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Interactions between Corticosteroids and 2-Agonists in Asthma and Chronic Obstructive Pulmonary Disease

Malcolm Johnson
GlaxoSmithKline Research and Development, Middlesex, United Kingdom

The combination of a long-acting 2-agonist (LABA) and an inhaled corticosteroid is more efficacious in asthma and chronic obstructive pulmonary disease (COPD) than other combination therapies or than either alone. Corticosteroids may regulate 2-receptor function by increasing expression of the receptor, restoring G-protein/ 2-receptor coupling, and inhibiting 2-receptor downregulation. LABAs may prime the glucocorticoid receptor and affect its nuclear localization by modulating glucocorticoid receptor phosphorylation. These mechanisms suggest the possibility of interactions between the two classes of drugs at the molecular and receptor levels. Indeed, there is evidence that corticosteroid/2-agonist combination therapy has complementary, additive, and synergistic inhibitory effects on proinflammatory signaling pathways, inflammatory mediator release, and recruitment and survival of inflammatory cells. In the asthmatic patient, this is reflected in enhanced antiinflammatory activity with combination therapy beyond that which can be achieved by either drug alone, or the potential for LABAs to provide a steroid-sparing effect. Corticosteroid/2-agonist combined activity may also overcome the reduced sensitivity to inhaled corticosteroids that has been reported in some patients with COPD. Keywords: combination therapy; inhaled corticosteroids; long-acting 2-agonists; mechanisms

The underlying pathophysiology of bronchial asthma and chronic obstructive pulmonary disease (COPD) is complex and multifactorial. Bronchial asthma is characterized by smooth-muscle dysfunction, airway inammation, and airway remodeling (1). Smoothmuscle dysfunction is evidenced by exaggerated bronchoconstriction, bronchial hyperresponsiveness, hyperplasia, and possibly hypertrophy of the airway smooth-muscle cells and their release of proinammatory mediators. In asthma, acute and chronic airway inammation (1) is the result of the recruitment and activation of a range of inammatory cells, including mast cells, eosinophils, and CD4 T lymphocytes, and the release of mediators that perpetuate the inammatory cycle, causing edema formation and epithelial damage. The remodeling process induces permanent changes to the airway structure, such as the proliferation of resident cells and thickening of the reticular layer of the basement membrane (1). COPD is a heterogeneous group of disorders involving the large airways, small airways, and lung parenchyma (2), consisting of airway inammation, structural changes, mucociliary dysfunction, and a systemic component, all of which contribute to the airow limitation characteristic of the disease. There are increased numbers of inammatory cells, such as CD8 T lymphocytes, monocytes/macrophages, and neutrophils, and elevated levels of in-

ammatory mediators, such as interleukin (IL)-8, tumor necrosis factor (TNF)-, leukotriene B4, and in exacerbations the chemokine, regulated upon activation, normal T-cell expressed and secreted (RANTES). Inammation in COPD is associated with many structural changes, such as goblet cell hyperplasia and submucosal glandular hypertrophy in the large airways, brosis with collagen deposition and thickening of airway smooth muscle in the small airways, and alveolar destruction in the parenchyma (2). Patients with COPD exhibit ciliary abnormalities, mucus hypersecretion, increased mucus viscosity, and reduced mucociliary transport, all of which promote bacterial colonization and mucosal damage (2). A number of clinical studies (36) have shown that patients with asthma and patients with COPD treated with long-acting 2-agonists (LABAs) and inhaled corticosteroids (ICSs) have better lung function, increased symptom control, and reduced exacerbations compared with those given either LABAs or higher doses of ICS alone. In asthma, ICSs inhibit many aspects of chronic inammation and may resolve some of the processes of airway remodeling, whereas LABAs have long-lasting effects on airway smooth muscle and attenuate components of acute inammation (7). In COPD, ICSs have been shown to affect key inammatory cells and mediators, and LABAs have additional effects on mucociliary dysfunction and may inuence elements of the systemic component of the disease (8). Furthermore, the combination of a LABA and an ICS provides greater clinical efcacy in both diseases than other combination therapies, suggesting the possibility of complementary interactions between LABAs and corticosteroids at the molecular and receptor levels.


