RESUMEN
BACKGROUND AND AIMS: Pulmonary hypertension (PH) is a heterogeneous disorder associated with poor prognosis. For the majority of patients, only limited therapeutic options are available. Thus, there is great interest to develop novel treatment strategies focusing on pulmonary vascular and right ventricular remodeling. Interleukin 9 (IL9) is a pleiotropic cytokine with pro- and anti-inflammatory functions. The aim of this study was to evaluate the therapeutic activity of F8IL9F8 consisting of IL9 fused to the F8 antibody, specific to the alternatively-spliced EDA domain of fibronectin, which is abundantly expressed in pulmonary vasculature and right ventricular myocardium in PH. METHODS: The efficacy of F8IL9F8 in attenuating PH progression in the monocrotaline mouse model was evaluated in comparison to an endothelin receptor antagonist (ERA) or an IL9 based immunocytokine with irrelevant antibody specificity (KSFIL9KSF). Treatment effects were assessed by right heart catheterization, echocardiography as well as histological and immunohistochemical tissue analyses. RESULTS: Compared to controls, systolic right ventricular pressure (RVPsys) was significantly elevated and a variety of right ventricular echocardiographic parameters were significantly impaired in all MCT-induced PH groups except for the F8IL9F8 group. Both, F8IL9F8 and ERA treatments lead to a significant reduction in RVPsys and an improvement of echocardiographic parameters when compared to the MCT group not observable for the KSFIL9KSF group. Only F8IL9F8 significantly reduced lung tissue damage and displayed a significant decrease of leukocyte and macrophage accumulation in the lungs and right ventricles. CONCLUSIONS: Our study provides first pre-clinical evidence for the use of F8IL9F8 as a new therapeutic agent for PH in terms of a disease-modifying concept addressing cardiovascular remodeling.
Asunto(s)
Anticuerpos/química , Hipertensión Pulmonar/terapia , Interleucina-9/uso terapéutico , Animales , Células CHO , Cricetulus , Citocinas/metabolismo , Modelos Animales de Enfermedad , Portadores de Fármacos , Ecocardiografía , Antagonistas de los Receptores de Endotelina/química , Hemodinámica , Hipertensión Pulmonar/inmunología , Inmunohistoquímica , Inflamación , Interleucina-9/administración & dosificación , Leucocitos/metabolismo , Pulmón/metabolismo , Macrófagos/metabolismo , Ratones , Unión Proteica , Función Ventricular DerechaRESUMEN
The endothelin (ET) axis plays a pivotal role in cardiovascular diseases. Enhanced levels of circulating ET-1 have been correlated with an inferior clinical outcome after myocardial infarction (MI) in humans. Thus, the evaluation of endothelin-A receptor (ETAR) expression over time in the course of myocardial injury and healing may offer valuable information toward the understanding of the ET axis involvement in MI. We developed an approach to track the expression of ETAR with a customized molecular imaging probe in a murine model of MI. The small molecular probe based on the ETAR-selective antagonist 3-(1,3-benzodioxol-5-yl)-5-hydroxy-5-(4-methoxyphenyl)-4-[(3,4,5-trimethoxyphenyl)methyl]-2(5H)-furanone (PD156707) was labeled with fluorescent dye, IRDye800cw. Mice undergoing permanent ligation of the left anterior descending artery (LAD) were investigated at day 1, 7, and 21 post surgery after receiving an intravenous injection of the ETAR probe. Cryosections of explanted hearts were analyzed by cryotome-based CCD, and fluorescence reflectance imaging (FRI) and fluorescence signal intensities (SI) were extracted. Fluorescence-mediated tomography (FMT) imaging was performed to visualize probe distribution in the target region in vivo. An enhanced fluorescence signal intensity in the infarct area was detected in cryoCCD images as early as day 1 after surgery and intensified up to 21 days post MI. FRI was capable of detecting significantly enhanced SI in infarcted regions of hearts 7 days after surgery. In vivo imaging by FMT localized enhanced SI in the apex region of infarcted mouse hearts. We verified the localization of the probe and ETAR within the infarct area by immunohistochemistry (IHC). In addition, neovascularized areas were found in the affected myocardium by CD31 staining. Our study demonstrates that the applied fluorescent probe is capable of delineating ETAR expression over time in affected murine myocardium after MI in vivo and ex vivo.
