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 b 2 -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 1 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.
The contribution of gene expression changes to the adverse and therapeutic effects of -adrenoceptor agonists in asthma was investigated using human airway epithelial cells as a therapeutically relevant target. Operational model-fitting established that the long-acting-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., ,, ,, ,, ,) encode proteins with proinflammatory activity and are annotated by several, enriched gene ontology (GO) terms, including ,, and The general enriched GO term was also associated with indacaterol-induced genes, and many of those, including ,, and 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 -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-adrenoceptor agonists in asthma.
BACKGROUND AND PURPOSEInhaled glucocorticoid (ICS)/long-acting β2-adrenoceptor agonist (LABA) combination therapy is a recommended treatment option for patients with moderate/severe asthma in whom adequate control cannot be achieved by an ICS alone. Previously, we discovered that LABAs can augment dexamethasone-inducible gene expression and proposed that this effect may explain how these two drugs interact to deliver superior clinical benefit. Herein, we extended that observation by analysing, pharmacodynamically, the effect of the LABA, indacaterol, on glucocorticoid receptor (GR)-mediated gene transcription induced by seven ligands with intrinsic activity values that span the spectrum of full agonism to antagonism. EXPERIMENTAL APPROACHBEAS-2B human airway epithelial cells stably transfected with a 2× glucocorticoid response element luciferase reporter were used to model gene transcription together with an analysis of several glucocorticoid-inducible genes. KEY RESULTSIndacaterol augmented glucocorticoid-induced reporter activation in a manner that was positively related to the intrinsic activity of the GR agonist. This effect was demonstrated by an increase in response maxima without a change in GR agonist affinity or efficacy. Indacaterol also enhanced glucocorticoid-inducible gene expression. However, the magnitude of this effect was dependent on both the GR agonist and the gene of interest. CONCLUSIONS AND IMPLICATIONSThese data suggest that indacaterol activates a molecular rheostat, which increases the transcriptional competency of GR in an agonist-and gene-dependent manner without apparently changing the relationship between fractional GR occupancy and BJP British Journal of Pharmacology
BACKGROUND AND PURPOSEInternational asthma guidelines recommend that inhaled glucocorticoids be used as a monotherapy in all patients with mild to moderate disease because of their ability to suppress airways inflammation. Current evidence suggests that the therapeutic benefit of glucocorticoids is due to the transactivation and transrepression of anti-inflammatory and pro-inflammatory genes respectively. However, the extent to which clinically relevant glucocorticoids are equivalent in their ability to modulate gene expression is unclear. EXPERIMENTAL APPROACHA pharmacodynamics investigation of glucocorticoid receptor (GR)-mediated gene transactivation in BEAS-2B human airway epithelial cells was performed using a glucocorticoid response element luciferase reporter coupled with an analysis of glucocorticoid-inducible genes encoding proteins with anti-inflammatory and adverse-effect potential. KEY RESULTSUsing transactivation as a functionally relevant output, a given glucocorticoid displayed a unique, gene expression 'fingerprint' where intrinsic efficacy and GR density were essential determinants. We showed that depending on the gene selected for analysis, a given glucocorticoid can behave as an antagonist, partial agonist, full agonist or even 'super agonist'. In the likely event that different, tissue-dependent gene expression profiles are reproduced in vivo, then the anti-inflammatory and adverse-effect potential of many glucocorticoids currently available as asthma therapeutics may not be equivalent. CONCLUSIONS AND IMPLICATIONSThe generation of gene expression 'fingerprints' in target and off-target human tissues could assist the rational design of GR agonists with improved therapeutic ratios. This approach could identify compounds that are useful in the management of severe asthma and other inflammatory disorders where systemic exposure is desirable. AbbreviationsCRISPLD2, cysteine-rich secretory protein LCCL (Limulus clotting factor C, Cochlin, Lgl1) domain-containing 2; DC, desisobutyrylciclesonide; Dex, dexamethasone; FF, fluticasone furoate; GILZ, glucocorticoid-inducible leucine zipper; GR, glucocorticoid receptor; GRE, glucocorticoid response element; GW, GW 870086X (6α,9α-difluoro-11β-hydroxy-16α-methyl-3-oxo-17α-(2,2,3,3-tetramethylcyclopropylcarbonyl)-oxo-androsta-1,4-diene-17β-carboxylic acid cyanomethylester); HC, hydrocortisone; HSD, hydroxysteroid dehydrogenase; Mif, mifepristone; Org, Org 34517 (11β-(1,3-benzodioxolo)-17β-hydroxy-17-(1-propynyl)-oestra-4,9-dien-3-one); p57 kip2 , kinase inhibitor protein 2 of 57 kDa; PDK, pyruvate dehydrogenase kinase; SFM, serum-free medium
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 A 2B -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 A 2B -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 A 2B -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 A 2B -receptor agonist and a glucocorticoid administered together may have utility in the treatment of inflammatory disorders that respond suboptimally to glucocorticoids as a monotherapy.
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