RESUMO
We investigated the anxiolytic effects and mechanism of action of a new anxiolytic drug, (R)-piperonyl-1,2,3,4-tetrahydro[1]benzothieno[2,3-c]pyridine-3- carboxamide hydrochloride (AP521). AP521 showed equal or more potent anxiolytic-like effects compared with diazepam, a benzodiazepine receptor agonist, or tandospirone, a partial 5-hydroxytryptamine (5-HT)1A receptor agonist, in three rat anxiety models; the Vogel-type conflict test, elevated plus maze test, and conditioned fear stress test. Although AP521 did not bind to the benzodiazepine receptor, it did bind to 5-HT1A, 5-HT1B, 5-HT1D, 5-HT5A and 5-HT7 receptors, and showed agonist activity for the human 5-HT1A receptor expressed in HEK293 cells. Tandospirone, which can stimulate the presynaptic 5-HT1A receptors in the raphe, tended to decrease extracellular 5-HT concentration in the medial prefrontal cortex (mPFC) in rats. In contrast, AP521 increased extracellular 5-HT concentration. In addition, AP521 enhanced the anti-freezing effect of citalopram, a selective serotonin reuptake inhibitor, in the fear conditioning model in rats and enhanced the citalopram-caused increase of the extracellular 5-HT concentration in the mPFC. These results suggest that AP521 exhibits potent anxiolytic effects by acting as a postsynaptic 5-HT1A receptor agonist and by enhancing serotonergic neural transmission in the mPFC by a novel mechanism of action.
Assuntos
Ansiolíticos/farmacologia , Piridinas/farmacologia , Serotonina/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Tiofenos/farmacologia , Animais , Ansiolíticos/uso terapêutico , Ansiedade/tratamento farmacológico , Células Cultivadas , Citalopram/farmacologia , Diazepam/farmacologia , Modelos Animais de Doenças , Sinergismo Farmacológico , Humanos , Isoindóis/farmacologia , Masculino , Piperazinas/farmacologia , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Piridinas/uso terapêutico , Pirimidinas/farmacologia , Ratos , Receptores de Serotonina/metabolismo , Agonistas do Receptor 5-HT1 de Serotonina/farmacologia , Tiofenos/uso terapêuticoRESUMO
We have designed a series of potent EP1 receptor antagonists. These antagonists are a series of 2-(1H-indazol-1-yl)-thiazoles in which the core structure was replaced with pyrazole-phenyl groups. In preliminary conscious rat cystometry experiments, two representative candidates, 2 and 22, increased bladder capacity. In particular, the increase using 22 was approximately 2-fold that of the baseline. More detailed profiling of this compound and further optimization of this series promises to provide a novel class of drug for treating overactive bladder (OAB).
Assuntos
Indazóis/química , Receptores de Prostaglandina E Subtipo EP1/antagonistas & inibidores , Tiazóis/química , Animais , Avaliação Pré-Clínica de Medicamentos , Meia-Vida , Humanos , Ligação Proteica , Ratos , Receptores de Prostaglandina E Subtipo EP1/metabolismo , Relação Estrutura-Atividade , Tiazóis/farmacocinética , Tiazóis/farmacologia , Tiazóis/uso terapêutico , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária Hiperativa/tratamento farmacológicoRESUMO
We investigated the effects of partial bladder outlet obstruction (BOO) on the function and gene expression of 5-hydroxytryptamine (5-HT) receptor subtypes in rat bladder. Isometric contractions of the isolated bladders from sham-operated control and BOO rats were examined. The contractile responses to 5-HT were significantly increased in BOO rat bladder strips, while the responses to KCl, carbachol, or phenylephrine were not different from the control. The 5-HT-induced hypercontraction in BOO rat bladder strips was inhibited by ketanserin, a 5-HT(2A) receptor antagonist. The contractile responses to 5-HT in bladder strips were not affected by urothelium removal from the intact bladder. The gene expression of 5-HT receptor subtypes in the bladders was analyzed by RT-PCR. The mRNA expression of the 5-HT(2A), 5-HT(2B), 5-HT(2C), 5-HT(4), and 5-HT(7) receptors was detected in both the control and BOO rat bladders. Quantitative RT-PCR analysis showed there was a significant increase of 5-HT(2A) receptor mRNA in the BOO rat bladder compared with the control bladder. On the other hand, the gene expression of the 5-HT(4) receptor was not changed in the BOO rat bladder. These results suggest that the increased contractile responses to 5-HT in BOO rat bladder may be partly caused by 5-HT(2A) receptor upregulation in the detrusor smooth muscles.