Asunto(s)
Dioxoles/metabolismo , Antagonistas de los Receptores de Endotelina/administración & dosificación , Colorantes Fluorescentes/administración & dosificación , Infarto del Miocardio/metabolismo , Receptores de Endotelina/metabolismo , Animales , Crioultramicrotomía , Dioxoles/química , Modelos Animales de Enfermedad , Antagonistas de los Receptores de Endotelina/análisis , Antagonistas de los Receptores de Endotelina/química , Femenino , Colorantes Fluorescentes/análisis , Inmunohistoquímica , Indoles/metabolismo , Ratones , Ratones Endogámicos C57BL , Infarto del Miocardio/diagnóstico por imagen , Neovascularización Fisiológica , Imagen Óptica , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismoRESUMEN
The endothelin axis and in particular the two receptor subtypes, ETA and ETB, are under investigation for the treatment of various diseases such as pulmonary arterial hypertension, fibrosis, renal failure and cancer. Previous work in our lab has shown that 1,3,6-trisubstituted-4-oxo-1,4-dihydroquinoline-2-carboxylic acid derivatives exhibit noteworthy endothelin receptor antagonist activity. A series of analogues with modifications centered around position 6 of the heterocyclic quinolone core and replacement of the aryl carboxylic acid group with an isosteric tetrazole ring was designed and synthesized to further optimize the structure activity relationship. The endothelin receptor antagonist activity was determined by in vitro Förster resonance energy transfer (FRET) using GeneBLAzer® assay technology. The most potent member of this series exhibited ETA receptor antagonist activity in the subnanomolar range with an IC50 value of 0.8nM, and was 1000-fold selective for the ETA receptor compared to the ETB receptor. Its activity and selectivity profile resembles that of the most recently approved drug, macitentan.
Asunto(s)
Ácidos Carboxílicos/química , Ácidos Carboxílicos/farmacología , Quinolinas/química , Quinolinas/farmacología , Receptor de Endotelina A/efectos de los fármacos , Animales , Ácidos Carboxílicos/síntesis química , Antagonistas de los Receptores de Endotelina/síntesis química , Antagonistas de los Receptores de Endotelina/química , Antagonistas de los Receptores de Endotelina/farmacología , Transferencia Resonante de Energía de Fluorescencia , Humanos , Concentración 50 Inhibidora , Relación Estructura-ActividadRESUMEN
A plethora of properties are typically studied during a medicinal chemistry program and many of these parameters may shape the cascade of compound selection. Given the task to discover a molecule with a profile superior to that of the dual endothelin receptor antagonist bosentan, we tailored our compound profiling cascade to the specific properties that were not optimal in bosentan, namely in vivo efficacy and safety. Contrary to conventional thinking, we therefore focused on corresponding in vivo experiments. In the following, we highlight and illustrate some key learnings of our approach that led to the discovery of macitentan (1), an orally available potent dual endothelin receptor antagonist approved for the treatment of pulmonary arterial hypertension.
Asunto(s)
Química Farmacéutica , Antagonistas de los Receptores de Endotelina/química , Pirimidinas/química , Sulfonamidas/química , Antagonistas de los Receptores de Endotelina/farmacología , Hipertensión Pulmonar , Pirimidinas/farmacología , Sulfonamidas/farmacologíaRESUMEN
In humans, two endothelin receptors, ETa and ETb, are activated by three endogenous 21-mer cyclic peptides, ET-1, ET-2, and ET-3, which control various physiological processes, including vasoconstriction, vasodilation, and stimulation of cell proliferation. The first stage of this study it to produce a stable solubilized and purified receptor in a monodisperse state. This article is focused on the engineering, expression, purification, and characterization of the endothelin receptor B for subsequent structural and functional studies.
