Keiji Miyata

Effect of (R)-2-(2-Aminothiazol-4-yl)-4′-{2-[(2-hydroxy-2-phenylethyl)amino]ethyl} Acetanilide (YM178), a Novel Selective β3-Adrenoceptor Agonist, on Bladder Function

We evaluated the pharmacological characteristics of (R)-2-(2-aminothiazol-4-yl)-4′-{2-[(2-hydroxy-2-phenylethyl)amino]-ethyl} acetanilide (YM178). YM178 increased cyclic AMP accumulation in Chinese hamster ovary (CHO) cells expressing human β3-adrenoceptor (AR). The half-maximal effective concentration (EC50) value was 22.4 nM. EC50 values of YM178 for human β1- and β2-ARs were 10,000 nM or more, respectively. The ratio of intrinsic activities of YM178 versus maximal response induced by isoproterenol (nonselective β-AR agonist) was 0.8 for human β3-ARs, 0.1 for human β1-ARs, and 0.1 for human β2-ARs. The relaxant effects of YM178 were evaluated in rats and humans bladder strips precontracted with carbachol (CCh) and compared with those of isoproterenol and 4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one hydrochloride (CGP-12177A) (β3-AR agonist). EC50 values of YM178 and isoproterenol in rat bladder strips precontracted with 10–6 M CCh were 5.1 and 1.4 μM, respectively, whereas those in human bladder strips precontracted with 10–7 M CCh were 0.78 and 0.28 μM, respectively. In in vivo study, YM178 at a dose of 3 mg/kg i.v. decreased the frequency of rhythmic bladder contraction induced by intravesical filling with saline without suppressing its amplitude in anesthetized rats. These findings suggest the suitability of YM178 as a therapeutic drug for the treatment of symptoms of overactive bladder such as urinary frequency, urgency, and urge incontinence.

Effect of YM178, a Novel Selective β3-Adrenoceptor Agonist, on Bladder Function

We evaluated the pharmacological characteristics of (R)-2-(2-aminothiazol-4-yl)-4′-{2-[(2-hydroxy-2-phenylethyl)amino]-ethyl} acetanilide (YM178). YM178 increased cyclic AMP accumulation in Chinese hamster ovary (CHO) cells expressing human β3-adrenoceptor (AR). The half-maximal effective concentration (EC50) value was 22.4 nM. EC50 values of YM178 for human β1- and β2-ARs were 10,000 nM or more, respectively. The ratio of intrinsic activities of YM178 versus maximal response induced by isoproterenol (nonselective β-AR agonist) was 0.8 for human β3-ARs, 0.1 for human β1-ARs, and 0.1 for human β2-ARs. The relaxant effects of YM178 were evaluated in rats and humans bladder strips precontracted with carbachol (CCh) and compared with those of isoproterenol and 4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one hydrochloride (CGP-12177A) (β3-AR agonist). EC50 values of YM178 and isoproterenol in rat bladder strips precontracted with 10–6 M CCh were 5.1 and 1.4 μM, respectively, whereas those in human bladder strips precontracted with 10–7 M CCh were 0.78 and 0.28 μM, respectively. In in vivo study, YM178 at a dose of 3 mg/kg i.v. decreased the frequency of rhythmic bladder contraction induced by intravesical filling with saline without suppressing its amplitude in anesthetized rats. These findings suggest the suitability of YM178 as a therapeutic drug for the treatment of symptoms of overactive bladder such as urinary frequency, urgency, and urge incontinence.