The target receptor for corticosteroids is the intracellular glucocorticoid receptor (GR). Under resting conditions, the inactive GR is largely located in the cytosol of the cell, associated with multiple chaperone proteins. The glucocorticoid molecule rst penetrates the cell membrane, then binds to the GR through the glucocorticoid-binding domain (9). This induces a conformational change in the receptor protein, dissociation of the chaperone proteins, and the formation of an active glucocorticoidGR complex. The complex may then form a dimer and translocate from the cytosol to the nucleus of the cell, where it binds to specic DNA sequences (glucocorticoid response elements, GREs) in the promoter region of target genes, leading to cofactor activation and either an increase or decrease in gene transcription. This process is termed transactivation (9). Alternatively, the active glucocorticoidGR complex, as a monomer, can interact directly with intracellular transcription factors such as activator protein-1 (AP-1) or nuclear factor (NF)-B, through a proteinprotein interaction, to attenuate the proinammatory processes mediated by those transcription factors. This process, termed transrepression, involves recruitment of histone deacetylases and modulation of chromatin structure (10).

(Received in original form February 18, 2004; accepted in final form April 13, 2004) Correspondence and requests for reprints should be addressed to Malcolm Johnson, Ph.D., GlaxoSmithKline Research & Development, Greenford Road, Middlesex UB6 OHE, UK. Email: Proc Am Thorac Soc Vol 1. pp 200206, 2004 DOI: 10.1513/pats.200402-010MS Internet address:


All 2-agonists exert their biological and therapeutic effects through cell-surface 2-adrenoceptors, which are members of

Johnson: Interactions with 2-Agonists


the 7-transmembrane, G-proteincoupled receptor family. After ligand binding to the active site of the receptor, the -component of the associated Gs-protein dissociates and activates adenylate cyclase. This process leads to the production of intracellular cyclic adenosine monophosphate (cAMP) and subsequent activation of protein kinase A (PKA), which then phosphorylates a number of intracellular regulatory proteins (11). 2-Agonists may also inuence gene transcription, through a cAMP mechanism, whereby there is increased translocation of the catalytic subunit of PKA to the nucleus and phosphorylation of CREB-binding protein, enhancing its DNA-binding and transactivation effects (12, 13). Recently, it has been reported that activation of the 2-receptor can also lead to coupling to Gi, resulting in stimulation of the extracellular signal-regulated kinase pathway and the p38 mitogen-activated protein kinase (MAPK) pathway (14). The mechanisms of action of the LABAs, salmeterol and formoterol, have been reported previously (1416). Briey, salmeterol partitions into the cell membrane and diffuses laterally to the 2-receptor. The side chain of the molecule then binds to a discrete, hydrophobic region of the fourth transmembrane domain, the exo-site. Binding to the exo-site prevents salmeterol from dissociating from the receptor, while the saligenin head of the molecule is free to engage and disengage the active site by ` re (hinge) principle, leading to a long, concentrationa Charnie independent duration of action (14). Formoterol, which is moderately lipophilic, is taken up into the cell membrane as a depot. The drug then progressively leaches out to activate the 2-receptor, imparting a prolonged, concentration-dependent action (16).

been depleted of corticosteroids by adrenalectomy, in contrast, lose the sensitivity of the 2-receptorcoupled adenylate cyclase system. Another study (21) has shown that proinammatory cytokines such as IL-1 and transforming growth factor (TGF)-1 regulate the coupling of 2-receptors to adenylate cyclase and receptor sensitivity. Corticosteroids, by reducing the concentration of such cytokines, may prevent or reverse these effects. This may have clinical relevance in preventing the development of tolerance to 2-agonists in patients with asthma and patients with COPD on chronic therapy.
Effects of LABAs on the GR


In discussing possible interactions between corticosteroids and LABAs, it is important to make clear distinctions between three similar terms. A complementary effect occurs when the response to two or more drugs combined is greater than the response to either drug alone. An additive effect occurs when the response of two or more drugs combined equals the sum of the responses to the individual drugs. A synergistic effect occurs when the response to two or more drugs combined is greater than the sum of the responses to the individual drugs.
Effects of Corticosteroids on 2-Receptors