Asunto(s)
Receptor de Endotelina B/química , Receptor de Endotelina B/aislamiento & purificación , Animales , Baculoviridae/genética , Compuestos de Bifenilo/química , Western Blotting , Dipéptidos/química , Antagonistas de los Receptores de Endotelina/química , Endotelinas/química , Ingeniería Genética/métodos , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Fragmentos de Péptidos/química , Desnaturalización Proteica , Estabilidad Proteica , Receptor de Endotelina B/genética , Receptor de Endotelina B/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Células Sf9 , TemperaturaRESUMEN
A series of phenoxybutanoic acid derivatives were synthesized and tested for their antagonistic activity on the contraction of the rat thoracic aortic ring induced by endothelin-1. Preliminary screening results showed that 6e and 6g with benzoheterocycles demonstrated significant antagonistic activities when compared to the reference compound BQ123. The results from additional assays for the binding affinity and selectivity for endothelin receptors showed that 6e was a selective ETA antagonist with a nanomolar IC50. Moreover, 6e was effective in relieving hypoxia-induced pulmonary arterial hypertension and right ventricular weight ratio. Therefore, 6e may have potential for further development as a therapeutic agent for the treatment of cardiovascular diseases.
Asunto(s)
Antihipertensivos/química , Antihipertensivos/farmacología , Butiratos/química , Butiratos/farmacología , Antagonistas de los Receptores de Endotelina/química , Antagonistas de los Receptores de Endotelina/farmacología , Vasoconstricción/efectos de los fármacos , Animales , Aorta Torácica/efectos de los fármacos , Aorta Torácica/fisiología , Descubrimiento de Drogas , Modelos Moleculares , Ratas , Ratas Sprague-Dawley , Receptores de Endotelina/metabolismo , Relación Estructura-ActividadRESUMEN
Glaucoma is the second leading cause of blindness in the world and is characterized by the loss of retinal ganglion cells (RGC) over a period of time, leading to complete blindness. Recently, endothelin has been identified as an important factor that influences intraocular pressure IOP, OBF, and direct RGC damage. Targeting the endothelin receptor signaling pathway in glaucoma is considered to be highly beneficial, as it can effectively modulate IOP, OBF, and RGC damage, the key factors which are essential to modulate the disease progression holistically. Currently, synthetic drugs like Bosentan, BQ-123, and prostaglandin analogues are available as endothelin receptor antagonists, which are extensively used in the treatment of cardiovascular and other conditions like systemic hypertension. However, the usage of these drugs in glaucoma is limited due to toxicity and poor bioavailability in the ocular milieu. Thus, there is a need for potential natural compounds as endothelin receptor antagonists that acts as dual inhibitors by targeting both ETA and ETB and are highly efficient with the least toxicity. Hence, this study is intended to prioritize endothelin receptor antagonists by structural bioinformatics approaches involving molecular modeling, molecular dynamics, and molecular docking studies. Subsequently, High throughput virtual screening (HTVS) vs. Natural compound databases targeting the optimal binding sites of both ETA and ETB. Following this, the common hits were subjected to binding free energy calculations (MMGBSA) and ADMETox analysis. Finally, the most potential hits were analyzed for MD based binding stability analysis and binding free energy. Similarly, the known synthetic inhibitors were also docked to the receptors and the results were analyzed. From this study, it was inferred that among the natural compounds dataset (8929 compounds), only 4 common compounds were identified as hits. Among these, only one compound ST075640 surpassed all the prioritization criteria including MMGBSA, ADMETox prediction, dual inhibitory potential (ETA & ETB), and also in structural comparative analysis with bosentan it showed similar efficiency. Thus, the validated hit shall prove to be effective in modulating endothelin mediated IOP, OBF, and RGC damage in glaucomatous condition.
Asunto(s)
Antagonistas de los Receptores de Endotelina/química , Antagonistas de los Receptores de Endotelina/farmacología , Glaucoma/tratamiento farmacológico , Ensayos Analíticos de Alto Rendimiento , Simulación del Acoplamiento Molecular , Receptor de Endotelina A/química , Receptor de Endotelina B/química , Secuencia de Aminoácidos , Glaucoma/metabolismo , Humanos , Conformación Proteica , Células Ganglionares de la Retina/efectos de los fármacos , Homología de SecuenciaRESUMEN
Human cytomegalovirus (HCMV) infection is an important cause of morbidity and mortality in immunocompromised patients and a major etiological factor for congenital birth defects in newborns. Ganciclovir and its pro-drug valganciclovir are the preferred drugs in use today for prophylaxis and treatment of viremic patients. Due to long treatment times, patients are at risk for developing viral resistance to ganciclovir and to other drugs with a similar mechanism of action. We earlier found that the endothelin receptor B (ETBR) is upregulated during HCMV infection and that it plays an important role in the life cycle of this virus. Here, we tested the hypothesis that ETBR blockade could be used in the treatment of HCMV infection. As HCMV infection is specific to humans, we tested our hypothesis in human cell types that are relevant for HCMV pathogenesis; i.e., endothelial cells, epithelial cells and fibroblasts. We infected these cells with HCMV and treated them with the ETBR specific antagonist BQ788 or ETR antagonists that are approved by the FDA for treatment of pulmonary hypertension; macitentan, its metabolite ACT-132577, bosentan and ambrisentan, and as an anti-viral control, we used ganciclovir or letermovir. At concentrations expected to be relevant in vivo, macitentan, ACT-132577 and BQ788 effectively inhibited productive infection of HCMV. Of importance, macitentan also inhibited productive infection of a ganciclovir-resistant HCMV isolate. Our results suggest that binding or signaling through ETBR is crucial for viral replication, and that selected ETBR blockers inhibit HCMV infection.
