RESUMEN
BACKGROUND: Astragalosidic acid (LS-102) is a new water-soluble derivative of astragaloside IV - a major effective component isolated from the Chinese herb Astragali Radix. Our previous study showed that LS-102 exhibited potent cardiovascular activity. PURPOSE: The objective of this study was to investigate the pharmacokinetic properties of LS-102 after single-dose, oral administration in beagle dogs by developing and validating an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method. METHOD AND RESULT: The chromatographic separation was performed on a Acquity HSS C18 column (100â¯mmâ¯×â¯2.1â¯mm, 1.8⯵m) by a gradient elution using a mobile phase consisting of water and acetonitrile at a flow rate of 0.35â¯ml/min. The analytes were detected with a triple quadrupole tandem mass spectrometry in multiple reaction monitoring mode. Method validation revealed a wide linearity over the range of 2.0-10,000â¯ng/ml together with satisfactory intra- and inter-day precision, accuracy, and recovery. Stability testing showed that LS-102 spiked into dog plasma was stable for 4â¯h at room temperature, for up to 2 weeks at -80⯰C, and during three freeze-thaw cycles. The method was effectively and successfully applied to the pharmacokinetics of LS-102 after oral administration (5, 10 and 20â¯mg/kg) to beagle dogs. Peak plasma concentrations are attained within approximately 2â¯h after oral administration with a half-life ranging from 1.55â¯h to 4.49â¯h. The plasma concentration-time curve of LS-102 after oral administration presents the phenomenon of a double-peak absorption phase. The peak concentration and area under the concentration-time curve of LS-102 seemed to increase with the increasing doses proportionally, that suggesting linear pharmacokinetics in dogs. Meanwhile, the doxorubicin (Dox)-injured H9c2 cell model was prepared by incubating the cells in 1 µM Dox for 24â¯h. MTT assay and LDH release measurement showed that LS-102 protected against Dox-induced cardiomyocyte death. CONCLUSION: The obtained results may help to guide the further pre-clinical research of LS-102 as a potentially novel cardioprotective agent.
Asunto(s)
Benzoxazoles/sangre , Benzoxazoles/farmacocinética , Cromatografía Liquida/métodos , Saponinas/química , Espectrometría de Masas en Tándem/métodos , Triazinas/sangre , Triazinas/farmacocinética , Triterpenos/química , Administración Oral , Animales , Astragalus propinquus , Benzoxazoles/administración & dosificación , Línea Celular , Cromatografía Líquida de Alta Presión/métodos , Perros , Doxorrubicina/efectos adversos , Estabilidad de Medicamentos , Medicamentos Herbarios Chinos/química , Femenino , Semivida , Masculino , Miocitos Cardíacos/efectos de los fármacos , Ratas , Reproducibilidad de los Resultados , Triazinas/administración & dosificaciónRESUMEN
It is an established fact that orexin plays an important role in regulating the reproductive axis and the secretions of gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH). However, its precise cellular and molecular mechanisms are not fully recognized. Accordingly, the aim of the present study is to find out whether the central injection of orexin A (OXA) and its antagonists, SB-334867 (as orexin receptor antagonist 1; OX1RA) and JNJ-10397049 (as orexin receptor antagonist 2; OX2RA), either alone or in combination, can leave any impact on the reproductive axis (either hormonal or behavioral) in the male Wistar rats. Furthermore, in order to see whether OXA signals can be relayed through the pathway of kisspeptin/neurokinin B/dynorphin (known as KNDy neurons, a neural network which works upstream of GnRH neurons) or not, the relative gene expression of these neuropeptides were measured. Overall, the data from radioimmunoassay revealed that OXA significantly decreases the mean serum level of LH and testosterone and, in a similar vein, its antagonists neutralize this impact. Moreover, data from real-time quantitative PCR indicated that OXA has significantly reduced the hypothalamic expression of Gnrh. In this line, the gene expressions of Kisspeptin and Neurokinin b decreased. However, OXA antagonists neutralize this impact. Also, the expression of Dynorphin gene was upregulated by the following application of the OXA. The results of this study are related to the impact of orexin on the reproductive axis. It is recommended that KNDy neurons as the interneural pathway relay the information of orexin to the GnRH neurons.