Corticosteroids can modulate 2-receptors and their function by several mechanisms: protection against desensitization and the development of tolerance, increased efciency of receptor coupling, and protection against inammation-induced receptor downregulation and uncoupling. Corticosteroids stimulate the transcription of 2-receptors via binding to GREs in the 5-noncoding promoter region of the 2-receptor gene (Figure 1). Dexamethasone has been shown to increase 2-receptor mRNA and protein in human lung tissue in vitro (17). This increase in transcription, which has also been shown in neutrophils and T cells, is both time- and dose-dependent, consistent with the later induction of receptor-binding activity. However, dexamethasone has not been found to alter the half-life of 2-receptor mRNA (17). Baraniuk and colleagues (18) reported that intranasal administration of beclomethasone dipropionate (100 g/day 3 days) signicantly increased the density of 2-receptors on the nasal mucosa. Both systemic corticosteroids and ICSs reversed 2-receptor downregulation after exposure to high doses of short-acting 2-agonists (19). Corticosteroids also reportedly modulate the efciency of coupling between the 2-receptor and Gs (20). As a result, 2-receptorstimulated adenylate cyclase activity and cAMP accumulation increase after corticosteroid treatment. Animals that have

LABAs prime the GR for subsequent corticosteroid binding by mitogen-activated protein kinase (MAPK)-dependent phosphorylation (22). After stimulation of the 2-receptor by salmeterol and dissociation of the -component of Gs, the residual -subunit of the G-protein initiates an intracellular signaling cascade involving the nonreceptor tyrosine kinase C-Src and the G-protein Ras, culminating in the stimulation of MAPK (23). This has been demonstrated in human embryonic kidney cells transfected with a MAPKluciferin/luciferase reporter construct, as well as by isolation of phospho-MAPK (24). The identity of the MAPK activated by salmeterol is unclear. Activation of the MAPK pathway by the LABA requires that the 2-receptor be phosphorylated, probably by PKA. MAPK then phosphorylates the GR at a number of proline-directed serine residues in the N-terminus region of the receptor. Indeed, phospho-GR has been detected in cells containing both 2-receptors and GR, when stimulated with salmeterol (25). It is possible that an increase in negative charge at the N-terminal domain of the receptor leads to a conformational change in the GR protein, leading in turn to the priming event and rendering the receptor more sensitive to steroid-dependent activation (Figure 1). Other studies have shown that translocation of the GR from the cell cytosol to the nucleus, a fundamental step in the antiinammatory activity of corticosteroids, is increased by the addition of a LABA (26, 27). Eickelberg and colleagues (26) showed that treatment of human lung broblasts and smooth muscle cells with uticasone and salmeterol resulted in increased translocation compared with uticasone treatment alone. Electrophoretic mobility shift assays conrmed that the LABA-activated nuclear GR actively bound to the GRE consensus sequence. The effects of salmeterol were mediated by the 2-receptor and by PKA, as receptor blockade with propranolol or the use of a PKA-inhibitor peptide abrogated GR activation (26). These in vitro ndings have now been conrmed in vivo in patients with asthma treated with LABA and corticosteroids. Usmani and colleagues (27) showed that inhalation of salmeterol (50 g) and uticasone (100 g) increased the nuclear translocation of the GR in sputum macrophages signicantly more than the same dose of uticasone alone. The results were equivalent to those observed with a vefold higher dose of the steroid. Whether these effects of salmeterol can be shown in the patient with COPD remains to be determined. In contrast, whereas budesonide (800 g) also signicantly increased the translocation of the GR from the cytosol into the nuclei of peripheral blood leukocytes, combination with formoterol (24 g) did not enhance this effect versus budesonide alone (28). The interactions between corticosteroids and LABAs may, therefore, be summarized as follows: corticosteroids increase 2-receptor synthesis, and LABAs prime the GR for steroiddependent activation. These mechanisms form a possible molecular and receptor basis for additive and synergistic interactions between the two classes of drugs in the treatment of asthma and COPD.