Assuntos
Receptor 5-HT2A de Serotonina/fisiologia , Obstrução do Colo da Bexiga Urinária/fisiopatologia , Bexiga Urinária Hiperativa/fisiopatologia , Bexiga Urinária/fisiopatologia , Animais , Feminino , Ketanserina/farmacologia , Contração Muscular/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Receptor 5-HT2A de Serotonina/biossíntese , Receptores de Serotonina/biossíntese , Serotonina/farmacologiaRESUMO
We describe a medicinal chemistry approach for generating a series of 2-(1H-pyrazol-1-yl)thiazoles as EP1 receptor antagonists. To improve the physicochemical properties of compound 1, we investigated its structure-activity relationships (SAR). Optimization of this lead compound provided small compound 25 which exhibited the best EP1 receptor antagonist activity and a good SAR profile.
Assuntos
Pirazóis/química , Pirazóis/farmacologia , Receptores de Prostaglandina E Subtipo EP1/antagonistas & inibidores , Tiazóis/química , Tiazóis/farmacologia , Humanos , Ligação Proteica/efeitos dos fármacos , Pirazóis/síntese química , Pirazóis/metabolismo , Receptores de Prostaglandina E Subtipo EP1/metabolismo , Relação Estrutura-Atividade , Tiazóis/síntese química , Tiazóis/metabolismoRESUMO
We describe a medicinal chemistry approach to generate a series of 2-(1H-pyrazol-1-yl)thiazole compounds that act as selective EP1 receptor antagonists. The obtained results suggest that compound 12 provides the best EP1 receptor antagonist activity and demonstrates good oral pharmacokinetics.
Assuntos
Pirazóis/farmacologia , Receptores de Prostaglandina E Subtipo EP1/antagonistas & inibidores , Tiazóis/farmacologia , Animais , Humanos , Camundongos , Modelos Moleculares , Pirazóis/farmacocinética , Receptores de Prostaglandina E Subtipo EP1/metabolismo , Relação Estrutura-Atividade , Tiazóis/química , Tiazóis/farmacocinéticaRESUMO
OBJECTIVE: Naftopidil is used clinically for the treatment of voiding disorders in benign prostatic hyperplasia. Previous in vivo experiments in which naftopidil was applied intrathecally abolished rhythmic bladder contraction, suggesting that naftopidil might inhibit a voiding reflex through interaction with spinal dorsal horn neurons. Here we aimed to clarify the mechanism of action of naftopidil on dorsal horn neurons. METHODS: Whole-cell patch-clamp recordings were performed using substantia gelatinosa neurons of adult rat spinal cord slices. Miniature or evoked inhibitor and excitatory postsynaptic currents (IPSCs and EPSCs, respectively) were analyzed. RESULTS: Bath-applied naftopidil increased the frequency but not the amplitude of miniature IPSCs (mIPSCs) in 38% of neurons tested; in contrast, the effect of naftopidil on miniature EPSCs (mEPSCs) were mild and observed in only 2 out of 19 neurons. Naftopidil enhanced the amplitude of both GABAergic and glycinergic evoked-IPSCs (eIPSCs) that were elicited by focal stimuli in the presence of either the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), or the NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (APV). CONCLUSIONS: Although naftopidil was developed as an alpha-1 adrenoceptor antagonist, our previous spinal cord slice experiments showed that the activation of an alpha-1 adrenoceptor in substantia gelatinosa increases the frequency of mIPSCs. This result suggested that, under our conditions, naftopidil may interact with a receptor(s) other than an alpha-1 adrenoceptor in the spinal dorsal horn. The present results suggested that naftopidil enhances the release of GABA and glycine by activating inhibitory interneuron terminals in the spinal dorsal horn via a receptor other than an alpha-1 adrenoceptor, thereby modulating sensory transmission in the substantia gelatinosa.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Naftalenos/farmacologia , Neurônios/efeitos dos fármacos , Piperazinas/farmacologia , Substância Gelatinosa/citologia , Animais , Relação Dose-Resposta a Droga , Técnicas In Vitro , Masculino , Neurotransmissores/farmacologia , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Bloqueadores dos Canais de Sódio/farmacologia , Corno Dorsal da Medula Espinal/anatomia & histologia , Tetrodotoxina/farmacologiaRESUMO
