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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.

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(1 H )-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(1 H )-ones bearing various less basic bicyclic motifs. Among these, a lead compound 6a bearing a 1 H -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 CF 3 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-1 H -benzimidazol-6-yl)pyridin-2(1 H )-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.

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.

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.

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.

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.

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.

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.

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.

近年，糖尿病，高血圧，高脂血症，肥満など，いわゆる生活習慣病が社会問題となっているが，これらの疾患は動脈硬化を引き起こし，脳卒中，心筋梗塞，神経障害，腎障害，網膜症などの血管合併症につながっていくため，生活習慣病さらにそれに引き続いて起こる合併症を改善する治療薬の要望は年々高まっている．本稿では，選択交配により作出された脳卒中易発症系高血圧自然発症ラット（SHRSP）を用いて，その脳血管に対する脆弱性という特徴を生かし，血管障害合併症の中で脳卒中後遺症および糖尿病網膜症に対する治療薬評価系を確立した．SHRSPに1%食塩水を飲用水として与えることによって，脳卒中発症期間の短縮，初発発作重篤度の均一化をはかり，さらに初発発作後に飲用水を水道水に戻すことにより，再発作や慢性的神経症状などの後遺症が認められる脳卒中後遺症モデルを作成した．後遺症評価として，神経症状のスコア化やMRIによる画像診断により，薬剤の治療効果が評価可能である．また，脳血管が脆弱なSHRSPの特徴から，脳血管と構造が類似する網膜血管の脆弱性が想定されたため，ストレプトゾトシン投与によって高血糖を惹起させて，血糖依存的な網膜障害を発症する糖尿病網膜症モデルを作成した．本モデルにおいて，動物モデルでは発現が難しかった網膜症につながる網膜変化（VEGF mRNA発現亢進と律動様小波の潜時延長）が認められ，これらの指標を用いて薬剤の治療効果を明らかにした．このように選択交配により確立された自然発症病態モデル動物の発現している特徴を生かすことにより，さらに有用な疾患モデルを作出することが可能である．

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.

We investigated the effects of the combination of idebenone, an energy metabolism enhancer, and manidipine 2HCl, a dihydropyridine-derivative calcium antagonist, on neurological deficits and histological changes in the brain and kidneys of stroke-prone spontaneously hypertensive rats (SHRSP) with cerebrovascular lesions (stroke). The SHRSP were kept on a 1% NaCl solution as their drinking water to synchronize the onset of stroke. After the onset of stroke symptoms, the salt solution was replaced with tap water. On the day following the onset of stroke, idebenone (50 mg/kg), manidipine 2HCl (2 mg/kg) or a combination of idebenone (50 mg/kg) and manidipine 2HCl (2 mg/kg) was administered orally once a day for 3 weeks. In the combination group and manidipine 2HCl-treated group, the neurological deficits after the onset of stroke were ameliorated during the entire experimentalperiod. Especially, the combination significantly decreased the number of days with severe neurological deficits as compared to the control group. The combination and manidipine 2HCl significantly recovered the decrease in body weight and ameliorated the increase of brain weight, which was mainly caused by edema, significantly as compared to the control group. Manidipine 2HCl ameliorated the histological changes in the brain. In the combination group, the histological changes in both the brain and the kidneys were ameliorated. In conclusion, the combination of idebenone and manidipine 2HCl significantly ameliorated the neurological deficits and the histological changes in the brain and the kidney of SHRSP with stroke as compared to each individual treatment. We concluded that manidipine 2HCl enhances the therapeutic effect of idebenone in the treatment of cerebrovascular diseases.

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 (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 Asn294 and Asp123 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.

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.

The effects of TDN-345 on mortality and ischemic neurological deficit following transient global cerebral ischemia in Mongolian gerbils and also the rate of local cerebral glucose utilization (LCGU) in stroke-prone spontaneously hypertensive rats (SHRSP) with cerebrovascular lesions were investigated. In Mongolian gerbils, ischemia was produced by clamping the bilateral common carotid arteries for 15 min. TDN-345 (0.1-1.0 mg/kg) dose-dependently decreased the mortality and ischemic neurological deficit score when administered orally twice, 60 min before ischemia and 90 min after recirculation. Additionally, TDN-345 (0.2 or 1.0 mg/kg, p.o. once daily for 3 weeks after the onset of stroke) decreased the mortality and recurrence of stroke in SHRSP. To determine the site of action of TDN-345 in the brain, the rate of LCGU in various brain regions in SHRSP with stroke was examined using a [14C]2-deoxy-D-glucose method. The rate of LCGU decreased significantly in all the brain regions in SHRSP with stroke compared with Wistar-Kyoto (WKY) control rats, whereas the reduction in the rate of LCGU in SHRSP with stroke was prevented by TDN-345 treatment, especially in the sensorimotor cortex and locus coeruleus. These results suggest that TDN-345 has therapeutic efficacy in the treatment of cerebrovascular disease.

Ordered neuronal connections in mature brains are thought to be sculpted from initially diffuse projections by elimination of inappropriate projections and strengthening of appropriate ones. Although evidence suggests that neuronal activity plays a role in these processes, the mechanism behind the modification of neuronal connections remains obscure. To gain insight into the mechanisms of axonal elimination and projection strengthening, we examined the morphology of individual axons that were to be eliminated as well as the consequences of partial denervation. While corticorubral projections in adult cats are thought to be uncrossed, early in postnatal development and after early unilateral lesions to the sensorimotor cortex, however, a significant amount of crossed corticorubral projections occurs. We examined the morphology of individual corticorubral axons in fetal cats and kittens from embryonic day 59 to postnatal day 48 and those that had received early unilateral lesions to the cortex, by serial reconstruction of Phaseolus-vulgaris-leucoagglutinin- or biocytin-labeled axons. For about 2 weeks during pre- and postnatal development, crossed axons remained simple in morphology, with few branches. Thereafter, they showed an increase in branch number, but then began to show fewer branches again. Axons and their collaterals were found in nonrestricted areas of the red nucleus (RN) throughout the period of observation, indicating that axons can sit at an inappropriate target for weeks but fail to ramify. In contrast, crossed corticorubral axons in kittens with cortical lesions showed terminal-arbor-like structures in the RN region that are in mirror symmetry to topographically appropriate areas in the ipsilateral RN, although some showed simple morphology without arbors. These complicated forms of morphology of individual axons during development and after partial denervation may not be explained by a simple activity-dependent mechanism.