RESUMEN
The effects of phosphodiesterase (PDE) 4 inhibitors on gene expression changes in BEAS-2B human airway epithelial cells are reported and discussed in relation to the mechanism(s) of action of roflumilast in chronic obstructive pulmonary disease (COPD). Microarray-based gene expression profiling failed to identify mRNA transcripts that were differentially regulated by the PDE4 inhibitor 6-[3-(dimethylcarbamoyl)benzenesulphonyl]-4-[(3-methoxyphenyl)amino]-8-methylquinoline-3-carboxamide (GSK 256066) after 1, 2, 6, or 18 hours of exposure. However, real-time polymerase chain reaction analysis revealed that GSK 256066 was a weak stimulus, and the negative microarray results reflected low statistical power due to small sample sizes. Furthermore, GSK 256066, roflumilast, and its biologically active metabolite roflumilast N-oxide generally potentiated gene expression changes produced by the long-acting ß 2-adrenoceptor agonists (LABAs) salmeterol, indacaterol, and formoterol. Many of these genes encode proteins with antiviral, anti-inflammatory, and antibacterial activities that could contribute to the clinical efficacy of roflumilast in COPD. RNA-sequencing experiments established that the sensitivity of genes to salmeterol varied by â¼7.5-fold. Consequently, the degree to which a PDE4 inhibitor potentiated the effect of a given concentration of LABA was gene-dependent. Operational model fitting of concentration-response curve data from cells subjected to fractional, ß 2-adrenoceptor inactivation determined that PDE4 inhibition increased the potency and doubled the efficacy of LABAs. Thus, adding roflumilast to standard triple therapy, as COPD guidelines recommend, may have clinical relevance, especially in target tissues where LABAs behave as partial agonists. Collectively, these results suggest that the genomic impact of roflumilast, including its ability to augment LABA-induced gene expression changes, may contribute to its therapeutic activity in COPD.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Indanos/farmacología , Pulmón/citología , Inhibidores de Fosfodiesterasa 4/farmacología , Quinolonas/farmacología , Agonistas de Receptores Adrenérgicos beta 2/farmacología , Aminopiridinas/farmacología , Aminoquinolinas/farmacología , Benzamidas/farmacología , Línea Celular , Ciclopropanos/farmacología , Sinergismo Farmacológico , Células Epiteliales/química , Células Epiteliales/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Pulmón/química , Pulmón/efectos de los fármacos , Análisis de Secuencia por Matrices de Oligonucleótidos , Xinafoato de Salmeterol/farmacología , Sulfonas/farmacologíaRESUMEN
The contribution of gene expression changes to the adverse and therapeutic effects of ß2-adrenoceptor agonists in asthma was investigated using human airway epithelial cells as a therapeutically relevant target. Operational model-fitting established that the long-acting ß2-adrenoceptor agonists (LABA) indacaterol, salmeterol, formoterol, and picumeterol were full agonists on BEAS-2B cells transfected with a cAMP-response element reporter but differed in efficacy (indacaterol ≥ formoterol > salmeterol ≥ picumeterol). The transcriptomic signature of indacaterol in BEAS-2B cells identified 180, 368, 252, and 10 genes that were differentially expressed (>1.5- to <0.67-fold) after 1-, 2-, 6-, and 18-hour of exposure, respectively. Many upregulated genes (e.g., AREG, BDNF, CCL20, CXCL2, EDN1, IL6, IL15, IL20) encode proteins with proinflammatory activity and are annotated by several, enriched gene ontology (GO) terms, including cellular response to interleukin-1, cytokine activity, and positive regulation of neutrophil chemotaxis The general enriched GO term extracellular space was also associated with indacaterol-induced genes, and many of those, including CRISPLD2, DMBT1, GAS1, and SOCS3, have putative anti-inflammatory, antibacterial, and/or antiviral activity. Numerous indacaterol-regulated genes were also induced or repressed in BEAS-2B cells and human primary bronchial epithelial cells by the low efficacy LABA salmeterol, indicating that this genomic effect was neither unique to indacaterol nor restricted to the BEAS-2B airway epithelial cell line. Collectively, these data suggest that the consequences of inhaling a ß2-adrenoceptor agonist may be complex and involve widespread changes in gene expression. We propose that this genomic effect represents a generally unappreciated mechanism that may contribute to the adverse and therapeutic actions of ß2-adrenoceptor agonists in asthma.