OBJECTIVE: To investigate changes in expression and activity of monoamine oxidase A (MAO-A) in rats with partial bladder outlet obstruction (pBOO). Previous studies suggested that monoamines, such as serotonin (5-hydroxytryptamine [5-HT]) and noradrenaline, are involved in bladder hyperactivity secondary to pBOO. However, little is known about the role of MAO-A, an enzyme oxidizing 5-hydroxytryptamine and noradrenalin, in the pathogenesis. MATERIALS AND METHODS: Female Sprague Dawley rats were subjected to sham or pBOO operations for 7 days, then their bladders were isolated. MAO-A protein levels in the bladder were examined by Western blotting. MAO-A activity was measured by the commercially available MAO-Glo Assay kit. In addition, MAO-A distribution in the bladder was examined by immunohistochemistry. RESULTS: Weights of the bladders from rats with pBOO were increased about 3.5-fold, compared with those from sham rats. Significant decreases in MAO-A protein and activity levels were observed in whole bladder of rats with pBOO compared with those of sham rats. By immunohistochemistry, it was firstly demonstrated that MAO-A was predominantly expressed in the detrusor layer of the sham rat bladders. The intensity of staining was decreased after pBOO operation. CONCLUSION: We demonstrated, for the first time, the distribution of MAO-A in the bladder and the pathologic changes in MAO-A protein and activity levels in rats with pBOO. This marked decrease in MAO-A potentially resulting in increased monoamine levels, especially in the detrusor of rat bladder, might be a key factor explaining the mechanism of bladder overactivity associated with pBOO.
Assuntos
Monoaminoxidase/biossíntese , Obstrução do Colo da Bexiga Urinária/enzimologia , Bexiga Urinária/enzimologia , Animais , Feminino , Ratos , Ratos Sprague-DawleyRESUMO
We previously discovered that indazole derivative 8 was a highly selective ß3-adrenergic receptor (ß3-AR) agonist, but it appeared to be metabolically unstable. To improve metabolic stability, further optimization of this scaffold was carried out. We focused on the sulfonamide moiety of this scaffold, which resulted in the discovery of compound 15 as a highly potent ß3-AR agonist (EC50 = 18 nM) being inactive to ß1-, ß2-, and α1A-AR (ß1/ß3, ß2/ß3, and α1A/ß3 > 556-fold). Compound 15 showed dose-dependent ß3-AR-mediated responses in marmoset urinary bladder smooth muscle, had a desirable metabolic stability and pharmacokinetic profile (Cmax and AUC), and did not obviously affect heart rate or mean blood pressure when administered intravenously (3 mg/kg) to anesthetized rats. Thus, compound 15 is a highly potent, selective, and orally available ß3-AR agonist, which may serve as a candidate drug for the treatment of overactive bladder without off-target-based cardiovascular side effects.
Assuntos
Agonistas de Receptores Adrenérgicos beta 3/farmacologia , Sistema Cardiovascular/efeitos dos fármacos , Indazóis/farmacologia , Administração Oral , Agonistas de Receptores Adrenérgicos beta 3/efeitos adversos , Agonistas de Receptores Adrenérgicos beta 3/química , Animais , Cães , Descoberta de Drogas , Humanos , Indazóis/efeitos adversos , Indazóis/química , RatosRESUMO
AIMS: For patients with benign prostatic hyperplasia (BPH), storage symptoms due to bladder dysfunction are bothersome, and that mechanism elucidation is needed. Piezo1, a mechanically activated ion channel, is believed to play a role in sensing bladder distension. To investigate the involvement of Piezo1 in bladder dysfunction, we examined the expression and distribution of Piezo1 and neurofilament (NF-L) to understand pathological alterations in rat bladders with partial bladder outlet obstruction (pBOO), an animal model of BPH. MAIN METHODS: Female Sprague-Dawley rats were subjected to sham or pBOO operations. On days 3, 7, and 14 after pBOO, Piezo1 mRNA levels in the bladder were examined by quantitative real-time PCR. The expression of light NF-L was also examined by western blotting. On day 7, the distributions of Piezo1 were examined by in situ hybridization. KEY FINDINGS: The expression levels of Piezo1 mRNA in whole bladder were significantly increased from days 3 to 14 after pBOO. On day 7 in pBOO rats, significant increases in Piezo1 mRNA were observed in the detrusor layer as well as the suburothelial layer, while the predominant distribution was observed in the urothelium of sham rats. Coinciding with the increase in Piezo1, the decreases in NF-L expression were observed in the bladder from pBOO rats. SIGNIFICANCE: The increase in Piezo1 in pBOO rat bladders might be involved in the compensatory mechanism associated with bladder denervation including the decrease in NF-L. Inhibition of Piezo-1 may be a new therapeutic approach to ameliorate the storage dysfunction shown in pBOO.