Asunto(s)
Citomegalovirus/fisiología , Antagonistas de los Receptores de Endotelina/farmacología , Pirimidinas/farmacología , Sulfonamidas/farmacología , Línea Celular , Citomegalovirus/efectos de los fármacos , Infecciones por Citomegalovirus/virología , Antagonistas de los Receptores de Endotelina/química , Ganciclovir/farmacología , Humanos , Concentración 50 Inhibidora , Metaboloma , Oligopéptidos , Piperidinas , Pirimidinas/química , Sulfonamidas/química , Virión/efectos de los fármacos , Virión/metabolismo , Internalización del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
Endothelin receptors (ETA and ETB) are G-protein-coupled receptors activated by endothelin-1 and are involved in blood pressure regulation. IRL2500 is a peptide-mimetic of the C-terminal tripeptide of endothelin-1, and has been characterized as a potent ETB-selective antagonist, which has preventive effects against brain edema. Here, we report the crystal structure of the human ETB receptor in complex with IRL2500 at 2.7 Å-resolution. The structure revealed the different binding modes between IRL2500 and endothelin-1, and provides structural insights into its ETB-selectivity. Notably, the biphenyl group of IRL2500 penetrates into the transmembrane core proximal to D2.50, thus stabilizing the inactive conformation. Using the newly-established constitutively active mutant, we clearly demonstrate that IRL2500 functions as an inverse agonist for the ETB receptor. The current findings will expand the chemical space of ETR antagonists and facilitate the design of inverse agonists for other class A GPCRs.
Asunto(s)
Compuestos de Bifenilo/química , Dipéptidos/química , Antagonistas de los Receptores de Endotelina/química , Receptor de Endotelina B/química , Sitios de Unión , Bosentán/química , Cristalización , Cristalografía por Rayos X , Diseño de Fármacos , Agonismo Inverso de Drogas , Endotelina-1/química , HumanosRESUMEN
In this study, we prepared carrier-based formulations for dry powder inhalers by mixing bosentan microparticles with carrier, prepared in three separate types of lactose. Spray-dried, milled and sieved lactose resulted in formulations with various shapes, surface morphology and particle size distributions. In the spray-dried lactose, the micronized bosentan particles were trapped and strongly interlocked in the rugged surface of spray-dried lactose, whereas in the milled and sieved lactose they exhibited lower binding affinity onto the smooth surface of carrier. In all of the carrier-based formulations, the flow properties were improved compared with bosentan microparticles alone, in the following order spray-dried, sieved and milled lactose. The aerodynamic characteristics of each were evaluated by particle image velocimetry and Andersen cascade impactor™. Depending on the lactose carrier type, particle dispersion showed different flow characteristics. In the spray-dried lactose, the formulation was dispersed fast in the only frontal direction, while the milled and sieved lactose formulations formed a relatively slower S-shaped and fountain-shaped flow stream, respectively. In addition, milled and sieved lactose formulations showed that the drug particles were readily liberated from the lactose carrier, and demonstrated significantly higher aerosol performance than spray-dried lactose.