Asunto(s)
Agonistas de Receptores Adrenérgicos beta 2/farmacología , Bronquios/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Indanos/farmacología , Quinolonas/farmacología , Receptores Adrenérgicos beta 2/metabolismo , Transcriptoma/efectos de los fármacos , Agonistas de Receptores Adrenérgicos beta 2/efectos adversos , Agonistas de Receptores Adrenérgicos beta 2/uso terapéutico , Línea Celular , AMP Cíclico/metabolismo , Humanos , Indanos/efectos adversos , Indanos/uso terapéutico , Quinolonas/efectos adversos , Quinolonas/uso terapéutico , Factores de TiempoRESUMEN
(R)-6-[(3-{[4-(5-{[2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethyl]amino}pent-1-yn-1-yl)phenyl] carbamoyl}phenyl)sulphonyl]-4-[(3-methoxyphenyl)amino]-8-methylquinoline-3-carboxamide trifluoroacetic acid (GS-5759) is a bifunctional ligand composed of a quinolinone-containing pharmacophore [ß2-adrenoceptor agonist orthostere (ß2A)] found in several ß2-adrenoceptor agonists, including indacaterol, linked covalently to a phosphodiesterase 4 (PDE4) inhibitor related to 6-[3-(dimethylcarbamoyl)benzenesulphonyl]-4-[(3-methoxyphenyl)amino]-8-methylquinoline-3-carboxamide (GSK 256066) by a pent-1-yn-1-ylbenzene spacer. GS-5759 had a similar affinity for PDE4B1 and the native ß2-adrenoceptor expressed on BEAS-2B human airway epithelial cells. However, compared with the monofunctional parent compound, ß2A, the KA of GS-5759 for the ß2-adrenoceptor was 35-fold lower. Schild analysis determined that the affinities of the ß-adrenoceptor antagonists, (2R,3R)-1-[(2,3-dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl) amino]-2-butanol (ICI 118551) and propranolol, were agonist-dependent, being significantly lower for GS-5759 than ß2A. Collectively, these data can be explained by "forced proximity," bivalent binding where the pharmacophore in GS-5759 responsible for PDE4 inhibition also interacts with a nonallosteric domain within the ß2-adrenoceptor that enhances the affinity of ß2A for the orthosteric site. Microarray analyses revealed that, after 2-hour exposure, GS-5759 increased the expression of >3500 genes in BEAS-2B cells that were highly rank-order correlated with gene expression changes produced by indacaterol and GSK 256066 in combination (Ind/GSK). Moreover, the line of regression began close to the origin with a slope of 0.88, indicating that the magnitude of most gene expression changes produced by Ind/GSK was quantitatively replicated by GS-5759. Thus, GS-5759 is a novel compound exhibiting dual ß2-adrenoceptor agonism and PDE4 inhibition with potential to interact on target tissues in a synergistic manner. Such polypharmacological behavior may be particularly effective in chronic obstructive pulmonary disease and other complex disorders where multiple processes interact to promote disease pathogenesis and progression.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Células Epiteliales/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Enfermedad Pulmonar Obstructiva Crónica/genética , Quinolonas/farmacología , Receptores Adrenérgicos beta 2/metabolismo , Sistema Respiratorio/patología , Sulfonas/farmacología , Agonistas de Receptores Adrenérgicos beta 2/farmacología , Agonistas de Receptores Adrenérgicos beta 2/uso terapéutico , Aminoquinolinas/farmacología , Línea Celular , Interacciones Farmacológicas , Células Epiteliales/metabolismo , Humanos , Indanos/farmacología , Inhibidores de Fosfodiesterasa 4/farmacología , Inhibidores de Fosfodiesterasa 4/uso terapéutico , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Enfermedad Pulmonar Obstructiva Crónica/patología , Quinolonas/uso terapéutico , Sulfonas/uso terapéuticoRESUMEN