PROCEEDINGS OF THE AMERICAN THORACIC SOCIETY VOL 1 2004 Figure 1. Synergistic interactions between long-acting 2agonists (LABA; salmeterol) and corticosteroids (fluticasone). Corticosteroids penetrate the cell membrane and bind to inactive glucocorticoid receptor (GR), forming an active receptor complex. This complex translocates to the nucleus and activates target genes such as the 2-adrenoceptor gene. 2Receptor protein is synthesized and inserted into the cell membrane. LABAs, through the 2receptor, stimulate an intracellular biochemical cascade, leading to priming of the GR. The primed GR requires less corticosteroids to form an active receptor complex. These interacting pathways provide a molecular basis for positive interactions between LABAs and corticosteroids.

Effects of Corticosteroid/LABA on Proinflammatory Signaling Pathways

Airway inammation is an important component of the pathophysiology of both asthma and COPD (1, 2). Proinammatory transcription factors such as NF-B and AP-1 are key to the signaling pathways involved. NF-B is retained in the cytoplasm by the inhibitory protein IB, but when this is phosphorylated by the IB kinase, IKK-2, NF-B is released for transport to the nucleus, where it promotes the transcription of proinammatory genes (29). TNF- induction of a NF-B reporter gene in airway epithelial cells is inhibited by uticasone, and this inhibition is enhanced in the presence of salmeterol (30). The mechanism of this effect may be mediated through reduced IB phosphorylation. Fluticasone administration (108 to 107 M) resulted in a concentration-dependent decrease in phospho-IB in T cells. Salmeterol (108 M) was without effect, but it increased the suppressive effect of uticasone so that the combined effect was greater than that obtained with a 10-fold higher concentration of uticasone alone, thus providing evidence for synergy at the molecular level (31). In vivo studies (32) have suggested that a LABA and a corticosteroid can also interact to enhance the nuclear export of the transcription factor, GATA-3. Nuclear GATA-3 expression was reduced in mononuclear cells 60 minutes after patients inhaled 500 g of uticasone but not after administration of 100 g. However, in the presence of salmeterol (50 g), the effect of 100 g of uticasone was signicantly enhanced, and there was no difference between the effects of the two doses of uticasone (32).
Effects of Corticosteroid/LABA on Inflammatory Mediator Release

In sputum and bronchoalveolar lavage uid, elevated levels of inammatory mediators, such as IL-4, IL-5, IL-10, IL-13, and granulocyte macrophage colony-stimulating factor (GM-CSF), have been detected in asthma; elevated levels of TNF-, IL-8, leukotriene B4, and RANTES have been detected in COPD in exacerbations (1, 2). The interactions between a corticosteroid

and a LABA result in complementary inhibitory effects on cytokine and chemokine release. For example, human airway epithelial cells stimulated with TNF- release GM-CSF. This release is partially inhibited by budesonide and also by formoterol (Figure 2). However, when budesonide and formoterol are combined, the level of cytokine inhibition is increased in an additive manner (33). The combination of uticasone (109 M) and salmeterol (109 M) synergistically inhibit rhinovirus-induced IL-8 and RANTES release (34) from bronchial epithelial cells (Figure 3). Such an effect may be of relevance to the efcacy of combination therapy in reducing COPD exacerbations. Pang and Knox (35) showed that TNF- stimulated release of the eosinophil chemoattractant eotaxin from human airway smooth-muscle cells, and that this release was inhibited to some extent by both salmeterol and uticasone. The combination of salmeterol and uticasone, however, further enhanced the inhibition of eotaxin. Human airway smooth-muscle cells also produce IL-8, a major neutrophil chemoattractant involved in the pathogenesis of COPD. TNF-induced IL-8 release from human airway smooth-muscle cells was markedly inhibited by uticasone, but was unaffected by salmeterol (36). However, the combination of uticasone and salmeterol synergistically enhanced the inhibition induced by the corticosteroid alone (Figure 4). Similarly, in human lung broblasts, both budesonide and formoterol inhibited IL-1induced expression of the vascular cell adhesion molecule (VCAM), but the level of inhibition was signicantly greater when the two drugs were combined (37). Peripheral blood monocytes stimulated with cigarette smoke extract release large quantities of IL-8. This increase is markedly inhibited by the combination of salmeterol and uticasone, but not by either drug alone (38). Similar results were reported in human alveolar macrophages isolated from the bronchoalveolar lavage uid of patients with COPD. Fluticasone (108 to 1012 M) inhibited IL-8 and TNF- production in a dose-dependent manner, whereas salmeterol was without effect. The combination of the two drugs resulted in greater inhibition of cytokine production than uticasone alone (39). Elevated numbers of eosinophils have been found in induced

