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Emerging pharmaceutical therapies for COPD

COPD, for which cigarette smoking is the major risk factor, remains a worldwide burden. Current therapies provide only limited short-term benefit and fail to halt progression. A variety of potential therapeutic targets are currently being investigated, including COPD-related proinflammatory mediators and signaling pathways. Other investigational compounds target specific aspects or complications of COPD such as mucus hypersecretion and pulmonary hypertension. Although many candidate therapies have shown no significant effects, other emerging therapies have improved lung function, pulmonary hypertension, glucocorticoid sensitivity, and/or the frequency of exacerbations. Among these are compounds that inhibit the CXCR2 receptor, mitogen-activated protein kinase/Src kinase, myristoylated alanine-rich C kinase substrate, selectins, and the endothelin receptor. Activation of certain transcription factors may also be relevant, as a large retrospective cohort study of COPD patients with diabetes found that the peroxisome proliferator-activated receptor γ (PPARγ) agonists rosiglitazone and pioglitazone were associated with reduced COPD exacerbation rate. Notably, several therapies have shown efficacy only in identifiable subgroups of COPD patients, suggesting that subgroup identification may become more important in future treatment strategies. This review summarizes the status of emerging therapeutic pharmaceuticals for COPD and highlights those that appear most promising.


Introduction
COPD affects ~200 million people worldwide 1 and is the third leading cause of death in the US, claiming ~150,000 lives in 2013. 2 Approximately 6.3% of US adults aged 18 years have COPD, 3 and its combined indirect and direct costs in 2010 surpassed $52 billion. More effective treatments for COPD are needed to address this serious health problem. This review summarizes the status of potential therapies currently or recently in clinical trials and highlights those that appear most promising.

Treatment of stable COPD
The American Thoracic Society and other leading pulmonary disease organizations recommend that COPD patients cease smoking and use one or more inhaled bronchodilators. 14 The wide choice of inhaled bronchodilators includes short-acting beta 2 -agonists (SABA) and long-acting beta 2agonists (LABA), short-acting muscarinic antagonists (SAMA) and long-acting muscarinic antagonists (LAMA), and combinations of β-agonists and antimuscarinic agents. Inhaled corticosteroids (ICS) and combinations of corticosteroids with other agents may also be used. Oral medications, including methylxanthines and a phosphodiesterase 4 (PDE4) inhibitor, may also be used as adjunctive agents or, in the case of systemic corticosteroids, for acute exacerbations.
The Global Initiative for Chronic Obstructive Lung Disease (GOLD) recently categorized COPD patients into four groups, in which the familiar spirometry-based grades (GOLD I-IV) are supplemented by patients' symptom burden (assessed by the modified British Medical Research Council questionnaire or COPD Assessment Test) and exacerbation history: spirometry and exacerbation history are jointly used to determine the risk of adverse health events. 5 Groups A and C have relatively mild symptoms, while Groups A and B have relatively low risk. GOLD recommends that Groups A and B receive SABA, LABA, SAMA, or LAMA bronchodilators. It recommends that Groups C and D also receive ICS or possibly the PDE4 inhibitor roflumilast. Despite symptomatic relief and reduced rates of exacerbations with current medications, long-term studies of treated COPD patients suggest that medications do not halt the decline of lung function 4 and resulting mortality, although there is limited evidence that fluticasone and/or salmeterol may slow the rate of decline. 15,16 Several groups are pursuing identification of biomarkers for COPD subgroups based on either the etiological agent/event triggering the exacerbation or the physiological targets that may indicate response to a given therapy. 6,17 For example, COPD patients with higher levels of exhaled nitric oxide (FeNO) had a greater probability of the enhanced eosinophilic infiltration 18 that is associated with a greater response to corticosteroids. [19][20][21] These studies carry significant implications both for current treatment and for the development of novel therapies that may be targeted to specific groups of patients.