Chronic obstructive pulmonary disease (COPD) is a neutrophilic inflammatory disorder that is weakly responsive to glucocorticoids. Identification of ways to enhance the anti-inflammatory activity of glucocorticoids is, therefore, a major research objective. Adenosine receptor agonists that target the A2B-receptor subtype are efficacious in several cell-based assays and preclinical models of inflammation. Accordingly, the present study was designed to determine if a selective A2B-receptor agonist, 2-[6-amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)phenyl]pyridin-2-ylsulphanyl]acetamide (Bay 60-6583), and a glucocorticoid, dexamethasone, in combination display putative anti-inflammatory activity that is superior to either drug alone. In BEAS-2B human airway epithelial cells stably transfected with cAMP-response element (CRE) and glucocorticoid response element (GRE) reporter constructs, Bay 60-6583 promoted CRE-dependent transcription and enhanced GRE-dependent transcription by an adenosine A2B-receptor-mediated mechanism that was associated with cAMP formation and abolished by an inhibitor of cAMP-dependent protein kinase. Analysis of the concentration-response relationship that described the enhancement of GRE-dependent transcription showed that Bay 60-6583 increased the magnitude of response without affecting the potency of dexamethasone. Bay 60-6583 and dexamethasone also induced a panel of genes that, collectively, could have benefit in COPD. These were categorized into genes that were induced in a positive cooperative manner (RGS2, p57(kip2)), an additive manner (TTP, BRL-1), or by Bay 60-6583 (CD200, CRISPLD2, SOCS3) or dexamethasone (GILZ) only. Thus, the gene induction "fingerprints" produced by Bay 60-6583 and dexamethasone, alone and in combination, were distinct. Collectively, through their actions on gene expression, an adenosine A2B-receptor agonist and a glucocorticoid administered together may have utility in the treatment of inflammatory disorders that respond suboptimally to glucocorticoids as a monotherapy.
Asunto(s)
Agonistas del Receptor de Adenosina A2/farmacología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Inflamación/genética , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Receptor de Adenosina A2B/fisiología , Receptores de Glucocorticoides/agonistas , Mucosa Respiratoria/citología , Mucosa Respiratoria/metabolismo , Adenosina/análogos & derivados , Adenosina/farmacología , Algoritmos , Aminopiridinas/farmacología , Calcio/metabolismo , Línea Celular , AMP Cíclico/metabolismo , Modulador del Elemento de Respuesta al AMP Cíclico/genética , Citosol/metabolismo , Dexametasona/farmacología , Humanos , Fenetilaminas/farmacología , Enfermedad Pulmonar Obstructiva Crónica/patología , ARN Mensajero/biosíntesis , ARN Mensajero/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa , Mucosa Respiratoria/efectos de los fármacos , TransfecciónRESUMEN
Post-hoc analysis of two phase III clinical studies found that the phosphodiesterase 4 (PDE4) inhibitor, roflumilast, reduced exacerbation frequency in patients with severe chronic obstructive pulmonary disease (COPD) who were taking inhaled corticosteroids (ICS) concomitantly, whereas patients not taking ICS derived no such benefit. In contrast, in two different trials also performed in patients with severe COPD, roflumilast reduced exacerbation rates in the absence of ICS, indicating that PDE4 inhibition alone is sufficient for therapeutic activity to be realized. Given that roflumilast is recommended as an "add-on" medication to patients with severe disease who will inevitably be taking a long-acting ß2-adrenoceptor agonist (LABA)/ICS combination therapy, we tested the hypothesis that roflumilast augments the ability of glucocorticoids to induce genes with anti-inflammatory activity. Using a glucocorticoid response element (GRE) luciferase reporter transfected into human airway epithelial cells [both bronchial epithelium + adenovirus 12 - SV40 hybrid (BEAS-2B) cells and primary cultures], roflumilast enhanced fluticasone propionate-induced GRE-dependent transcription. Roflumilast also produced a sinistral displacement of the concentration-response curves that described the augmentation of GRE-dependent transcription by the LABA formoterol. In BEAS-2B cells and primary airway epithelia, roflumilast interacted with formoterol in a positive cooperative manner to enhance the expression of several glucocorticoid-inducible genes that have anti-inflammatory potential. We suggest that the ability of roflumilast and formoterol to interact in this way supports the concept that these drugs together may impart clinical benefit beyond that achievable by an ICS alone, a PDE4 inhibitor alone, or an ICS/LABA combination therapy. Roflumilast may, therefore, be especially effective in patients with severe COPD.