Assuntos
Proteínas de Membrana/genética , Regulação para Cima , Obstrução do Colo da Bexiga Urinária/genética , Bexiga Urinária/patologia , Animais , Feminino , Proteínas de Membrana/análise , RNA Mensageiro/genética , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Bexiga Urinária/metabolismo , Obstrução do Colo da Bexiga Urinária/patologiaRESUMO
INTRODUCTION: Benign prostatic hyperplasia causes partial bladder outlet obstruction (pBOO), and many patients with pBOO are affected by not only voiding symptoms but also storage symptoms. We previously suggested that enhancement of 5-hydroxytryptamine (5-HT)-induced bladder contraction in the pBOO bladder may be one cause of storage symptoms. However, little is known about the presence of 5-HT in rat bladders. In this study, we hypothesized that mast cells are a source of 5-HT and investigated the distribution of mast cells and 5-HT in the bladders of rats with pBOO. METHODS: The bladders of female Sprague-Dawley rats were subjected to pBOO and sham operations for 1 week, were isolated, and were fixed for light or electron microscopy. Mast cells and 5-HT in the bladders were detected by toluidine blue staining and immunohistochemical staining, respectively. The mast cells were counted under a light microscope. Degranulated mast cells were observed under an electron microscope and counted under a light microscope. RESULTS: Mast cells were present in the mucosa/submucosa region in sham rat bladders. Their number was increased in the detrusor muscle/subserosa/serosa region, especially the subserosal layer, in pBOO rat bladders. The localization of mast cells almost matched that of 5-HT-positive cells in consecutive sections. Degranulated mast cells were present in sham and pBOO rat bladders, but the proportion of degranulated mast cells was significantly increased in every region in pBOO rat bladders compared with that in sham rat bladders. CONCLUSION: These results suggest that mast cells contain 5-HT and are more abundant locally in the subserosal layer of pBOO rat bladders. 5-HT released from mast cells could stimulate 5-HT2 receptors on the detrusor muscle, and this may underlie storage symptoms. FUNDING: Asahi Kasei Pharma Corp.
Assuntos
Degranulação Celular , Sintomas do Trato Urinário Inferior/diagnóstico , Mastócitos/fisiologia , Obstrução do Colo da Bexiga Urinária , Bexiga Urinária/patologia , Animais , Modelos Animais de Doenças , Feminino , Imuno-Histoquímica , Ratos , Ratos Sprague-Dawley , Obstrução do Colo da Bexiga Urinária/etiologia , Obstrução do Colo da Bexiga Urinária/patologia , Obstrução do Colo da Bexiga Urinária/fisiopatologiaRESUMO
Serotonin (5-hydroxytryptamine; 5-HT)-induced bladder contraction is enhanced after partial bladder outlet obstruction (pBOO) in rats. We investigated time-dependent changes in bladder contraction and expression of 5-HT(2A) and 5-HT(2B) receptor mRNA in bladder tissue to elucidate the mechanism of this enhancement. On day 3 and 7 after pBOO, contractile responses of isolated rat bladder strips to 5-HT were increased compared with that in sham-operated rats; on day 14, the response had decreased to the same level as that in sham rat bladders. In contrast, carbacholinduced contraction was not enhanced by pBOO at any time point. In sham rats, 5-HT(2A) receptor mRNA was expressed in the urothelium, and 5-HT(2B) receptor mRNA was expressed in the detrusor muscle layer. In pBOO rats, both receptor mRNAs were increased in the detrusor muscle and subserosal layers, but not in the urothelium. The increase of 5-HT(2A) receptor mRNA was maintained from day 3 to day 14 after pBOO, and 5-HT(2B) receptor mRNA was increased on day 7 after pBOO. These results suggested that pBOO induced up-regulation of the 5-HT(2A) and 5-HT(2B) receptors in the detrusor muscle and subserosal layers of the bladder, and such up-regulation may be related to the enhanced bladder contractile response to 5-HT.