Inflammation in COPD
Smoke, from cigarettes, biomass fuel, or air pollution, is a common inducer of inflammation in COPD. The inhaled irritants provoke significant oxidative stress and activate inflammatory cells. 22,23 Excess neutrophils and macrophages, along with T cells, B cells, and dendritic cells, infiltrate the peripheral airways, [24][25][26] concurrent with the narrowing or obliteration of small bronchioles. 27 A number of inflammatory markers are significantly elevated in the serum of patients with COPD, notably including CD40 ligand, epidermal growth factor (EGF), brain-derived neurotrophic factor (BDNF), a variety of acute-phase proteins, and neutrophilassociated proteins. 28 These molecules may represent targets for future COPD therapies.

Glucocorticoid (GC) resistance
COPD is relatively resistant to modulation by corticosteroids, as even high doses of GCs do not delay or inhibit COPD progression. Histological analysis of lung tissue removed during lung volume reduction surgery of COPD patients indicated that GC treatment significantly reduced the number of airways with lymphoid follicles but had no long-term effect on luminal occlusion or airway wall thickening. 29 Corticosteroids suppress inflammation both by activating transcription of anti-inflammatory and suppressing transcription of proinflammatory genes. 30 Binding of corticosteroids to the classic glucocorticoid receptor (GRα) leads to activation, which allows release from its cytoplasmic anchor. The receptor is then acetylated, allowing binding to response elements in the promoter elements of target genes, and recruitment of coactivators such as CREBbinding protein, with consequent transcriptional activation. GC-mediated gene repression requires histone deacetylase 2 (HDAC2) at two distinct steps. 31 First, this acetyl group must be removed by HDAC2 to allow binding to proinflammatory transcription factors such as nuclear factor-κB (NF-κB). Second, activated GR recruits HDAC2, which then deacetylates histones and thus inactivates transcription. Alveolar macrophages (AMs), lungs, and bronchi of COPD patients express lower levels of HDAC2 than those in healthy controls, 32 however, and theophylline treatment, which enhances HDAC activity in AMs, also restores AM GC sensitivity. 33 These effects may underlie the reported ability of theophylline treatment (200-400 mg/day, depending on weight), added to the LABA formoterol and the GC budesonide in a 50-patient (study completion) singleblinded, placebo-controlled study, to improve 6-minute walk distance and forced expiratory volume in 1 second (FEV 1 ). 34 However, a double-blind, placebo-controlled study in 46 per-protocol patients found no effect of low-dose (100 mg bid) theophylline added to fluticasone plus salmeterol on HDAC activity, inflammatory biomarkers, or frequency of exacerbation. 35 Observed COPD-associated reductions in HDAC2 are believed to result from oxidative and nitrosative stress, 32 and levels of the antioxidant transcription factor NF-E2-related factor 2 (Nrf2) are similarly reduced in AMs of COPD patients. 36 Treatment with the Nrf2 activator sulforaphane, similar to theophylline treatment, increases HDAC2 activity and restores GC sensitivity. 37 A number of other mechanisms of GC resistance have also been suggested. 30 Among these may be increased expression of the translationally inactive GRβ, although this remains controversial 30 and GR expression has been found to be reduced in lungs of healthy smokers and COPD patients. 38 Another possibility is posttranslational modifications of GRα by phosphorylation or nitrosylation. 39 Such phosphorylation may be due to p38 mitogen-activated protein kinase (MAPK) activation, and p38 MAPK inhibitors have been shown to reduce the GC resistance seen in some patients with severe asthma. 40 Restoration of GC sensitivity is an attractive strategy for the development of novel pharmaceutical therapies. As previously noted, the ability of theophylline treatment to accomplish this is controversial. 34,35 However, roflumilast has been shown ex vivo to improve GC sensitivity of neutrophils from COPD patients, 41 and subgroup analysis of two clinical trials showed that patients receiving ICS were among those particularly likely to experience a reduction of exacerbations in roflumilast treatment. 42 A subsequent 1-year trial showed that roflumilast treatment reduced exacerbations in patients also receiving ICS and LABA. 13

