RESUMEN

Activation of anaplastic lymphoma receptor tyrosine kinase (ALK) is involved in the pathogenesis of several carcinomas, including non-small cell lung cancer (NSCLC). Echinoderm microtubule-associated protein like 4 (EML4)-ALK, which is derived from the rearrangement of ALK and EML4 genes, has been validated as a therapeutic target in a subset of patients with NSCLC. Here, we investigated the effects of ASP3026, a novel small-molecule ALK inhibitor, against ALK-driven NSCLC. ASP3026 inhibited ALK activity in an ATP-competitive manner and had an inhibitory spectrum that differed from that of crizotinib, a dual ALK/MET inhibitor. In mice xenografted with NCI-H2228 cells expressing EML4-ALK, orally administered ASP3026 was well absorbed in tumor tissues, reaching concentrations >10-fold higher than those in plasma, and induced tumor regression with a wide therapeutic margin between efficacious and toxic doses. In the same mouse model, ASP3026 enhanced the antitumor activities of paclitaxel and pemetrexed without affecting body weight. ASP3026 also showed potent antitumor activities, including tumor shrinkage to a nondetectable level, in hEML4-ALK transgenic mice and prolonged survival in mice with intrapleural NCI-H2228 xenografts. In an intrahepatic xenograft model using NCI-H2228 cells, ASP3026 induced continuous tumor regression, whereas mice treated with crizotinib showed tumor relapse after an initial response. Finally, ASP3026 exhibited potent antitumor activity against cells expressing EML4-ALK with a mutation in the gatekeeper position (L1196M) that confers crizotinib resistance. Taken together, these findings indicate that ASP3026 has potential efficacy for NSCLC and is expected to improve the therapeutic outcomes of patients with cancer with ALK abnormality.

Asunto(s)

Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Sulfonas/farmacología , Triazinas/farmacología , Células 3T3 , Quinasa de Linfoma Anaplásico , Animales , Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Sinergismo Farmacológico , Glutamatos/farmacología , Guanina/análogos & derivados , Guanina/farmacología , Humanos , Immunoblotting , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Ratones Transgénicos , Estructura Molecular , Paclitaxel/farmacología , Pemetrexed , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacocinética , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Sulfonas/química , Sulfonas/farmacocinética , Análisis de Supervivencia , Triazinas/química , Triazinas/farmacocinética , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Constipation is a major gastrointestinal motility disorder with clinical need for effective drugs. We previously reported that transient receptor potential ankyrin 1 (TRPA1) is highly expressed in enterochromaffin (EC) cells, which are 5-hydroxytryptamine (5-HT)-releasing cells, and might therefore be a novel target for constipation. Here, we examined the effects of ASP7663, a novel and selective TRPA1 agonist, in constipation models as well as an abdominal pain model. ASP7663 activated human, rat, and mouse TRPA1 and released 5-HT from QGP-1 cells, and oral but not intravenous administration of ASP7663 significantly improved the loperamide-induced delay in colonic transit in mice. While pretreatment with the TRPA1 antagonist HC-030031 and vagotomy both inhibited the ameliorating effect of oral ASP7663 on the colonic transit, both orally and intravenously administered ASP7663 significantly inhibited colorectal distension (CRD)-induced abdominal pain response in rats. Taken together, these results demonstrate that ASP7663 exerts both anti-constipation and anti-abdominal pain actions, the former is likely triggered from the mucosal side of the gut wall via activation of vagus nerves while the latter is assumed to be provoked through systemic blood flow. We conclude that ASP7663 can be an effective anti-constipation drug with abdominal analgesic effect.

Asunto(s)

Dolor Abdominal/tratamiento farmacológico , Analgésicos/uso terapéutico , Estreñimiento/tratamiento farmacológico , Ácidos Indolacéticos/uso terapéutico , Proteínas del Tejido Nervioso/agonistas , Canales Catiónicos TRPC/agonistas , Canales de Potencial de Receptor Transitorio/agonistas , Dolor Abdominal/fisiopatología , Analgésicos/farmacología , Animales , Calcio/metabolismo , Canales de Calcio , Clonidina , Colon/efectos de los fármacos , Colon/fisiología , Estreñimiento/inducido químicamente , Estreñimiento/fisiopatología , Tránsito Gastrointestinal/efectos de los fármacos , Células HEK293 , Humanos , Ácidos Indolacéticos/farmacología , Loperamida , Masculino , Ratones , Ratas , Ratas Wistar , Serotonina/metabolismo , Canal Catiónico TRPA1 , Dolor Visceral/tratamiento farmacológico , Dolor Visceral/fisiopatología