Bone regeneration by recombinant human bone morphogenetic protein-2 and a novel biodegradable carrier in a rabbit ulnar defect model

The effects of recombinant human bone morphogenetic protein (rhBMP)-2 and a novel carrier, PLGA-coated gelatin sponge (PGS), on bone defect repair was examined. A 1.5 cm unilateral segmental bone defect was created in the ulnar diaphysis of a Japanese white rabbit. In an initial study, defects were either treated with PGS impregnated with various concentrations of rhBMP-2 (0, 0.1, 0.4 and 1 mg/cm(3)) or left untreated. Defect healing was assessed by radiographic union rate, and biomechanical properties of regenerated bones were determined at 16 weeks postoperatively. In a second study, defects were implanted with PGS with or without rhBMP-2, and histologically observed at postoperative weeks 8 and 16. Radiographic union rate increased the dose-dependently at an early time point. All defects treated with rhBMP-2 (0.4 and 1 mg/cm(3)) were radiographically repaired. Mechanical properties of regenerated bones were restored in a dose-dependent manner. Neither ulnae left untreated nor implanted PGS alone showed radiographic union. Longitudinal alignment of lamellar structure was observed histologically at 16 weeks, indicating that remodeling of regenerated bone was complete. Implanted PGS was almost completely resorbed by 8 weeks, and no abnormalities were observed in the surrounding soft tissue. These results suggest that PGS is a promising carrier for rhBMP-2.

Compounds possessing 5-HT3 receptor antagonistic activity inhibit intestinal propulsion in mice

The role of 5-HT3 receptors in the control of intestinal propulsive activity was investigated in mice by a simple method in which the time taken for excretion of the head of an orally administered non-absorbable marker (whole gut transit time) was measured. Selective 5-HT3 receptor antagonists ramosetron (YM060) at 0.01-0.3 mg/kg s. c. and ondansetron at 0.1-1 mg/kg s.c. dose-dependently prolonged the whole gut transit time. Prokinetic benzamides, such as renzapride (0.3-10 mg/kg s.c.), zacopride (0.01-0.3 mg/kg s.c.) and cisapride (0.1-3 mg/kg s.c.), which have been reported to possess 5-HT3 receptor blocking properties, also dose-dependently prolonged it. These results indicate that activation of 5-HT3 receptors seems to be one factor that underlies the physiological control of intestinal propulsive activity in mice. In contrast to their beneficial therapeutic effects on gastroduodenal dysmotility, prokinetic benzamides, at least those which have 5-HT3 receptor antagonistic activity, may be unsuitable in the treatment of impaired lower intestinal propulsive activity.

Muscarinic receptor binding, plasma concentration and inhibition of salivation after oral administration of a novel antimuscarinic agent, solifenacin succinate in mice.

1 A novel muscarinic receptor antagonist, solifenacin succinate, inhibited specific binding of [N‐methyl‐3H]‐scopolamine ([3H]‐NMS) in the mouse bladder, submaxillary gland and heart in a concentration‐dependent manner. This inhibitory effect was greatest in the submaxillary gland, followed by the bladder and heart. 2 After oral administration of oxybutynin (76.1 μmol kg−1) or solifenacin (62.4, 208 μmol kg−1), a significant dose‐ and time‐dependent increase in KD values for specific [3H]‐NMS binding was seen in the bladder, prostate, submaxillary gland, heart, colon and lung, compared with control values. The increase in KD induced by oxybutynin in each tissue reached a maximum 0.5 h after oral administration and then rapidly declined, while that induced by solifenacin was greatest 2 h after administration and was maintained for at least 6 or 12 h, depending on the dose. The muscarinic receptor binding of oral solifenacin was slower in onset and of a longer duration than that of oxybutynin. 3 Plasma concentrations of oxybutynin and its active metabolite (N‐desethyl‐oxybutynin, DEOB) were maximum 0.5 h after its oral administration and then declined rapidly. Oral solifenacin persisted in the blood for longer than oxybutynin. 4 Pilocarpine‐induced salivary secretion in mice was significantly reduced by oral administration of solifenacin and was completely abolished 0.5 h after oral oxybutynin. Although the suppression induced by solifenacin was more persistent than that due to oxybutynin, the antagonistic effect of solifenacin on the dose–response curves to pilocarpine was significantly weaker than that of oxybutynin. It is concluded that oral solifenacin persistently binds to muscarinic receptors in tissues expressing the M3 subtype, such as the bladder.

