pyridin-2(1H)-one derivatives and mitigation of mutagenicity in Ames test.

To develop non-basic melanin-concentrating hormone receptor 1 (MCHR1) antagonists with a high probability of target selectivity and therapeutic window, we explored neutral bicyclic motifs that could replace the previously reported imidazo[1,2-a]pyridine or 1H-benzimidazole motif. The results indicated that the binding affinity of a chemically neutral 2H-indazole derivative 8a with MCHR1 (hMCHR1: IC50=35nM) was comparable to that of the imidazopyridine and benzimidazole derivatives (1 and 2, respectively) reported so far. However, 8a was positive in the Ames test using TA1537 in S9- condition. Based on a putative intercalation of 8a with DNA, we introduced a sterically-hindering cyclopropyl group on the indazole ring to decrease planarity, which led to the discovery of 1-(2-cyclopropyl-3-methyl-2H-indazol-5-yl)-4-{[5-(trifluoromethyl)thiophen-3-yl]methoxy}pyridin-2(1H)-one 8l without mutagenicity in TA1537. Compound 8l exerted significant antiobesity effects in diet-induced obese F344 rats and exhibited promising safety profile.

Melanin-Concentrating Hormone Receptor 1 Antagonists Lacking an Aliphatic Amine: Synthesis and Structure-Activity Relationships of Novel 1-(Imidazo[1,2-a]pyridin-6-yl)pyridin-2(1H)-one Derivatives.

Aiming to discover melanin-concentrating hormone receptor 1 (MCHR1) antagonists with improved safety profiles, we hypothesized that the aliphatic amine employed in most antagonists reported to date could be removed if the bicyclic motif of the compound scaffold interacted with Asp123 and/or Tyr272 of MCHR1. We excluded aliphatic amines from our compound designs, with a cutoff value of pK(a) < 8, and explored aliphatic amine-free MCHR1 antagonists in a CNS-oriented chemical space limited by four descriptors (TPSA, ClogP, MW, and HBD count). Screening of novel bicyclic motifs with high intrinsic binding affinity for MCHR1 identified the imidazo[1,2-a]pyridine ring (represented in compounds 6a and 6b), and subsequent cyclization of the central aliphatic amide linkage led to the discovery of a potent, orally bioavailable MCHR1 antagonist 4-[(4-chlorobenzyl)oxy]-1-(2-cyclopropyl-3-methylimidazo[1,2-a]pyridin-6-yl)pyridin-2(1H)-one 10a. It exhibited low potential for hERG inhibition and phospholipidosis induction as well as sufficient brain concentration to exert antiobesity effects in diet-induced obese rats.

Synthesis, structure-activity relationship, and pharmacological studies of novel melanin-concentrating hormone receptor 1 antagonists 3-aminomethylquinolines: reducing human ether-a-go-go-related gene (hERG) associated liabilities.

Recently, we discovered 3-aminomethylquinoline derivative 1, a selective, highly potent, centrally acting, and orally bioavailable human MCH receptor 1 (hMCHR1) antagonist, that inhibited food intake in F344 rats with diet-induced obesity (DIO). Subsequent investigation of 1 was discontinued because 1 showed potent hERG K(+) channel inhibition in a patch-clamp study. To decrease hERG K(+) channel inhibition, experiments with ligand-based drug designs based on 1 and a docking study were conducted. Replacement of the terminal p-fluorophenyl group with a cyclopropylmethoxy group, methyl group introduction on the benzylic carbon at the 3-position of the quinoline core, and employment of a [2-(acetylamino)ethyl]amino group as the amine portion eliminated hERG K(+) channel inhibitory activity in a patch-clamp study, leading to the discovery of N-{3-[(1R)-1-{[2-(acetylamino)ethyl]amino}ethyl]-8-methylquinolin-7-yl}-4-(cyclopropylmethoxy)benzamide (R)-10h. The compound (R)-10h showed potent inhibitory activity against hMCHR1 and dose-dependently suppressed food intake in a 2-day study on DIO-F344 rats. Furthermore, practical chiral synthesis of (R)-10h was performed to determine the molecule's absolute configuration.

Melanin-concentrating hormone receptor 1 antagonists. Synthesis and structure-activity relationships of novel 3-(aminomethyl)quinolines.

It was found that 3-(aminomethyl)quinoline derivatives showed high binding affinities for melanin-concentrating hormone receptor 1 (MCHR1) with reduced affinity for serotonin receptor 2c (5-HT2c) when the dihydronaphthalene nucleus of compound 1 (human MCHR1, IC(50) = 1.9 nM; human 5-HT2c receptor, IC(50) = 0.53 nM) was replaced by other bicyclic core scaffolds. Among the synthesized compounds, 8-methylquinoline derivative 5v especially showed high binding affinity (IC(50) = 0.54 nM), potent in vitro antagonistic activity (IC(50) = 2.8 nM) for MCHR1, and negligible affinity for 5-HT2c receptor (IC(50) > 1000 nM). Oral administration of 5v significantly and dose-dependently suppressed nocturnal food intake in diet-induced obese rats and did not affect food intake in MCHR1-deficient mice. These results and rat pharmacokinetic study findings suggested that compound 5v is a highly potent, orally bioavailable, and centrally acting nonpeptide MCHR1 antagonist.