RESUMEN

Hypofunction of brain N-methyl-d-aspartate (NMDA) receptors has been implicated in psychiatric disorders such as schizophrenia and Alzheimer's disease. Inhibition of glycine transporter-1 (GlyT1) is expected to increase glycine, a co-agonist of the NMDA receptor and, consequently, to facilitate NMDA receptor function. We have identified ASP2535 (4-[3-isopropyl-5-(6-phenyl-3-pyridyl)-4H-1,2,4-triazol-4-yl]-2,1,3-benzoxadiazole) as a novel GlyT1 inhibitor, and here describe our in vitro and in vivo characterization of this compound. ASP2535 potently inhibited rat GlyT1 (IC(50)=92 nM) with 50-fold selectivity over rat glycine transporter-2 (GlyT2). It showed minimal affinity for many other receptors except for µ-opioid receptors (IC(50)=1.83 µM). Oral administration of ASP2535 dose-dependently inhibited ex vivo [(3)H]-glycine uptake in mouse cortical homogenate, suggesting good brain permeability. This profile was confirmed by pharmacokinetic analysis. We then evaluated the effect of ASP2535 on animal models of cognitive impairment in schizophrenia and Alzheimer's disease. Working memory deficit in MK-801-treated mice and visual learning deficit in neonatally phencyclidine (PCP)-treated mice were both attenuated by ASP2535 (0.3-3mg/kg, p.o. and 0.3-1mg/kg, p.o., respectively). ASP2535 (1-3mg/kg, p.o.) also improved the PCP-induced deficit in prepulse inhibition in rats. Moreover, the working memory deficit in scopolamine-treated mice and the spatial learning deficit in aged rats were both attenuated by ASP2535 (0.1-3mg/kg, p.o. and 0.1mg/kg, p.o., respectively). These studies provide compelling evidence that ASP2535 is a novel and centrally-active GlyT1 inhibitor that can improve cognitive impairment in animal models of schizophrenia and Alzheimer's disease, suggesting that ASP2535 may satisfy currently unmet medical needs for the treatment of these diseases.

Asunto(s)

Enfermedad de Alzheimer/tratamiento farmacológico , Trastornos del Conocimiento/tratamiento farmacológico , Proteínas de Transporte de Glicina en la Membrana Plasmática/antagonistas & inhibidores , Oxadiazoles/farmacología , Esquizofrenia/tratamiento farmacológico , Triazoles/farmacología , Administración Oral , Enfermedad de Alzheimer/fisiopatología , Animales , Encéfalo/metabolismo , Trastornos del Conocimiento/etiología , Trastornos del Conocimiento/fisiopatología , Modelos Animales de Enfermedad , Maleato de Dizocilpina/toxicidad , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Concentración 50 Inhibidora , Masculino , Trastornos de la Memoria/tratamiento farmacológico , Trastornos de la Memoria/fisiopatología , Ratones , Oxadiazoles/administración & dosificación , Oxadiazoles/farmacocinética , Permeabilidad , Ratas , Ratas Wistar , Esquizofrenia/fisiopatología , Triazoles/administración & dosificación , Triazoles/farmacocinética

RESUMEN

We describe the preparation and evaluation of a novel series of glycine transporter 1 (GlyT1) inhibitors derived from a high-throughput screening hit. The SAR studies resulted in the discovery of 3-biphenyl-4-yl-4-(2-fluorophenyl)-5-isopropyl-4H-1,2,4-triazole (6p). A pharmacokinetic study was also conducted and revealed that 6p had excellent oral bioavailability and ameliorated learning impairment in passive avoidance tasks in mice.

Asunto(s)

Compuestos de Bifenilo/síntesis química , Proteínas de Transporte de Glicina en la Membrana Plasmática/antagonistas & inhibidores , Nootrópicos/síntesis química , Triazoles/síntesis química , Animales , Reacción de Prevención/efectos de los fármacos , Disponibilidad Biológica , Compuestos de Bifenilo/farmacocinética , Compuestos de Bifenilo/farmacología , Encéfalo/metabolismo , Permeabilidad de la Membrana Celular , Ratones , Actividad Motora/efectos de los fármacos , Nootrópicos/farmacocinética , Nootrópicos/farmacología , Relación Estructura-Actividad , Triazoles/farmacocinética , Triazoles/farmacología