Asunto(s)
Portadores de Fármacos , Antagonistas de los Receptores de Endotelina/química , Lactosa/química , Sulfonamidas/química , Administración por Inhalación , Aerosoles , Bosentán , Composición de Medicamentos , Inhaladores de Polvo Seco , Antagonistas de los Receptores de Endotelina/administración & dosificación , Tamaño de la Partícula , Polvos , Reología , Sulfonamidas/administración & dosificación , Propiedades de Superficie , Tecnología Farmacéutica/métodos , Factores de TiempoRESUMEN
Endothelin receptors (ETRs) have crucial roles in vascular control and are targets for drugs designed to treat circulatory-system diseases and cancer progression. The nonpeptide dual-ETR antagonist bosentan is the first oral drug approved to treat pulmonary arterial hypertension. Here we report crystal structures of human endothelin ETB receptor bound to bosentan and to the ETB-selective analog K-8794, at 3.6-Å and 2.2-Å resolution, respectively. The K-8794-bound structure reveals the detailed water-mediated hydrogen-bonding network at the transmembrane core, which could account for the weak negative allosteric modulation of ETB by Na+ ions. The bosentan-bound structure reveals detailed interactions with ETB, which are probably conserved in the ETA receptor. A comparison of the two structures shows unexpected similarity between antagonist and agonist binding. Despite this similarity, bosentan sterically prevents the inward movement of transmembrane helix 6 (TM6), and thus exerts its antagonistic activity. These structural insights will facilitate the rational design of new ETR-targeting drugs.
Asunto(s)
Antagonistas de los Receptores de Endotelina/química , Antagonistas de los Receptores de Endotelina/metabolismo , Receptor de Endotelina B/química , Receptor de Endotelina B/metabolismo , Sulfonamidas/química , Sulfonamidas/metabolismo , Bosentán , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Unión Proteica , Conformación ProteicaRESUMEN
In this study, we reported a strategy to improve delivery efficiency of a long-circulation biomimetic photothermal nanoagent for enhanced photothermal therapy through selectively dilating tumor vasculature. By using a simply nanocoating technology, a biomimetic layer of natural red blood cell (RBC) membranes was camouflaged on the surface of photothermal polypyrrole nanoparticles (PPy@RBC NPs). The erythrocyte-mimicking PPy NPs inherited the immune evasion ability from natural RBC resulting in superior prolonged blood retention time. Additionally, excellent photothermal and photoacoustic imaging functionalities were all retained attributing to PPy NPs cores. To further improve the photothermal outcome, the endothelin A (ETA) receptor antagonist BQ123 was jointly employed to regulate tumor microenvironment. The BQ123 could induce tumor vascular relaxation and increase blood flow perfusion through modulating an ET-1/ETA transduction pathway and blocking the ETA receptor, whereas the vessel perfusion of normal tissues was not altered. Through our well-designed tactic, the concentration of biomimetic PPy NPs in tumor site was significantly improved when administered systematically. The study documented that the antitumor efficiency of biomimetic PPy NPs combined with specific antagonist BQ123 was particularly prominent and was superior to biomimetic PPy NPs (P < 0.05) and PEGylated PPy NPs with BQ123 (P < 0.01), showing that the greatly enhanced photothermal treatment could be achieved with low-dose administration of photothermal agents. Our findings would provide a promising procedure for other similar enhanced photothermal treatment by blocking ETA receptor to dramatically increase the delivery of biomimetic photothermal nanomaterials.
Asunto(s)
Antagonistas de los Receptores de Endotelina/uso terapéutico , Hipertermia Inducida/métodos , Nanopartículas/uso terapéutico , Neoplasias/terapia , Péptidos Cíclicos/uso terapéutico , Fototerapia/métodos , Polímeros/uso terapéutico , Pirroles/uso terapéutico , Animales , Materiales Biomiméticos/química , Materiales Biomiméticos/uso terapéutico , Materiales Biocompatibles Revestidos/química , Materiales Biocompatibles Revestidos/uso terapéutico , Antagonistas de los Receptores de Endotelina/química , Membrana Eritrocítica/química , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Nanopartículas/química , Neoplasias/irrigación sanguínea , Péptidos Cíclicos/química , Polímeros/química , Pirroles/química , Células RAW 264.7RESUMEN
Traditional Chinese medicine (TCM) preparations have significant effects on some refractory diseases; however, these compositions are complex and their mechanisms are unknown. Identification of the active components in these preparations is essential. The mortality rate for heart failure (HF) has been increasing in recent years, and myocardial dysfunction (MD) has been proved to be the pathological basis of HF. Yixinshu Capsule (YXSC) is a multi-component oral drug with therapeutic effects on HF. However, the key active components are still unclear. In this study, YXSC intestinal absorption liquid (IAL) was used and 62 compounds were identified by an analytical chemistry approach. Then, a compound - target - function network was established with a bioinformatics analysis tool. Finally, a cell model of MD on human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) was used to verify the therapeutic effects of the active components of YXSC. Schisandrin A (Sch A) and schisandrin B (Sch B) were demonstrated to be the active components of YXSC by attenuating endothelin-1 (ET-1)-induced contraction dysfunction, brain natriuretic peptide (BNP) content elevation, and the morphological changes of hiPS-CMs. For the first time, our data illustrate the potent protective effects of Sch A and Sch B on ET-1-induced dysfunctional hiPS-CMs and revealed their effective targets and pathways. The integrative approach used in our study was applied to identify active components in TCM preparations and excavate the possible mechanisms.