Proinflammatory cytokines and chemokines
Numerous proinflammatory cytokines and chemokines are significantly higher in COPD patients than in healthy controls, 43 and multiple biologics and small molecules that target these mediators are under investigation (Tables 1  and 2). Chemokines significantly contribute to inflammation  47,48 (NCT01215279) had no effect. IL-1 expression was significantly higher in lung and sputum from COPD patients compared to non-COPD controls. 49 However, treatment with a monoclonal antibody (MAb) inhibiting IL-1β (canakinumab) 50 remains an open question in COPD patients ( Table 2). IL-5 is largely produced by eosinophils and is therefore low in typical COPD. A subset of COPD patients have elevated eosinophil numbers and IL-5 levels in their blood and airways; these patients are at increased risk for acute exacerbations. 9,51 Two MAbs targeted to IL-5 have been investigated for their ability to reduce exacerbation rate in COPD patients, but one showed no evidence of efficacy and results for the other are not yet available ( Table 2).
Secretion of IL-17A is a canonical marker of T helper 17 (Th17) lymphocytes, which are distinct from Th1 and Th2 cells. However, most of the cells secreting IL-17 in patients with very severe COPD were identified as mast  52 This study also found that levels of IL-17A in lungs of COPD patients correlated with functional decline, with elevations becoming significant in patients with severe and very severe disease. Another study found that the numbers of CD4 + IL-17 + cells in the alveolar walls and small airways of COPD patients and CD4 + IL-17 + numbers in small airways positively correlated with airflow limitations. 53 These results are compatible with the finding that Th17 cells were present in the lungs of patients with emphysema but not normal controls and that IL-17A-induced secretion of the elastin-degrading enzyme matrix metalloprotease 12 by lung macrophages. 54 The levels of IL-17A were also elevated in the sputum of patients with acute COPD exacerbations associated with Haemophilus influenzae infection but not other acute exacerbations. 55 Other studies have not similarly distinguished among causes of acute exacerbations, likely accounting for findings that the ratio of regulatory T cells to IL-17 levels in peripheral blood was similar in COPD patients with and without current acute exacerbations, although exacerbations significantly increased levels of transforming growth factor β (TGF-β). 56 Indeed, one study was unable to detect IL-17 in sputum or serum of COPD patients with or without exacerbations. 57 Another study found that the sputum of patients with severe COPD had significantly higher levels of IL-8 but 4.8-fold lower levels of IL-17 compared to that of patients with mild COPD and healthy controls. 58 T cells from many COPD patients have also been reported to produce less IL-17A and IL-22 (also a signature cytokine of Th17 cells) than those of most normal smokers. 59 These complex and apparently contradictory findings underline the uncertainty of the role of IL-17 in COPD. Nevertheless, an IL-17 modulator is currently in clinical trials for COPD (Table 2). Based on an IL-18-overexpressing transgenic mouse model that develops emphysema and airway remodeling, 60 Kang et al 60 and Nakajima and Owen 61 proposed that IL-18 is a master regulator of lung pathology in COPD. A phase I safety and tolerability clinical trial (NCT01322594) of the MAb MEDI2338 (targeted to IL-18) in COPD patients found no serious adverse events, but there have been no efficacy studies. 62 Tumor necrosis factor α (TNF-α) plays multiple roles in COPD inflammatory pathology, and the levels of interferon γ (IFNγ) and TNF-α in the intraepithelial T cells from bronchi of COPD patients with GOLD II-III disease showed a significant negative correlation with FEV 1 . 63 Nevertheless, studies with infliximab showed no clinical benefits on FEV 1 , dyspnea, or exacerbations and were associated with higher rates of pneumonia and malignancy (Table 2). Similarly, treatment with etanercept, a fusion protein that competitively binds TNF-α, was not superior to prednisone in COPD exacerbations and in fact was less effective among patients with eosinophilia (Table 2).
Taken together, these data show that an increased level of a specific cytokine or chemokine during COPD exacerbations or stable COPD does not necessarily predict the efficacy of its specific inhibitor in COPD patients. Whether modulators of specific cytokines or chemokines can provide improved efficacy in a subgroup of patients is a possibility and warrants further investigation. 6