Asunto(s)
Agonistas de Receptores Adrenérgicos beta 2/administración & dosificación , Aminopiridinas/administración & dosificación , Benzamidas/administración & dosificación , Etanolaminas/administración & dosificación , Glucocorticoides , Inhibidores de Fosfodiesterasa 4/administración & dosificación , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Células Cultivadas , Ciclopropanos/administración & dosificación , Sinergismo Farmacológico , Quimioterapia Combinada , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Femenino , Fumarato de Formoterol , Glucocorticoides/metabolismo , Humanos , Masculino , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/patología , Mucosa Respiratoria/efectos de los fármacos , Mucosa Respiratoria/metabolismo , Transcripción Genética/efectos de los fármacos , Transcripción Genética/fisiología , Resultado del TratamientoRESUMEN
A novel bacterial consortium (TJ-2) for mineralization of aromatic amines resulting from decolorization of azo dyes was developed. Three bacterial strains were identified as Pseudomonas pseudoalcaligenes (TJ-21,EU072476), Pseudomonas citronellolis (TJ-22,EU072477) and Pseudomonas testosterone (TJ-23,EU072477) by 16S rRNA gene sequence analysis. Aromatic amine mineralization under aerobic conditions was observed to be significantly higher with the consortium as compared to pure strains indicating complementary interactions among these strains. It was observed that more than 90% mineralization of aromatic amines was achieved within 18h for different initial aromatic amines concentrations. It was also observed that aromatic amine mineralization depends upon the structure of aromatic amine. Para- and meta-hydroxy substituted aromatic amine were easily mineralized as compared to ortho-substituted which undergoes autoxidation when exposed to oxygen. The consortium was capable of mineralizing other aromatic amines, thus, conferring the possibility of application of TJ-2 for the treatment of industrial wastewaters containing aromatic amines.
Asunto(s)
Bacterias/metabolismo , Minerales/metabolismo , Microbiología del Suelo , Ácidos Sulfanílicos/metabolismo , Aerobiosis , Bacterias/genética , Bacterias/crecimiento & desarrollo , Bacterias/aislamiento & purificación , Biodegradación Ambiental , Biomasa , Datos de Secuencia Molecular , Filogenia , ARN Ribosómico 16S/genéticaRESUMEN
A novel bacterial consortium (TJ-1), which could decolorize Acid Orange 7 (AO7) and manyother azo dyes, was developed. In TJ-1 three bacterial strains were identified as Aeromonas caviae, Proteus mirabilis and Rhodococcus globerulus by 16S rRNA gene sequence analysis. AO7 decolorization was significantly higher with the use of consortium as compared to the use of individual strains, indicating complementary interactions among these strains. AO7 decolorization was observed under microaerophilic condition in the presence of organic carbon source. Either yeast extract (YE) alone or a combination of YE and glucose resulted in much higher decolorization of AO7 as compared to glucose alone, peptone or starch. Kinetic studies with different initial AO7 concentrations showed that more than 90% decolorization could be achieved even at 200mg/l within 16h. Fed-batch studies showed that AO7 decolorization required 10h during the first cycle and 5h in the second and third cycles, showing that bacterial cells could be used for multiple cycles. The consortium also decolorized fifteen other azo dyes individually as well as a simulated wastewater containing a mixture of all the sixteen azo dyes, thus, conferring the possibility of application of TJ-1 for the treatment of industrial wastewaters.