Asunto(s)
Cardiotónicos/farmacología , Ciclooctanos/farmacología , Medicamentos Herbarios Chinos/química , Antagonistas de los Receptores de Endotelina/farmacología , Lignanos/farmacología , Miocitos Cardíacos/efectos de los fármacos , Compuestos Policíclicos/farmacología , Actinina/antagonistas & inhibidores , Actinina/genética , Actinina/metabolismo , Animales , Bosentán , Cardiotónicos/química , Cardiotónicos/aislamiento & purificación , Diferenciación Celular , Línea Celular , Ciclooctanos/química , Ciclooctanos/aislamiento & purificación , Medicamentos Herbarios Chinos/farmacología , Antagonistas de los Receptores de Endotelina/química , Antagonistas de los Receptores de Endotelina/aislamiento & purificación , Endotelina-1/antagonistas & inhibidores , Endotelina-1/farmacología , Regulación de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/efectos de los fármacos , Lignanos/química , Lignanos/aislamiento & purificación , Masculino , Medicina Tradicional China , Redes y Vías Metabólicas/efectos de los fármacos , Metaboloma , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Péptido Natriurético Encefálico/antagonistas & inhibidores , Péptido Natriurético Encefálico/genética , Péptido Natriurético Encefálico/metabolismo , Compuestos Policíclicos/química , Compuestos Policíclicos/aislamiento & purificación , Ratas , Ratas Sprague-Dawley , Sulfonamidas/farmacología , Troponina T/antagonistas & inhibidores , Troponina T/genética , Troponina T/metabolismoRESUMEN
Bosentan, the first-in-class drug used in treatment of pulmonary arterial hypertension, is principally metabolized by the cytochromes P450, and it is responsible for cytochromes induction and drug-drug interaction events with moderate to severe consequences. A strategy to reduce drug-drug interactions consists of increasing the metabolic stability of the perpetrator, and fluorinated analogues are often designed to block the major sites of metabolism. In this paper bosentan analogues were synthesized, and their metabolism and biological activity were evaluated. All synthesized compounds showed an improved metabolic stability towards CYP2C9, with one maintaining a moderate antagonist effect towards the ETA receptor.
Asunto(s)
Antagonistas de los Receptores de Endotelina/metabolismo , Antagonistas de los Receptores de Endotelina/farmacología , Sulfonamidas/metabolismo , Sulfonamidas/farmacología , Animales , Bosentán , Células CHO , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cricetinae , Cricetulus , Citocromo P-450 CYP2C9/química , Citocromo P-450 CYP2C9/metabolismo , Antagonistas de los Receptores de Endotelina/química , Modelos Moleculares , Conformación Proteica , Receptores de Endotelina/agonistas , Receptores de Endotelina/metabolismo , Sulfonamidas/químicaRESUMEN
Little is understood of skeletal muscle tissue in terms of oxidative stress and inflammation. Endothelin-1 is an endogenous, vasoconstrictive peptide which can induce overproduction of reactive oxygen species and proinflammatory cytokines. The aim of this study was to evaluate whether BQ123, an endothelin-A receptor antagonist, influences the level of TNF-α, IL-6, SOD-1, HO-1, Nrf2 mRNA, and NF-κB subunit RelA/p65 mRNA in the femoral muscle obtained from endotoxemic rats. Male Wistar rats were divided into 4 groups (n = 6) and received iv (1) saline (control), (2) LPS (15 mg/kg), (3) BQ123 (1 mg/kg), (4) BQ123 (1 mg/kg), and LPS (15 mg/kg, resp.) 30 min later. Injection of LPS led to significant increase in levels of RelA/p65 mRNA, TNF-α, and IL-6, while content of SOD-1, HO-1, and Nrf2 mRNA was unchanged. Administration of BQ123 prior to LPS challenge resulted in a significant reduction in RelA/p65 mRNA, TNF-α, and IL-6 levels, as well as markedly elevated concentrations of SOD-1, HO-1, and Nrf2 mRNA. BQ123 appears to enhance antioxidant defense and prevent production of TNF-α and IL-6 in skeletal muscle of LPS-treated rat. In conclusion, endothelin-A receptor antagonism exerts significant impact on the skeletal muscle favouring anti-inflammatory effects and protection against oxidative stress.