Signaling molecules
Multiple signaling molecules help regulate inflammation and airway remodeling and represent plausible targets for the development of therapeutic candidates. Candidate drugs include inhibitors of p38 MAPK and related kinases, phosphoinositide kinase δ (PI3Kδ), leukotriene B 4 , selectins, and vasoactive intestinal peptide (Table 3). Although several oral and inhaled p38 MAPK inhibitors have been discontinued, the inhaled narrow spectrum kinase (p38α + Src family) inhibitor JNJ49095397 (previously RV568) shows promising activity in COPD patients; conference presentations have indicated that RV568 significantly increased FEV 1 and inhibited IL-1β (90% at 800 µg dose) and CXCL8 expression (73%). 64 However, a recent conference report performed in over 200 COPD patients (half placebo, half 400 µg dose) showed no benefit with RV568 on lung function or EXACT-PRO. 65 PI3Kδ participates in many functions of lymphoid and myeloid cells: B-cell development, migration and activation of natural killer (NK) cells and T cells, neutrophil oxidative burst, macrophage activation triggered by immune complexes, and degranulation and maturation of mast cells. 66 Specific PI3Kδ inhibitors are being developed, 67 and studies on the effects of such inhibitors in COPD are in progress. Efficacy data remain limited, however (Table 3). Selectins are essential for migration of inflammatory cells from the bloodstream into pulmonary tissue; the selectin modulator bimosiamose reduced inflammation in COPD patients and thus warrants further testing (Table 3). 68 Similar to the glucocorticoid receptor, peroxisome proliferator-activated receptor γ (PPARγ), also a member of the nuclear hormone receptor superfamily, exerts potent antioxidant and anti-inflammatory effects via multiple mechanisms, including downregulation of NF-κB and other proinflammatory transcription factors. Lung tissue and bronchial epithelial cells from COPD patients express significantly lower levels of PPARγ than those of nonsmoking controls. 69 In vitro treatment of COPD patient and control bronchial epithelial cells  with PPARγ agonists including 10-nitro-oleic acid (a possible endogenous ligand) or rosiglitazone (a thiazolidinedione) blocked cigarette smoke-induced production of cytokines, chemokines, and ROS, and accompanying suppression of HDAC2 levels. 69 The PPARγ agonist rosiglitazone dose dependently inhibited LPS-induced production of TNF-α and CCL5 by AMs of COPD patients, smokers, and never-smokers, shifting them toward an anti-inflammatory M2 phenotype, while both rosiglitazone and pioglitazone attenuated pulmonary inflammation in a tobacco smoke mouse model. 70 PPARγ also appears to play a central role in the development of emphysema. PPARγ is downregulated in lung myeloid dendritic cells of smokers with emphysema and endogenous PPARγ agonist activity is reduced in plasma of those with emphysema, while treatment with the thiazolidinedione ciglitazone reverses emphysema in a mouse model. 71 A retrospective epidemiological study of veterans with both diabetes and COPD indicated that the 7,887 veterans treated with a PPARγ agonist for their diabetes (97.1% rosiglitazone) significantly reduced the risk of COPD exacerbations in comparison to those receiving other diabetes medications (n=42,347; incidence rate ratio [IRR]=0.85; CI: 0.80-0.91) ( Table 4). 72 Taken together, these data support further studies of PPARγ agonists in COPD patients with early signs of emphysema. COPD also involves progressive increase in pulmonary arterial pressure, and 20%-91% (depending on definition, COPD severity, and method of measurement) of COPD patients have progressed to pulmonary hypertension. 73 Endothelin signaling plays a major role in vascular remodeling and hence in development of pulmonary hypertension. 74,75 An 18-month treatment with the

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Lakshmi et al endothelin antagonist bosentan improved measures of pulmonary hypertension compared to those at baseline, especially in GOLD grade III and IV COPD patients, while pulmonary hypertension worsened in placebo-treated patients (Table 4). 74 However, bosentan can actually worsen hypoxemia in COPD patients without pulmonary hypertension. 76 Although statins are known to have anti-inflammatory properties, simvastatin treatment (40 mg/day) of COPD patients at high risk for exacerbation did not increase the time to first exacerbation nor reduce the number of exacerbations. 77 However, rosuvastatin (12-week treatment) improved endothelium-dependent vascular function in a prespecified subgroup (patients with supramedian circulating high-sensitivity C-reactive protein [hsCRP]) but not in the total COPD population in a double-blind, placebo-controlled trial. 78 This study found no statistically significant effect on pulmonary function parameters, however.