Mechanisms of cisplatin- and m-chlorophenylbiguinide-induced emesis in ferrets

We investigated the involvement of peripheral and central serotonin (5-HT)3 receptors in cisplatin- and 5-HT3 receptor agonist-induced emesis in ferrets. Cisplatin (10 mg/kg i.v.)-induced emesis was inhibited by intravenous YM060 (0.003-0.1 microgram/kg). A highly selective and potent 5-HT3 receptor agonist, m-chlorophenylbiguanide (1-10 mg/kg i.p.), dose dependently elicited emesis an effect which was inhibited by YM060 (0.003-0.3 microgram/kg i.v.). Vagotomy markedly reduced this emesis, and the combination of abdominal vagotomy and greater splanchnicectomy abolished emesis. Lesion of greater splanchnic nerves alone did not markedly inhibit emesis. Intracerebroventricularly (4th ventricle) administered YM060 inhibited cisplatin- and m-chlorophenylbiguanide-induced emesis only at higher doses (0.01-0.1 and 0.01-0.03 microgram, respectively). Intracerebroventricularly (4th ventricle) administered m-chlorophenylbiguanide (30-100 micrograms) produced only a weak retching response. These results indicate that stimulation of abdominal vagal afferent nerves via peripheral 5-HT3 receptors is important for triggering cisplatin- and m-chlorophenylbiguanide-induced emesis in ferrets.

Agonist diversity in 5-HT2C receptor-mediated weight control in rats

RationaleFood intake and energy expenditure are the two main determinants of body weight. Given that 5-HT2C receptor agonists are reported to have effects on both energy expenditure and food intake, this strongly suggests that 5-HT2C receptor agonists have excellent potential for development as antiobesitiy drugs. One important issue in antiobesity drug development is whether the effects of the compound are maintained during chronic drug treatment.ObjectivesThe purpose of the present study was to investigate the effect of repeated oral administration of three 5-HT2C receptor agonists, m-chlorophenylpiperazine (mCPP), d(S)-2-(6-chloro-5-fluoroindol-1-yl)-1-methylethylamine (RO60-0175) and (S)-2-(7-ethyl-1H-furo[2,3-g]indazol-1-yl)-1-methylethylamine (YM348), on food intake and energy expenditure in rats.ResultsIn the food intake study, mCPP, RO60-0175 and YM348 decreased food intake in a dose-dependent manner on day 1 of administration. On day 14 of repeated administration, the hypophagic effect of YM348 was lost and that of mCPP was reduced. In contrast, the hypophagic effect of RO60-0175 was maintained even after repeated administration. The hypophagic effects of all agonists were significantly inhibited by a 5-HT2C receptor antagonist, SB242084. In contrast to the hypophagic effects, no drug tolerance developed with respect to the hyperthermic effects of mCPP, RO60-0175, and YM348. The hyperthermic effects of these drugs were also inhibited by SB242084.ConclusionsTogether, the difference between compounds in their hypophagic effects and the similarity in their hyperthermic effects suggest a diversity in agonists in 5-HT2C receptor-mediated weight control in rats.

Interleukin-11 induces osteoblast differentiation and acts synergistically with bone morphogenetic protein-2 in C3H10T1/2 cells

Interleukin-11 (IL-11) is a pleiotropic cytokine that supports various types of hematopoietic cell growth and is involved in bone resorption. We report here the involvement of recombinant human IL-11 (rHuIL-11) in osteoblast differentiation in mouse mesenchymal progenitor cells, C3H10T1/2. rHuIL-11 alone increased alkaline phosphatase (ALP) activity and upregulated expression levels of osteocalcin (OC), bone sialo protein (BSP), and parathyroid hormone receptor (PTHR) mRNA. rHuIL-11 had no effect on expression of type II collagen, peroxisome proliferator-activated receptor-gamma2 (PPAR-gamma2), adipocyte fatty acid-binding protein P2 (aP2), and myogenic MyoD protein (MyoD). Recombinant human bone morphogenetic protein (rHuBMP)-2 increased ALP activity and mRNA expression of these genes except for MyoD. The expression patterns of ALP activity and osteoblast-specific or chondrocyte-specific genes suggest that rHuIL-11 may be involved in early differentiation of osteoblasts at a step earlier than that which is affected by rHuBMP-2. In support of this hypothesis, combined treatment with rHuIL-11 and rHuBMP-2 synergistically increased ALP activity and mRNA expression of OC and type II collagen, rHuIL-11 also abrogated the increased levels of PPAR-gamma2, aP2 mRNA caused by rHuBMP-2. Our results suggest that rHuIL-11 alone and in combination with rHuBMP-2 can induce osteoblastic differentiation of progenitor cells and plays an important role in osteogenesis.