RESUMEN

Although it is known that both clonidine and loperamide cause delayed colonic transit in mice, these models of drug-induced experimental constipation have not yet been fully characterized. Therefore, the aims of this study were to validate the clonidine- and loperamide-induced delays of colonic transit in mice as models of atonic and spastic constipation, respectively, and to evaluate the effect of mustard oil, a TRPA1 agonist, in both models. Colonic transit was evaluated in mice by determining the time needed to evacuate a bead inserted into the distal colon. Both loperamide and clonidine dose-dependently prolonged the evacuation time. Clonidine (10 microg/kg) and loperamide (0.3 mg/kg) tripled the evacuation time compared to controls. These delays were antagonized by the administration of yohimbine and naloxone, respectively. Tegaserod, a gastrointestinal motor-stimulating drug, reversed the delay in both models, but the effects were diminished at high doses. Atropine, an antispastic drug, improved the loperamide-induced delay, but did not affect the clonidine-induced delay. Mustard oil accelerated the colonic transit dose-dependently in both models of drug-induced constipations. These results indicate that clonidine- and loperamide-induced delays in colonic transit are models of atonic and spastic constipation, respectively, and that mustard oil may be effective on both types of constipation.

Asunto(s)

Estreñimiento/inducido químicamente , Estreñimiento/tratamiento farmacológico , Tránsito Gastrointestinal/efectos de los fármacos , Aceites de Plantas/uso terapéutico , Animales , Atropina/farmacología , Clonidina , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Indoles/farmacología , Loperamida , Masculino , Ratones , Ratones Endogámicos , Planta de la Mostaza , Naloxona/farmacología , Canal Catiónico TRPA1 , Canales de Potencial de Receptor Transitorio/agonistas , Yohimbina/farmacología

RESUMEN

Our recent study found that TRPA1 is highly expressed in enterochromaffin cells and that stimulation of these cells with TRPA1 agonists enhances 5-hydroxytryptamine (5-HT) secretion in vitro. Here, to demonstrate the 5-HT-releasing effect of TRPA1 agonists in vivo, we examined the effect of TRPA1 agonists on gastric emptying in rats. The results showed that TRPA1 agonists dose-dependently delayed gastric emptying. Further, the effects of TRPA1 agonists on this delay were abolished in rats treated with a TRPA1 antagonist, an inhibitor of tryptophan hydroxylase, or a 5-HT(3) receptor antagonist. Taken together, these results indicate that TRPA1 agonists delay in vivo gastric emptying through serotonergic pathways.

Asunto(s)

Acroleína/análogos & derivados , Canales de Calcio/efectos de los fármacos , Células Enterocromafines/efectos de los fármacos , Vaciamiento Gástrico/efectos de los fármacos , Isotiocianatos/farmacología , Serotonina/metabolismo , Acroleína/farmacología , Animales , Ancirinas , Canales de Calcio/metabolismo , Relación Dosis-Respuesta a Droga , Células Enterocromafines/metabolismo , Inhibidores Enzimáticos/farmacología , Fenclonina/farmacología , Granisetrón/farmacología , Masculino , Ratas , Ratas Wistar , Receptores de Serotonina 5-HT3/metabolismo , Rojo de Rutenio/farmacología , Antagonistas del Receptor de Serotonina 5-HT3 , Antagonistas de la Serotonina/farmacología , Canal Catiónico TRPA1 , Canales Catiónicos TRPC , Triptófano Hidroxilasa/antagonistas & inhibidores , Triptófano Hidroxilasa/metabolismo

RESUMEN

Transient receptor potential ankyrin1 (TRPA1) is a non-selective cation channel activated by cold stimuli under 17 degrees C, mechanosensation, and pungent irritants such as allyl isothiocyanates (AITC) and cinnamaldehyde (CA). In this study, we cloned the dog orthologue of TRPA1 for the first time and induced its heterologous expression in HEK293 cells to investigate its functional properties using a fluorescence imaging plate reader-based Ca(2+) influx assay. Moreover, we examined the effect of AITC on gastrointestinal motility in dogs. At the amino acid level, the sequence of dog TRPA1 was 82-83% identical to that of human, mouse, and rat orthologues. TRPA1 is strongly expressed in the brain, cerebellum, stomach, pancreas, and small and large intestine of dogs. Like other mammalian orthologues, TRPA1 agonists, including AITC, CA, allicin, and diallyl disulfide, evoked a concentration-dependent increase in intracellular Ca(2+) influx in dog TRPA1-expressing cells. AITC stimulated gastric antrum and jejunum motility and induced the occurrence of giant migrating contractions in the colon of fasted dogs. The effects of AITC were inhibited by ruthenium red, a TRPA1 antagonist. These results indicate that AITC stimulated the gastrointestinal motility through TRPA1 in conscious dogs.