PDE inhibitors
The 11-membered phosphodiesterase enzyme family (PDE1-11) differentially hydrolyzes cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), which regulate many cellular processes including release of inflammatory mediators and relaxation of smooth muscles. 10 PDE3 appears to be involved in bronchoconstriction since its inhibitors induce bronchodilation in humans. 10 PDE4 is expressed in most inflammatory cell types and is a main target for emerging COPD therapies (Table 5), with the PDE4 inhibitor roflumilast having been approved by the FDA as a COPD treatment. In vitro studies of low-dose combinations of PDE4 and PI3Kδ inhibitors significantly reduced cigarette smoke extract-induced apoptosis of lung epithelial cells, neutrophil elastase production, and macrophage secretion of TNF-α, phosphorylated protein kinase B, and matrix metalloproteinase 9 (MMP-9). 79 Gastrointestinal adverse effects can be a significant problem with roflumilast, but PDE4 inhibitors with inhaled or nebulized (eg, RPL554, GSK-256066) formulations appear to have more tolerable side effect profiles than orally administered roflumilast. 80,81 The severity of emphysema and small airways disease is associated with higher expression of multiple MMPs. 82 However, despite promising results in an animal model, 83 an inhibitor of MMP-9 and -12 has not shown significant benefit for COPD patients in a clinical trial (Table 6).

Oxidative stress and antioxidants
Oxidants, both from inhaled pollutants and produced as part of the inflammatory response, are major contributors to COPD pathophysiology. Furthermore, inflammationassociated production of ROS and reactive nitrogen species (RNS) is accompanied by downregulation of the antioxidant transcription factor Nrf 2. ROS activates the proinflammatory transcription factors activator protein 1 (AP-1) and NF-κB, with consequent production of inflammatory proteins and mediators. Excess ROS in COPD patients also directly contributes to airspace epithelial injury and inactivates antiproteases that help prevent emphysema. 84 Therapeutic agents that activate Nrf2 and thus neutralize the excess oxidants may prove beneficial to COPD treatment. 85 The antioxidant transcription factor Nrf 2, the primary mechanism for limiting oxidative stress, is reduced in COPD patients. 36 Kelch-like ECH-associated protein 1 (Keap1) sequesters Nrf 2 in the cytoplasm and under healthy conditions targets Nrf 2 to Cullin-3 for ubiquitination and degradation. 86 Keap1 monitors oxidative stress through its multiple cysteines with distinct stressors binding one or more

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Lakshmi et al specific cysteines. Following binding of these stress-related compounds, release of Nrf2 from Keap1 occurs, which transfers to the nucleus and activates multiple antioxidant enzymes and phase II proteins that counteract oxidative stress. 86 This makes the Nrf2/Keap1 system an attractive therapeutic target in COPD and other inflammatory diseases. The natural product sulforaphane activated Nrf2 in AMs isolated from COPD patients, denitrosylated HDAC2, and restored sensitivity to the glucocorticoid dexamethasone in a glutathione-dependent manner. 37 A study of the effect of two doses of sulforaphane on Nrf2 expression in 89 COPD patients was recently completed, and sulforaphane administered for four weeks to patients with COPD did not induce the expression of Nrf2 target genes or have an effect on oxidative stress, airway inflammation, or lung function. (NCT01335971). 87 Erdosteine and N-acetylcysteine directly scavenge ROS via their thiol groups (of the metabolite in the case of erdosteine) and also have mucolytic activity. High-dose (900 mg/day) erdosteine increased the ability of salbutamol to improve %FEV 1 reversibility, 88 and long-term treatment reduces exacerbations and improves quality of life. 89 Highdose (600 mg bid) N-acetylcysteine reduces the number of exacerbations patients experience; 12,90,91 reported effects with a lower dose (600 mg/day) have been inconsistent. [92][93][94]