Melanin-concentrating hormone receptor 1 antagonists: synthesis, structure-activity relationship, docking studies, and biological evaluation of 2,3,4,5-tetrahydro-1H-3-benzazepine derivatives.

Melanin-concentrating hormone receptor 1 (MCHR1) antagonists have been studied as potential agents for the treatment of obesity. Initial structure-activity relationship studies of in-house hit compound 1a and subsequent optimization studies resulted in the identification of tetrahydroisoquinoline derivative 23, 1-(2-acetyl-1,2,3,4-tetrahydroisoquinolin-7-yl)-4-[4-(4-chlorophenyl)piperidin-1-yl]butan-1-one, as a potent hMCHR1 antagonist. A homology model of hMCHR1 suggests that these compounds interact with Asn 294 and Asp 123 in the binding site of hMCHR1 to enhance binding affinity. Oral administration of compound 23 dose-dependently reduced food intake in diet-induced obesity (DIO)-F344 rats.

Discovery, synthesis, and structure-activity relationship of 6-aminomethyl-7,8-dihydronaphthalenes as human melanin-concentrating hormone receptor 1 antagonists.

Human melanin-concentrating hormone receptor 1 (hMCHR1) antagonists are promising targets for obesity treatment. We identified the tetrahydronaphthalene derivative 1a with modest binding affinity for hMCHR1 by screening an in-house G protein-coupled receptor (GPCR) ligand library. We synthesized a series of 6-aminomethyl-5,6,7,8-tetrahydronaphthalenes and evaluated their activity as hMCHR1 antagonists. Modification of the biphenylcarbonylamino group revealed that the biphenyl moiety played a crucial role in the interaction of the antagonist with the receptor. The stereoselective effect of the chiral center on binding affinity generated the novel 6-aminomethyl-7,8-dihydronaphthalene scaffold without a chiral center. Optimization of the amino group led to the identification of a potent antagonist 2s (4'-fluoro-N-[6-(1-pyrrolidinylmethyl)-7,8-dihydro-2-naphthalenyl][1,1'-biphenyl]-4-carboxamide), which significantly inhibited the nocturnal food intake in rats after oral administration. Pharmacokinetic analysis confirmed that 2s had good oral bioavailability and brain penetrance. This antagonist appears to be a viable lead compound that can be used to develop a promising therapy for obesity.

Changes in glycated haemoglobin levels in diabetic rats measured with an automatic affinity HPLC.

1. The level of glycated haemoglobin (GHb) in diabetic rats was measured using a newly developed automatic high-performance liquid chromatography (HPLC) with a boronate affinity column that requires only 2.5 min per sample for analysis. 2. Levels of GHb were 2.7% in normal 7-week-old Sprague-Dawley rats. These levels increased gradually following the abrupt induction of hyperglycaemia by intravenous injection of streptozotocin (STZ), reaching a maximal level of 10.1% after 6 weeks. 3. Glycosylated haemoglobin (HbA1) levels measured by cation-exchange chromatography were also increased by STZ treatment, although HbA1 values were lower than GHb measured by affinity column HPLC. 4. In Wistar fatty rats, GHb levels declined gradually over 5 weeks following the administration of pioglitazone (0.75 or 2.25 mg/kg per day) as a food admixture, which reduced plasma glucose (PG) levels to normal levels within 1 week. Glycated haemoglobin levels after 5 weeks treatment with pioglitazone correlated better with the area under the curve for PG over the entire 5 week treatment period than with the PG level at the end of treatment. 5. In addition, GHb determined by affinity column HPLC correlated well with HbA1 measured by cation-exchange chromatography, although the GHb value was higher than the HbA1 value. 6. Glycated haemoglobin levels in db/db and KKAy mice were higher than those in control normoglycaemic animals and were also higher than HbA1 values measured by the cation-exchange method, although the two values did show good correlation. 7. These results indicate that the newly developed affinity column HPLC system is useful for evaluating total GHb levels in rats as an index of antidiabetic treatment.

[Ameliorative effects on retinal disorder in diabetic SHRSP (stroke-prone spontaneously hypertensive rat)].

The results of the EUCLID highlighted the importance of the renin-angiotensin system in the pathogenesis of diabetic retinopathy. We aimed to evaluate the effectiveness of candesartan cilexetil(TCV-116), a potent angiotensin II receptor antagonist, in ameliorating retinal disorders in stroke-prone spontaneously hypertensive rats(SHRSP) with storeptozotocin(STZ)-induced diabetes. Retinal VEGF mRNA expression was significantly higher and the latencies of oscillatory potentials were significantly elongated in STZ-treated SHRSP compared with a non-treated SHRSP group matched for age. Treatment with TCV-116(3 mg/kg) significantly diminished retinal VEGF mRNA expression and the latencies of oscillatory potentials, but had no effect on plasma glucose concentrations. These results suggest that TCV-116 is effective in preventing the development of diabetic retinopathy already in the early stages.