Asunto(s)
Antioxidantes/metabolismo , Músculo Esquelético/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Péptidos Cíclicos/química , Animales , Citocinas , Antagonistas de los Receptores de la Endotelina A/química , Antagonistas de los Receptores de Endotelina/química , Hemo Oxigenasa (Desciclizante)/metabolismo , Inflamación , Interleucina-6/metabolismo , Lipopolisacáridos/química , Masculino , Músculo Esquelético/efectos de los fármacos , Estrés Oxidativo , Ratas , Ratas Wistar , Superóxido Dismutasa-1/metabolismo , Regulación hacia ArribaRESUMEN
The endothelin axis and in particular the two endothelin receptors, ETA and ETB, are targets for therapeutic intervention in human diseases. Endothelin-receptor antagonists are in clinical use to treat pulmonary arterial hypertension and have been under clinical investigation for the treatment of several other diseases, such as systemic hypertension, cancer, vasospasm, and fibrogenic diseases. In this Perspective, we review the molecules that have been evaluated in human clinical trials for the treatment of pulmonary arterial hypertension, as well as other cardiovascular diseases, cancer, and fibrosis. We will also discuss the therapeutic consequences of receptor selectivity with regard to ETA-selective, ETB-selective, or dual ETA/ETB antagonists. We will also consider which chemical characteristics are relevant to clinical use and the properties of molecules necessary for efficacy in treating diseases against which known molecules displayed suboptimal efficacy.
Asunto(s)
Antagonistas de los Receptores de Endotelina/uso terapéutico , Hipertensión Pulmonar/tratamiento farmacológico , Neoplasias/tratamiento farmacológico , Receptores de Endotelina/metabolismo , Animales , Enfermedades Cardiovasculares/tratamiento farmacológico , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/patología , Ensayos Clínicos como Asunto , Antagonistas de los Receptores de Endotelina/efectos adversos , Antagonistas de los Receptores de Endotelina/química , Antagonistas de los Receptores de Endotelina/farmacología , Fibrosis , Humanos , Hipertensión Pulmonar/metabolismo , Hipertensión Pulmonar/patología , Neoplasias/metabolismo , Neoplasias/patologíaRESUMEN
A stability-indicating reversed-phase high-performance liquid chromatography (HPLC) method was developed for the determination of ambrisentan, a drug used in the treatment of pulmonary hypertension. The desired chromatographic separation was achieved on a Kromasil C18 column (250 × 4.6 mm, 5 µm) using the mobile phase acetonitrile-ammonium formate (pH 3.0; 0.02 M) in gradient mode. The flow rate was set at 1.0 mL/min, and chromatograms were extracted at 262 nm using a photodiode array detector. The method was successfully validated in accordance to International Conference on Harmonization (ICH) guidelines acceptance criteria for linearity, accuracy, precision, robustness and forced degradation studies, which further proved the stability-indicating power. Linearity of ambrisentan peak area responses was demonstrated within the concentration range of 25-200 µg/mL. The limits of detection and quantitation were 0.2 and 0.6 µg/mL, respectively. Forced degradation studies were performed on ambrisentan bulk drug samples as per ICH guidelines to demonstrate the stability-indicating power of the HPLC method. Significant degradation was observed during acidic hydrolysis, neutral hydrolysis and oxidative stress. The degradation products were well resolved from primary peak of ambrisentan, indicating that the method is specific and stability indicating. The newly developed method is applicable for assay determination of active pharmaceutical ingredient.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Cromatografía de Fase Inversa/métodos , Antagonistas de los Receptores de Endotelina/análisis , Fenilpropionatos/análisis , Piridazinas/análisis , Estabilidad de Medicamentos , Antagonistas de los Receptores de Endotelina/química , Fenilpropionatos/química , Piridazinas/química , Reproducibilidad de los ResultadosRESUMEN