Asunto(s)

Estado de Conciencia , Motilidad Gastrointestinal/efectos de los fármacos , Isotiocianatos/farmacología , Canales de Potencial de Receptor Transitorio/genética , Canales de Potencial de Receptor Transitorio/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Clonación Molecular , Perros , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Ratas , Transfección , Canales de Potencial de Receptor Transitorio/agonistas , Canales de Potencial de Receptor Transitorio/química

RESUMEN

Recently, we discovered that transient receptor potential ankyrin1 channel (TRPA1) is highly expressed in human and rat enterochromaffin (EC) cells, and those TRPA1 agonists such as allyl isothiocyanates (AITC) and cinnamaldehyde (CA) enhance the release of serotonin (5-hydroxytryptamine; 5-HT) from EC cells in vitro. In this study, QGP-1 cells, a human pancreatic endocrine cell line, were found to highly express TRPA1 and EC cell marker genes, such as tryptophan hydroxylase 1 (TPH1), chromogranin A (CgA), synaptophysin, ATP-dependent vesicular monoamine transporter 1 (VMAT1), metabotropic glutamate receptor 4 (mGluR4), beta1-adrenergic receptor (ADB1), muscarinic 4 acetylcholine receptor (ACM4), substance P, serotonin transporter (SERT), and guanylin. Furthermore, the TRPA1 agonists AITC, CA, and acrolein concentration dependently evoked an increase in intracellular Ca(2+) influx and the release of 5-HT in QGP-1 cells. The effects of these TRPA1 agonists were inhibited by ruthenium red, a TRPA1 antagonist, and TRPA1-specific siRNA. These results indicate that the Ca(2+) influx increase and 5-HT release induced by AITC, CA and acrolein in QGP-1 cells were mediated by TRPA1, and that the QGP-1 cell line could be a new model for the investigation of TRPA1 function in the human EC cell.

Asunto(s)

Canales de Calcio/metabolismo , Células Enterocromafines/metabolismo , Modelos Biológicos , Proteínas del Tejido Nervioso/metabolismo , Serotonina/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismo , Acroleína/análogos & derivados , Acroleína/farmacología , Bioensayo , Biomarcadores/metabolismo , Calcio/metabolismo , Canales de Calcio/genética , Línea Celular , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Activación del Canal Iónico/efectos de los fármacos , Isotiocianatos/farmacología , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Canal Catiónico TRPA1 , Canales de Potencial de Receptor Transitorio/antagonistas & inhibidores , Canales de Potencial de Receptor Transitorio/genética , Triptófano Hidroxilasa/genética , Triptófano Hidroxilasa/metabolismo

RESUMEN

Serotonin (5-hydroxytryptamine; 5-HT) is abundantly present throughout the gastrointestinal tract and stored mostly in enterochromaffin (EC) cells, which are located on the mucosal surface. 5-HT released from EC cells stimulate both intrinsic and extrinsic nerves, which results in various physiological and pathophysiological responses, such as gastrointestinal contractions. EC cells are believed to have the ability to respond to the chemical composition of the luminal contents of the gut; however, the underlying molecular and cellular mechanisms have not been identified. Here, we demonstrate that the transient receptor potential (TRP) cation channel TRPA1, which is activated by pungent compounds or cold temperature, is highly expressed in EC cells. We also found that TRPA1 agonists, including allyl isothiocyanate and cinnamaldehyde, stimulate EC cell functions, such as increasing intracellular Ca(2+) levels and 5-HT release, by using highly concentrated EC cell fractions and a model of EC cell function, the RIN14B cell line. Furthermore, we showed that allyl isothiocyanate promotes the contraction of isolated guinea pig ileum via the 5-HT(3) receptor. Taken together, our results indicate that TRPA1 acts as a sensor molecule for EC cells and may regulate gastrointestinal function.

Asunto(s)

Canales de Calcio/metabolismo , Células Enterocromafines/metabolismo , Motilidad Gastrointestinal , Proteínas del Tejido Nervioso/metabolismo , Serotonina/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismo , Animales , Ancirinas , Calcio/metabolismo , Canales de Calcio/genética , Línea Celular , Regulación de la Expresión Génica/genética , Cobayas , Humanos , Masculino , Ratones , Proteínas del Tejido Nervioso/genética , Ratas , Canal Catiónico TRPA1 , Canales Catiónicos TRPC , Canales de Potencial de Receptor Transitorio/genética