Effects of serotonin 5-HT3 receptor antagonists on stress-induced colonic hyperalgesia and diarrhoea in rats: a comparative study with opioid receptor agonists, a muscarinic receptor antagonist and a synthetic polymer.

Abstract  In this study, we examined the effects of serotonin (5‐HT)3 receptor antagonists (5‐HT3RAs) including ramosetron, alosetron, and cilansetron on colonic nociceptive threshold in rats. Furthermore, we established a restraint stress‐induced colonic hyperalgesia model in rats, and compared the inhibitory effects of 5‐HT3RAs on restraint stress‐induced colonic hyperalgesia and diarrhoea with those of loperamide, trimebutine, tiquizium and polycarbophil. The colonic nociceptive threshold was measured as the balloon pressure at the time the rat showed a nociceptive response during colonic distension by an intrarectally inserted balloon. Oral administration of ramosetron (3–30 μg kg−1), alosetron (30–300 μg kg−1), or cilansetron (30–300 μg kg−1) increased the colonic nociceptive threshold in a dose‐dependent manner in non‐stressed rats. Restraint stress for 1 h significantly decreased the colonic nociceptive threshold, but ramosetron (0.3–3 μg kg−1), alosetron (3–30 μg kg−1), cilansetron (3–30 μg kg−1) and trimebutine (100–1000 mg kg−1) significantly inhibited the decrease in the threshold. Loperamide (3–30 mg kg−1), tiquizium (100–1000 mg kg−1) and polycarbophil (1000 mg kg−1) did not affect the restraint stress‐induced decrease in the colonic nociceptive threshold. All drugs tested in this study showed dose‐dependent inhibition of restraint stress‐induced diarrhoea in rats. These results indicate that, unlike existing antidiarrhoeal and spasmolytic agents, 5‐HT3RAs have inhibitory effects on colonic nociception, and prevented restraint stress‐induced both diarrhoea and hyperalgesia at almost the same doses in rats. This suggests that the 5‐HT3RAs may be useful in ameliorating both colonic hyperalgesia and diarrhoea in patients with irritable bowel syndrome.

Involvement of the 5-HT3 receptor in CRH-induced defecation in rats

We evaluated the possibility that serotonin (5-HT) mediates defecation induced by corticotropin-releasing hormone (CRH) exogenously administered or released from the central nervous system by stress via the 5-HT3 receptor in rats. Intracerebroventricular (i.c.v.) injection of CRH (1, 3, and 10 micrograms/rat) dose dependently increased the number of stools excreted in rats, whereas intravenous (i.v.) injection of up to 100 micrograms/kg CRH did not affect defecation. alpha-Helical CRH-(9-41) and 5-HT3 receptor antagonists ramosetron and azasetron inhibited CRH (10 micrograms i.c.v.)-induced defecation in a dose-dependent manner with ED50 values of 4.3 micrograms/kg i.v., 3.8 micrograms/kg p.o., and 70.4 micrograms/kg p.o., respectively. alpha-Helical CRH-(9-41) also inhibited CRH-induced defecation by i.c.v. injection with an ED50 value of 0.078 microgram/rat. In contrast, ramosetron and azasetron injected i.c.v. had no effect on CRH-induced defecation. alpha-Helical CRH-(9-41), ramosetron, and azasetron reduced defecation caused by restraint stress with ED50 values of 0.32, 3.6, and 19.7 micrograms/kg i.v., respectively. These results indicate that CRH exogenously administered or released from the central nervous system by stress peripherally promotes the release of 5-HT, which in turn stimulates defecation through the 5-HT3 receptor.