Johnson: Interactions with 2-Agonists


Figure 2. Additive inhibitory effect of budesonide and formoterol on granulocytemacrophage colony-stimulating factor (GM-CSF) release from human airway epithelial cells. Tumor necrosis factor- (TNF-) induced GM-CSF release was measured in the presence of budesonide (B, 108 M), formoterol (F, 1010 M), and the combination. BL indicates baseline. Adapted by permission from Ref. 33.

Figure 4. Synergistic inhibitory effect of fluticasone plus salmeterol on interleukin-8 (IL-8) release from human airway smooth-muscle cells. TNF-induced release of IL-8 was measured in the presence of fluticasone propionate (FP, 108 M), salmeterol (SALM, 107 M), and the combination. Adapted by permission from Ref. 36.

sputum, bronchial biopsies, and the lamina propria of individuals with asthma and patients with COPD, particularly during exacerbations. Both formoterol (1 to 10 nM) and budesonide (1 to 100 nM) inhibited superoxide generation from peripheral blood eosinophils (40). Even though a low concentration of budesonide (0.1 nM) had little inhibitory effect alone, the combination of budesonide and formoterol (1 nM) inhibited eosinophilic oxidative burst to a level that exceeded an additive effect (40).
Effects of Corticosteroid/LABA on Inflammatory Cells

In vitro studies. A key element in the anti-inammatory effects of corticosteroids in asthma is their ability to inhibit the recruitment of inammatory cells and shorten their survival in airway

tissue. Fluticasone inhibits eosinophil adhesion to intercellular cell-adhesion molecule-1, an important step in cell inltration, recruitment and tissue retention and this inhibition is synergistically increased in the presence of salmeterol (41). Similarly, uticasone produces a concentration-dependent increase in apoptosis in eosinophils obtained by bronchoalveolar lavage uid from individuals with asthma (42). Salmeterol alone has little effect but increases the potency of uticasone by approximately threefold. The addition of salmeterol enhances apoptosis of T-lymphocytes in response to uticasone, at both low and higher concentrations, and this increase is associated with greater GR nuclear translocation (43). The epithelium may be an additional target for the interaction

Figure 3. Synergistic inhibitory effect of fluticasone plus salmeterol on regulated on activation, normal T cell expressed and secreted (RANTES) release from human airway epithelial cells. Human rhinovirusinduced RANTES release was measured in the presence of fluticasone propionate (FP, 109 and 108 M) and the combination of FP (109 M) and salmeterol (SM, 109 M). * p .05. ** p .01. Adapted by permission from Ref. 34.



Figure 5. Synergistic effect of corticosteroid/LABA therapy on airway angiogenesis in asthma. In patients with asthma, being treated with an ICS, the effect of adding additional steroid (fluticasone propionate [FP] 100 g BID) or salmeterol (50 g BID) on angiogenesis (subepithelial vascularity as assessed by collagen IV staining) was assessed over 3 months. * p 0.004. ** p 0.04. Adapted by permission from Ref. 49.