Asunto(s)
Dinorfinas/metabolismo , Hipotálamo/efectos de los fármacos , Kisspeptinas/metabolismo , Neuroquinina B/metabolismo , Neuronas/efectos de los fármacos , Orexinas/farmacología , Reproducción/efectos de los fármacos , Animales , Benzoxazoles/administración & dosificación , Benzoxazoles/farmacología , Dioxanos/administración & dosificación , Dioxanos/farmacología , Dinorfinas/genética , Regulación de la Expresión Génica/efectos de los fármacos , Hormona Liberadora de Gonadotropina/metabolismo , Hipotálamo/citología , Inyecciones Intraventriculares , Kisspeptinas/genética , Hormona Luteinizante/sangre , Masculino , Naftiridinas , Neuroquinina B/genética , Neuronas/metabolismo , Orexinas/administración & dosificación , Orexinas/antagonistas & inhibidores , Compuestos de Fenilurea/administración & dosificación , Compuestos de Fenilurea/farmacología , Distribución Aleatoria , Ratas , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa , Conducta Sexual Animal/efectos de los fármacos , Testosterona/sangre , Urea/administración & dosificación , Urea/análogos & derivados , Urea/farmacologíaRESUMEN
Sorafenib, an oral multikinase inhibitor of Raf, VEGF and PDGF receptor signaling is approved for advanced hepatocellular carcinoma (HCC). One strategy to improve HCC therapy is to combine agents that target key signaling pathways. Aberrant mesenchymal-epithelial transition factor (c-Met) activation is associated with a variety of human malignancies and therefore represents a target for therapy. In this study, we investigated a novel c-Met inhibitor, DE605, together with sorafenib in hepatocellular carcinoma cells in vitro and in vivo. DE605 and sorafenib synergistically induced apoptosis in hepatocellular carcinoma cells. Mechanistically, DE605 activated the FGFR3/Erk pathway, which in turn was inhibited by sorafenib, resulting in synergism. Finally, DE605 and sorafenib significantly inhibited growth of PLC/PRF/5 hepatocellular carcinoma tumor xenografts in athymic nude mice. Importantly, no obvious weight loss (toxicity) was detected. Thus in combination, DE605 and sorafenib target complementary anti-apoptotic pathways and synergistically suppress HCC, providing the rationale for clinical studies with this novel combination.
Asunto(s)
Aminopiridinas/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Benzoxazoles/administración & dosificación , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/patología , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Línea Celular Tumoral , Sinergismo Farmacológico , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Ratones , Niacinamida/administración & dosificación , Niacinamida/análogos & derivados , Compuestos de Fenilurea/administración & dosificación , Inhibidores de Proteínas Quinasas/farmacología , Reacción en Cadena en Tiempo Real de la Polimerasa , Sorafenib , Transfección , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The colorectal cancer is the leading contributor of cancer-related mortality. Mammalian target of rapamycin (mTOR), existing in 2 complexes (mTORC1/2), is frequently dysregulated and constitutively activated in colorectal cancers. It represents an important drug target. Here we found that INK-128, the novel ATP-competitive kinase inhibitor of mTOR, blocked both mTORC1 and mTORC2 activation in colorectal cancer cells (both primary and transformed cells). The immunoprecipitation results showed that the assembly of mTORC1 (mTOR-Raptor association) and mTORC2 (mTOR-Rictor-Sin1 association) was also disrupted by INK-128. INK-128 inhibited colorectal cancer cell growth and survival, and induced both apoptotic and non-apoptotic cancer cell death. Further, INK-128 showed no effect on Erk/MAPK activation, while MEK/Erk inhibition by MEK-162 enhanced INK-128-induced cytotoxicity in colorectal cancer cells. Meanwhile, INK-128 downregulated Fascin1 (FSCN1)/E-Cadherin expressions and inhibited HT-29 cell in vitro migration. In vivo, daily INK-128 oral administration inhibited HT-29 xenograft growth in mice, which was further enhanced by MEK-162 administration. Finally, we found that INK-128 sensitized 5-fluorouracil-(5-FU)-mediated anti-HT-29 activity in vivo and in vitro. Thus, our preclinical studies strongly suggest that INK-128 might be investigated for colorectal cancer treatment in clinical trials.