Mucus hypersecretion
Mucus hypersecretion can be modulated by blocking its overproduction and/or by inhibiting its secretion (Table 7). Hypothetically, a rebound effect after cessation of an inhibitor of mucin secretion may involve a rapid release of produced but unsecreted mucin. In contrast, the rebound effect after cessation of an inhibitor of mucin production may involve a lag phase for mucin production and a more gradual increase of mucin secretion. Multiple mucins comprise the gel-forming layer of normal airway mucus. Numerous signals can promote  95 and thus can promote hyperplasia of mucin-producing goblet cells. 97 Supporting this concept, nuclear HIF-1α was expressed in the majority of goblet cells in areas of remodeled airway tissues showing goblet cell hyperplasia from COPD patients but not in subjects without COPD. 97 IL-13, an essential component of COPD-associated inflammation, promotes goblet cell production of mucins. 98 After the mucins are expressed, glycosylated, and packaged in mucin granules, myristoylated alanine-rich C kinase substrate (MARCKS) mediates movement of intracellular mucin granules to the goblet cell apical membrane and is therefore essential for mucin exocytosis and secretion. 98

Summary
The heterogeneity of COPD presentation augments the challenges in identifying and developing therapeutic compounds for the treatment of COPD patients. It also emphasizes the importance of tailoring therapy to individual patients and their disease status, which may involve considerations beyond the standard GOLD categories. Several distinct emerging therapies have shown efficacy in at least some COPD patients. The length of treatment (3 months) and the inhaled route of administration can be associated with a higher probability of observing a positive effect on COPD variables and a reduced side effect profile, respectively. Although some of the emerging compounds showed significant activity in the total COPD population tested, 45,68,74,88,99 sometimes only in connection with assessed biomarkers rather than clinically significant outcomes, subgroup analysis showed that most compounds were significantly affected in one or more of the following subgroups: smokers or ex-smokers, 44,99 patients with chronic bronchitis or emphysema, 80,99 use of standard COPD therapies, 99 patients with alteration of relevant biomarkers, 78 or at different COPD grades. 74 These data suggest that subgroup analyses and the possibility of individualized therapy can benefit developers of emerging therapies by identification of the patients most likely to benefit from a new therapy.

Acknowledgments
This work was supported by a Merit Review award from the US Department of Veterans Affairs and the National Institutes of Health grants HL093196 and AI125338 (RCR). We gratefully acknowledge the scientific and writing expertise of KL Molnar-Kimber, PhD. The contents in this article do not represent the views of the US Department of Veterans Affairs or the United States Government.

Disclosure
The authors report no conflicts of interest in this work. AZD9668 AZD6553 AZD9668 (12-week treatment) showed no effect on pulmonary function or quality of life when combined with tiotropium (NCT00949975) or budesonide/formoterol (NCT01023516); there was likewise no effect on airway remodeling (NCT01054170) and studies found no decrease in degradation as assessed by urinary desmosine. AZD6553 clinical trial (NCT01068184) was terminated due to PK inconsistent with pharmaceutical properties.

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Matrix metalloproteinases (MMPs) Higher levels of multiple MMPs in lungs of COPD patients; involved in matrix breakdown and tissue remodeling.
AZD1236 (anti-MMP-9 and -12); GS-5745 (anti-MMP-9) In a 6-week DBPCRT (NCT00758706) of AZD1236 (anti-MMP-9 and -12) in moderateto-severe COPD patients, reduction in urinary desmosine did not reach statistical significance and there was no effect on COPD clinical symptoms. Another study (NCT00758459) has completed but statistical analysis not released. A 28-day safety and PK study of GS-5745 in COPD patients (NCT02077465) has been completed.

BIBw 2948 (inhibits eGFR autophosphorylation)
Inhalation of BIBw 2948 (4-week treatment) in DBPCRT (NCT00423137) reduced internalization of eGFR but did not reduce mucin stores; BIBw 2948 treatment was associated with higher discontinuation rate (24%) than placebo (4.3%). Fev 1 in the higher dose group significantly declined by visit 5 but returned to baseline by visit 7.

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Myristoylated alaninerich C kinase substrate (MARCKS) Mediates movement of mucin granules to the apical membrane as part of mucin exocytosis.