between LABAs and corticosteroids in COPD. Dowling and colleagues (44) reported that incubation of human respiratory mucosa with uticasone or salmeterol signicantly inhibited Pseudomonas aeruginosainduced epithelial damage and loss of ciliated cells in a concentration-dependent manner. However, when a combination of uticasone and salmeterol was used, the loss of cilia from the epithelial surface was signicantly reduced compared with the results obtained after treatment with uticasone or salmeterol alone (44). Fibrotic changes in the airway in asthma and COPD have been shown to be associated with increased numbers of activated broblasts, many of which have the phenotypic characteristics of myobroblasts (e.g., they express -smooth muscle actin, -SMA). Giuliani and coworkers (45) showed that TGF-1, a cytokine present in increased quantities in COPD, induced a dose-dependent increase in the number of SMA() cells in primary human airway broblast culture. This TGF-1induced myobroblast differentiation was effectively downregulated by uticasone and salmeterol. When the two agents were administered together, they produced an inhibitory effect signicantly greater than that produced by either agent alone (45). In vivo studies. Two studies have examined the possibility of complementary interactions between LABAs and corticosteroids on airway inammation in patients with asthma. In patients previously treated with ICSs, the addition of salmeterol (50 g twice daily) for 12 weeks led to a reduction in the total number of activated eosinophils in the lamina propria (46). Sue-Chu and associates (47) evaluated treatment with uticasone (200 g twice daily), uticasone (200 g twice daily) plus salmeterol (50 g twice daily), or higher-dose uticasone (500 g twice daily) for 3 months in patients previously uncontrolled on an ICS. Analysis of biopsy tissue obtained before and after treatment indicated ongoing inammation, with an increase in mast cells, CD3() cells, and CD4() cells, in the low-dose uticasone group. These increases were not observed in either the group receiving combination therapy or in the higher-dose uticasone group. Changes in airway inammation were associated with a signicant reduction in symptoms of clinical disease (47). Bronchial hyperreactivity is considered a clinical surrogate for airway inammation in asthma. Swenson and colleagues (48)

investigated the effect of uticasone/salmeterol (100/50 g twice daily) combination therapy on antigen-induced bronchial hyperresponsiveness to methacholine in mild asthmatics. After 28 days, there was a signicantly greater level of bronchoprotection (approximately 2.5 doubling dilutions) compared with the same doses of uticasone and salmeterol administered alone (48). Similar evidence of the synergy of these drugs was observed in a 1-year study of uticasone (500 g twice daily), salmeterol (50 g twice daily), or the combination in COPD patients (5). By Week 52, pre-dose FEV1 in the patients with more severe disease (FEV1 50% predicted) receiving combination therapy had increased by 110 L compared with 20 L in the salmeterol group and 40 L in the uticasone group (p 0.0001 versus placebo, salmeterol, FP). The median percentage of days without reliever medication was 0% for placebo, 3% for salmeterol, 2% for uticasone, and 14% for combination therapy (p 0.004). Airway remodeling occurs in both asthma and COPD. There is evidence of increased airway vascularity (angiogenesis) despite the use of ICSs. Orsida and colleagues (49) investigated the effect of adding salmeterol (50 g twice daily) to an ICS regimen, compared with increasing the steroid dose, on the degree of submucosal angiogenesis as assessed by immunohistochemistry for collagen IV. There was a signicant reduction in vascularity in the salmeterol/ICS group during the 3 months of treatment, whereas no effect was observed after high-dose ICS treatment (Figure 5). This raises the possibility that combination therapy with a corticosteroid and a LABA may be able to alleviate some components of airway remodeling in asthma and COPD.

Complementary interactions between corticosteroids and 2agonists, particularly LABAs, may be relevant in both asthma and COPD. Corticosteroids may regulate 2-receptor function by increasing expression of the receptor and, importantly, by restoring G-protein/2-receptor coupling and inhibiting 2-receptor downregulation. LABAs in turn may prime the GR and affect GR nuclear localization by modulating GR phosphorylation. These molecular and receptor mechanisms presumably lead to additive and/or synergistic effects on the inammatory pro-

Johnson: Interactions with 2-Agonists

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cesses involved in both diseases. In the patient with asthma, this is reected in enhanced anti-inammatory activity with combination therapy beyond that which can be achieved by either drug alone, or the potential for LABAs to provide a steroid-sparing effect. In COPD, corticosteroid/LABA combined activity may overcome the reduced sensitivity to ICSs that has been reported in some patients. The complementary interactions between corticosteroids and LABAs may explain the substantial clinical benets that have been consistently observed when they are used together in the treatment of asthma and COPD.
Conflict of Interest Statement : M.J. is employed by GlaxoSmithKline, who supported the meeting on which the manuscript is based through an educational grant and has an interest in the subject matter detailed in the manuscript, and he owns in stock in GSK.






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