Asunto(s)
Antineoplásicos/farmacología , Benzoxazoles/farmacología , Neoplasias Colorrectales/metabolismo , Complejos Multiproteicos/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Animales , Antineoplásicos/administración & dosificación , Apoptosis/efectos de los fármacos , Benzoxazoles/administración & dosificación , Cadherinas/metabolismo , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/patología , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Sinergismo Farmacológico , Activación Enzimática/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Fluorouracilo/farmacología , Células HT29 , Humanos , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina , Diana Mecanicista del Complejo 2 de la Rapamicina , Ratones , Proteínas de Microfilamentos/metabolismo , Inhibidores de Proteínas Quinasas/administración & dosificación , Pirimidinas/administración & dosificación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Glycolysis and the pentose phosphate pathway (PPP) are preferentially activated in cancer cells. Accumulating evidence indicated the significance of the altered glucose metabolism in cancer, but the implication for oncotherapy remains unclear. Here we report that the synthesis of glycolytic and PPP enzymes is almost ubiquitously augmented in colorectal carcinoma (CRC) specimens. The mammalian target of rapamycin (mTOR) inhibitor INK128 (300 nM) and phytochemical Avemar (1 mg/ml) inhibited the synthesis of PPP enzymes in CRC cell lines. INK128 (150-600 nM) and resveratrol (75-300 µM) inhibited aerobic glycolysis in the cell lines. INK128 (300 nM) and Avemar (1 mg/ml) decreased the NADPH/NADP(+) ratio as well as the GSH/GSSG ratio in the cell lines. Finally, per os administration of INK128 (0.8 mg/kg) or Avemar (1 g/kg) suppressed tumor growth and delayed tumor formation by transplantable CRC specimens derived from patients. Taken together, pharmacological inhibition of the mTOR-PPP axis is a promising therapeutic strategy against CRCs.
Asunto(s)
Antineoplásicos/farmacología , Benzoxazoles/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , Vía de Pentosa Fosfato/efectos de los fármacos , Extractos Vegetales/farmacología , Pirimidinas/farmacología , Estilbenos/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Administración Oral , Animales , Antineoplásicos/administración & dosificación , Benzoxazoles/administración & dosificación , Western Blotting , Línea Celular Tumoral , Neoplasias Colorrectales/enzimología , Femenino , Glucosa/metabolismo , Disulfuro de Glutatión/metabolismo , Glucólisis/efectos de los fármacos , Humanos , Japón , Ácido Láctico/metabolismo , Ratones , Ratones Desnudos , Extractos Vegetales/administración & dosificación , Pirimidinas/administración & dosificación , Receptores de Péptidos/metabolismo , Resveratrol , Estilbenos/administración & dosificación , Trasplante Heterólogo , Resultado del TratamientoRESUMEN
The posterior hypothalamus (PH) is known to reduce nociceptive pain, but the effect of PH stimulation on neuropathic pain is not known. Because neurons containing the neurotransmitter orexin-A are located in the PH in some strains of rat and intrathecal injection of orexin-A produces antinociception in a neuropathic pain model, we hypothesized that orexin-A from neurons in the PH modifies nociception in the spinal cord dorsal horn. To test this hypothesis, the cholinergic agonist carbachol or normal saline was microinjected into the PH of lightly anesthetized female Sprague-Dawley rats with chronic constriction injury (CCI) and foot withdrawal latencies (FWL) were measured. Carbachol-induced PH stimulation produced dose dependent antinociception as shown by significantly increased FWL compared to saline controls. To investigate the role of orexin-A in PH-induced antinociception, the orexin-1 receptor antagonist SB-334867 or dimethyl sulfoxide (DMSO) for control, was given intrathecally following carbachol-induced PH stimulation. SB-334867 decreased FWL compared to DMSO controls. These data are suggestive that stimulating the PH produces antinociception in a neuropathic pain model and that the antinociceptive effect is mediated in part by orexin-1 receptors in the spinal cord dorsal horn.