Assuntos
Expressão Gênica , Músculo Liso/metabolismo , Receptor 5-HT2A de Serotonina/genética , Receptor 5-HT2B de Serotonina/genética , Obstrução do Colo da Bexiga Urinária/genética , Animais , Modelos Animais de Doenças , Feminino , Hibridização In Situ , Contração Muscular/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Receptor 5-HT2A de Serotonina/metabolismo , Receptor 5-HT2B de Serotonina/metabolismo , Serotonina/farmacologia , Obstrução do Colo da Bexiga Urinária/metabolismoRESUMO
Novel indazole derivatives were prepared and evaluated for their biological activity and cardiovascular safety profile as human ß3-adrenergic receptor (AR) agonists. Although the initial hit compound 5 exhibited significant ß3-AR agonistic activity (EC50 = 21 nM), it also exhibited agonistic activity at the α1A-AR (EC50 = 219 nM, selectivity: α1A/ß3 = 10-fold). The major metabolite of 5, which was an oxidative product at the indazole 3-methyl moiety, gave a clue to a strategy for improvement of the selectivity for ß3-AR agonistic activity versus α1A-AR agonistic activity. Thus, modification of the 3-substituent of the indazole moiety effectively improved the selectivity to develop compound 11 with potent ß3-AR agonistic activity (EC50 = 13 nM) and high selectivity (α1A/ß3 = >769-fold). Compound 11 was also inactive toward ß1 and ß2-ARs and showed dose dependent ß3-AR mediated relaxation of marmoset urinary bladder smooth muscle, while it did not obviously affect heart rate or blood pressure (iv, 3 mg/kg) in anesthetized rats.
Assuntos
Agonistas de Receptores Adrenérgicos beta 3/química , Agonistas de Receptores Adrenérgicos beta 3/farmacologia , Indazóis/química , Indazóis/farmacologia , Agonistas de Receptores Adrenérgicos beta 3/efeitos adversos , Animais , Sistema Cardiovascular/efeitos dos fármacos , Humanos , Indazóis/efeitos adversos , Ratos , Relação Estrutura-AtividadeRESUMO
Naftopidil is an α(1D) and α(1A) subtype-selective α(1)-adrenoceptor antagonist that has been used to treat lower urinary tract symptoms of benign prostatic hyperplasia. In this study, we investigated the effects of naftopidil on 5-hydroxytryptamine (5-HT)-induced rat bladder contraction (10(-8)-10(-4) M). Naftopidil (0.3, 1, and 3 µM) inhibited 5-HT-induced bladder contraction in a concentration-dependent manner. On the other hand, other α(1)-adrenoceptor antagonists, tamsulosin, silodosin or prazosin, did not inhibit 5-HT-induced bladder contraction. The 5-HT-induced bladder contraction was inhibited by both ketanserin and 4-(4-fluoronaphthalen-1-yl)-6-propan-2-ylpyrimidin-2-amine (RS127445), serotonin 5-HT(2A) and 5-HT(2B) receptor antagonists, respectively. In addition, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) and α-methyl-5-HT, 5-HT(2A) and 5-HT(2) receptor agonists, respectively, induced bladder contraction. The 5-HT-induced bladder contraction was not inhibited by N-[2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl]-N-pyridin-2-yl-cyclohexanecarboxamide (WAY-100635), [1-[2[(methylsulfonyl)amino]ethyl]-4-piperidinyl]methyl-1-methyl-1H-indole-3-carboxylate (GR113808) or (R)-3-[2-[2-(4-methylpiperidin-1-yl)ethyl]pyrrolidine-1-sulphonyl]phenol (SB269970), 5-HT(1A), 5-HT(4) and 5-HT(7) receptor antagonists, respectively. Naftopidil inhibited both the 5-HT(2A) and 5-HT(2) receptor agonists-induced bladder contractions. Naftopidil binds to the human 5-HT(2A) and 5-HT(2B) receptors with pKi values of 6.55 and 7.82, respectively. These results suggest that naftopidil inhibits 5-HT-induced bladder contraction via blockade of the 5-HT(2A) and 5-HT(2B) receptors in rats. Furthermore, 5-HT-induced bladder contraction was enhanced in bladder strips obtained from bladder outlet obstructed rats, with this contraction inhibited by naftopidil. The beneficial effects of naftopidil on storage symptoms such as urinary frequency and nocturia in patients with benign prostatic hyperplasia may be due, in part, to the blockade of the 5-HT(2A) and 5-HT(2B) receptors in the bladder.