PURPOSE OF REVIEW: Excessive production of fibrosis is a feature of hypertension-induced renal injury. Activation of RhoA/Rho-kinase (ROCK) axis has been shown in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. We assessed whether selective endothelin receptor blockers can attenuate renal fibrosis by inhibiting RhoA/ROCK axis in DOCA-salt rats. METHODS: At 4 weeks after the start of DOCA-salt treatment and uninephrectomization, male Wistar rats were randomized into three groups for 4 weeks: vehicle, ABT-627 (endothelin-A receptor inhibitor) and A192621 (endothelin-B receptor inhibitor). RESULTS: DOCA-salt was characterized by increased blood pressure, decreased renal function, increased proteinuria, increased glomerulosclerosis and tubulointerstitial fibrosis with myofibroblast accumulation, increased renal endothelin-1 levels and RhoA activity along with increased expression of connective tissue growth factor at both mRNA and protein levels as compared with uninephrectomized control male Wistar rats. Treatment with a selective mineralocorticoid receptor antagonist, eplerenone, ameliorated proteinuria. Impaired renal function and histological changes were overcome by treatment with ABT-627, but not with A192621. The beneficial effects of bosentan, a nonspecific endothelin receptor blocker, on proteinuria, RhoA activity, and connective tissue growth factor levels were similar to ABT-627. Furthermore, in an isolated perfuse kidney, a RhoA inhibitor, C3 exoenzyme, and two ROCK inhibitors, fasudil and Y-27632, significantly attenuated connective tissue growth factor levels. CONCLUSIONS: These results indicate that DOCA-salt elevates renal endothelin-1 levels and RhoA activity via activation of mineralocorticoid receptor, resulting in renal fibrosis and proteinuria. Endothelin-A receptor blockade can attenuate DOCA-salt-induced renal fibrosis probably through the inhibition of RhoA/ROCK activity and connective tissue growth factor expression.
Asunto(s)
Acetato de Desoxicorticosterona/química , Antagonistas de los Receptores de Endotelina/química , Regulación de la Expresión Génica , Hipertensión/tratamiento farmacológico , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Animales , Atrasentán , Presión Sanguínea , Endotelina-1/metabolismo , Fibrosis/prevención & control , Hipertensión/inducido químicamente , Riñón/lesiones , Masculino , Proteinuria/metabolismo , Pirrolidinas/química , Distribución Aleatoria , Ratas , Ratas Wistar , Cloruro de Sodio DietéticoRESUMEN
The competitive endothelin receptor antagonists (ERA) bosentan and ambrisentan, which have long been approved for the treatment of pulmonary arterial hypertension, are characterized by very short (1 min) occupancy half-lives at the ET(A) receptor. The novel ERA macitentan, displays a 20-fold increased receptor occupancy half-life, causing insurmountable antagonism of ET-1-induced signaling in pulmonary arterial smooth muscle cells. We show here that the slow ET(A) receptor dissociation rate of macitentan was shared with a set of structural analogs, whereas compounds structurally related to bosentan displayed fast dissociation kinetics. NMR analysis showed that macitentan adopts a compact structure in aqueous solution and molecular modeling suggests that this conformation tightly fits into a well-defined ET(A) receptor binding pocket. In contrast the structurally different and negatively charged bosentan-type molecules only partially filled this pocket and expanded into an extended endothelin binding site. To further investigate these different ET(A) receptor-antagonist interaction modes, we performed functional studies using ET(A) receptor variants harboring amino acid point mutations in the presumed ERA interaction site. Three ET(A) receptor residues significantly and differentially affected ERA activity: Mutation R326Q did not affect the antagonist activity of macitentan, however the potencies of bosentan and ambrisentan were significantly reduced; mutation L322A rendered macitentan less potent, whereas bosentan and ambrisentan were unaffected; mutation I355A significantly reduced bosentan potency, but not ambrisentan and macitentan potencies. This suggests that--in contrast to bosentan and ambrisentan--macitentan-ET(A) receptor binding is not dependent on strong charge-charge interactions, but depends predominantly on hydrophobic interactions. This different binding mode could be the reason for macitentan's sustained target occupancy and insurmountable antagonism.