Asunto(s)
Hipotálamo/fisiopatología , Neuronas/fisiología , Dolor/fisiopatología , Células del Asta Posterior/fisiopatología , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropéptido/metabolismo , Neuropatía Ciática/complicaciones , Analgésicos/administración & dosificación , Animales , Benzoxazoles/administración & dosificación , Carbacol/administración & dosificación , Agonistas Colinérgicos/administración & dosificación , Relación Dosis-Respuesta a Droga , Femenino , Hipotálamo/efectos de los fármacos , Naftiridinas , Neuronas/efectos de los fármacos , Receptores de Orexina , Dolor/tratamiento farmacológico , Dolor/etiología , Dimensión del Dolor , Células del Asta Posterior/efectos de los fármacos , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores de Neuropéptido/antagonistas & inhibidores , Neuropatía Ciática/fisiopatología , Médula Espinal/efectos de los fármacos , Médula Espinal/fisiopatología , Urea/administración & dosificación , Urea/análogos & derivadosRESUMEN
We have previously shown that hypercapnic chemoreflex in prepro-orexin knockout mice (ORX-KO) is attenuated during wake but not sleep periods. In that study, however, hypercapnic stimulation had been chronically applied for 6 h because of technical difficulty in changing the composition of the inspired gas mixture without distorting the animal's vigilance states. In the present study we examined possible involvement of orexin in acute respiratory chemoreflex during wake periods. Ventilation was recorded together with electroencephalography and electromyography before and after intracerebroventricular administration of orexin or an orexin receptor antagonist, SB-334867. A hypercapnic (5 or 10% CO(2)) or hypoxic (15 or 10% O(2)) gas mixture was introduced into the recording chamber for 5 min. Respiratory parameters were analyzed only for quiet wakefulness. When mice breathed normal room air, orexin-A and orexin-B but not vehicle or SB-334867 increased minute ventilation in both ORX-KO and wild-type (WT) mice. As expected, hypercapnic chemoreflex in vehicle-treated ORX- KO mice (0.22 +/- 0.03 mlxmin(-1)xg(-1)x% CO(2)(-1)) was significantly blunted compared with that in WT mice (0.51 +/- 0.05 mlxmin(-1)xg(-1)x% CO(2)(-1)). Supplementation of orexin-A or -B (3 nmol) partially restored the hypercapnic chemoreflex in ORX-KO mice (0.28 +/- 0.03 mlxmin(-1).g(-1)x% CO(2)(-1) for orexin-A and 0.32 +/- 0.04 mlxmin(-1)xg(-1)x% CO(2)(-1) for orexin-B). In addition, injection of SB-334867 (30 nmol) in WT mice decreased the hypercapnic chemoreflex (0.39 +/- 0.04 mlxmin(-1)xg(-1)x% CO(2)(-1)). On the other hand, hypoxic chemoreflex in vehicle-treated ORX-KO and SB-334867-treated WT mice was not different from that in corresponding controls. Our findings suggest that orexin plays a crucial role in CO(2) sensitivity at least during wake periods in mice.
Asunto(s)
Dióxido de Carbono/sangre , Células Quimiorreceptoras/metabolismo , Hipercapnia/metabolismo , Hipotálamo/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neuropéptidos/metabolismo , Ventilación Pulmonar , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropéptido/metabolismo , Reflejo , Animales , Benzoxazoles/administración & dosificación , Células Quimiorreceptoras/efectos de los fármacos , Células Quimiorreceptoras/fisiopatología , Modelos Animales de Enfermedad , Electroencefalografía , Electromiografía , Hipercapnia/fisiopatología , Hipotálamo/efectos de los fármacos , Hipotálamo/fisiopatología , Hipoxia/metabolismo , Hipoxia/fisiopatología , Inyecciones Intraventriculares , Péptidos y Proteínas de Señalización Intracelular/administración & dosificación , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Actividad Motora , Naftiridinas , Neuropéptidos/administración & dosificación , Neuropéptidos/deficiencia , Neuropéptidos/genética , Receptores de Orexina , Orexinas , Ventilación Pulmonar/efectos de los fármacos , Receptores Acoplados a Proteínas G/efectos de los fármacos , Receptores de Neuropéptido/efectos de los fármacos , Reflejo/efectos de los fármacos , Factores de Tiempo , Urea/administración & dosificación , Urea/análogos & derivados , VigiliaRESUMEN
A novel class of potent and stable mGlu5 receptor antagonists was developed by combining information from a high-throughput screening campaign with the structure of the known anxiolytic fenobam. Representative compounds from this class show favorable pharmacokinetic properties and are active in an in vivo model of anxiety.
Asunto(s)
Ansiolíticos/síntesis química , Benzoxazoles/síntesis química , Benzoxazoles/farmacocinética , Receptores de Glutamato Metabotrópico/antagonistas & inhibidores , Animales , Ansiolíticos/administración & dosificación , Ansiolíticos/farmacocinética , Ansiedad/tratamiento farmacológico , Benzoxazoles/administración & dosificación , Diseño de Fármacos , Evaluación Preclínica de Medicamentos/métodos , Imidazoles/química , Modelos Animales , Farmacocinética , Unión Proteica , Ratas , Receptor del Glutamato Metabotropico 5 , Relación Estructura-ActividadRESUMEN
An orexin-1 receptor antagonist decreases food intake whereas orexin-A selectively induces hyperphagia to a high-fat diet. In the present study, we evaluated the effect of an orexin antagonist in two strains of rats that differ in their sensitivity to becoming obese while eating a high-fat diet. Male Osborne-Mendel (OM) and S5B/Pl (S5B) rats were treated acutely with an orexin-1 receptor antagonist (SB-334867), after adaptation to either a high-fat (56% fat energy) diet or a low-fat (10% fat energy) diet that were equicaloric for protein (24% energy). Ad libitum fed rats were injected intraperitoneally with SB-334867 at doses of 3, 10 or 30 mg/kg, or vehicle at the beginning of the dark cycle, and food intake and body weight were measured. Hypothalamic prepro-orexin and orexin-1 receptor mRNA expression were analyzed in OM and S5B rats fed at a high-fat or low-fat diet for two weeks. SB-334867 significantly decreased food intake in both strains of rats eating the high-fat diet but only in the OM rats eating the low fat diet. The effect was greatest at 12 and 24 h. Body weight was also reduced in OM rats 1d after injection of SB-334867 but not in the S5B rats. Prepro-orexin and orexin-1 receptor expression levels did not differ between strains or diets. These experiments demonstrate that an orexin antagonist (SB-334867) reduces food intake and has a greater effect in a rat strain that is susceptible to dietary-induced obesity, than in a resistant strain.
Asunto(s)
Benzoxazoles/administración & dosificación , Peso Corporal/efectos de los fármacos , Dieta/efectos adversos , Ingestión de Alimentos/efectos de los fármacos , Hipotálamo/metabolismo , Obesidad/metabolismo , Precursores de Proteínas/biosíntesis , Receptores de Neuropéptido/biosíntesis , Urea/análogos & derivados , Animales , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Naftiridinas , Receptores de Orexina , Precursores de Proteínas/antagonistas & inhibidores , ARN Mensajero/biosíntesis , Ratas , Receptores Acoplados a Proteínas G , Receptores de Neuropéptido/antagonistas & inhibidores , Especificidad de la Especie , Urea/administración & dosificaciónRESUMEN
Topical application of Benzoxazole to the inner surface of the ear of the rabbit induced a considerable and histologically verified follicular keratosis. This effect could be intensified by a preceding treatment of the animal with androgens. Vitamin A acid, applied topically, is able to eliminate this follicular keratosis.