Current Medicinal Chemistry, 2009, 16, 867-883 867

0929-8673/09 $55.00+.00 © 2009 Bentham Science Publishers Ltd.

Bronchial Asthma, Chronic Obstructive Pulmonary Disease and NF- B

Nikolaos Charokopos1, Nikolaos Apostolopoulos1, Maria Kalapodi2, Michael Leotsinidis3, Nikolaos Karamanos4 and Athanasia Mouzaki*,5

1,2Departments of Pulmonology and Internal Medicine, General Hospital of Pirgos, Ilia,

3Laboratory of Public Health,

Medical School, University of Patras, 4Laboratory of Biochemistry, Department of Chemistry, University of Patras,

5Division of Hematology, Department of Internal Medicine, Medical School, University of Patras, Patras, Greece

Abstract: Respiratory diseases place a considerable burden on global health. Bronchial asthma describes many heteroge-neous clinical phenotypes that result in chronic bronchial inflammation. Chronic obstructive pulmonary disease (COPD) is one of the most common adult respiratory disorders characterized by chronic airflow limitation that is not fully reversi-ble and is associated with an abnormal inflammatory response of the lungs to noxious particles and gases. Recognition of the global importance and rising prevalence of these diseases and the absence of effective treatments has led to concerted efforts to improve the efficacy of the existing drugs and develop new ones that target cellular and molecular mechanisms that underlie disease pathogenesis. The transcription factor nuclear factor kappa B (NF- B) regulates the expression of a wide array of genes that are involved in the molecular pathobiology of the lung by regulating cellular immune responses, cell adhesion, differentiation, proliferation, angiogenesis and apoptosis.

In this work, we review published clinical and experimental studies that link the inhibition of NF- B activity with the treatment of asthma and COPD. Our end point is to help identify pathway-specific inhibitors of NF- B that can be used for the treatment of specific human ailments.

INTRODUCTION

Chronic obstructive pulmonary disease (COPD) and bronchial asthma are leading causes of morbidity and mortal-ity worldwide. While there have been major advances in the understanding and management of asthma, COPD has re-mained somewhat neglected, and there are no current thera-pies that reduce the inevitable progression of this disease. However, because of escalating healthcare costs and the enormous burden of COPD and asthma, there is now a re-newed interest in the underlying cellular and molecular

mechanisms and a search for new therapies, resulting in a reevaluation of these diseases [1].

Although the pattern of inflammation clearly differs be-tween bronchial asthma and COPD, involving different cells and mediators, both are characterised by an increased ex-pression of genes, some of which are common to all inflam-matory diseases, whereas others are more specific to a par-ticular disease. Essential transcription factors (TFs) that control the expression of these genes include: NF- B, acti-vated through TNF- R, its prototype receptor. Activator protein (AP)-1, activated through cytokine binding to their cellular receptors. AP-1 cross-talks with GATA binding protein 3 (GATA-3) an essential TF for the expression of the cytokines IL-4, IL-5 and IL-13 which perpetuate allergic inflammation. Nuclear factor of activated T-cells (NF-AT) is an important TF that regulates the activation of T-cells. JAK/STAT3 TF is essential for IL-6 transcription. These TFs are activated in all inflammatory diseases and play a critical role in both the cellular stress response and the con-trol of intracellular levels of reactive oxygen species (ROS), which amplify and perpetuate or down regulate the inflam-matory process. The molecular pathways involved in the regulation of inflammatory gene expression are currently

*Address correspondence to this author at the Division of Hematology, Department of Internal Medicine, Medical School, University of Patras, Rion, Patras GR-26500, Greece; Tel/Fax: +30-2610-969123; E-mail: mouzaki@med.upatras.gr

under investigation, and it is becoming clear that chromatin remodeling plays a critical role in the transcriptional control of these genes [2-4].

NF- B is activated in the airways of both asthmatic and COPD patients. It was identified 20 years ago [5] as a nu-clear factor that binds the light chain enhancer in B-cells, hence the name NF- B, and it was shown to play roles in both the innate and adaptive immune responses. More re-cently, its role in many other cellular processes has become apparent. Not surprisingly, deregulated activity of the NF- B pathway has been observed and linked to the progression of several human ailments, including cancer. Research in the last two decades has identified the major mechanisms of activation of this pathway and documented the roles of the key players. Over 200 physiological stimuli are known to activate NF- B. These include bacterial and viral products, cellular receptors and ligands, mitogens, growth factors, and physical and biochemical stress inducers. The major cellular targets of NF- B are chemokines, immune receptors, adhe-sion molecules, stress response genes, regulators of apopto-sis, transcription factors, growth factors, enzymes and cell cycle regulators [6,7].

NF- B transcriptionally upregulates cytokines, chemoki-nes, immunity effector molecules and pro-survival factors. The pro-survival effects of NF- B can counter apoptotic signals from cytokine receptors, such as TNF receptor I, and they can also protect stressed cells from apoptosis, which can limit the effectiveness of cancer chemotherapeutic agents. Mutations that inactivate NF- B are generally lethal because of the essential role of this protein in cell survival. Partial loss of function results in varying degrees of immunodefi-ciency: humans with such mutations have variable levels of immunodeficiency, and many show a poor inflammatory response and lack some types of antibodies. These symptoms reflect the roles of NF- B in the innate immunity to bacteria (presumably via TLRs), inflammatory gene expression and B cell antigen receptor signaling [8].

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NF- B is an important TF for several viral pro-moter/enhancers (e.g., HIV-1 and CMV). Given that such a large number of stimuli can activate NF- B, which, in turn, activates an equally large number of target genes, it is a major challenge to understand how specificity is achieved within this pleiotropic signaling framework. However, a thorough understanding of the mechanisms of specificity is needed to design pathway-specific inhibitors of NF- B, developed to treat specific human ailments [7].

THE MOLECULAR BASIS OF INFLAMMATION

Chronic inflammatory diseases, such as asthma and COPD, involve the infiltration and activation of many in-flammatory and immune cells, which release multiple in-flammatory mediators that interact and activate structural cells at the site of inflammation.

The nature of both the inflammation and the involved immune cells is very different between the two conditions. While inflammation in asthma is characterized by an in-crease in CD4+ T (Th2) cells and eosinophils, increased numbers of neutrophils, macrophages and CD8+ T (Tc1) cells are observed in COPD [9].

The prominent feature of COPD is the elevated concen-trations of macrophages and Tc1 cells throughout the lumi-nal epithelium of the respiratory tract airway, which are positively related to the severity of the disease. Peripheral airway obstruction (bronchioles<2mm i.d) and a degree of damage to the alveolar wall and pulmonary vessels are ob-served [10].

Smoking may directly affect the epithelial cells and macrophages that generate reactive oxygen and reactive nitrogen species (RNS) [11, 12]. This oxidative stress may enhance inflammation by upregulating redox-sensitive tran-scription factors, such as NF- B and AP-1. The release of oxidants can result in a release of proinflammatory media-tors, such as IL-8, IL-1 and NO, all of which increase gene expression of inflammatory mediators and, also, binding and activation of NF- B, which, in turn, creates more oxidative stress [13].

Proinflammatory cytokines, such as TNF- , IL-1 , IL-6 and IL-8, released from epithelial and inflammatory cells, amplify the inflammation and might contribute to some of the systemic effects of COPD. Previous studies, performed in patients with mild to moderate COPD, have reported an increased number of T-lymphocytes and macrophages, as well as an increased presence of ICAM-1 expression in the airway epithelium, endothelial leukocyte adhesion molecule-1 expression in the endothelium and macrophage inflamma-tory protein-1b expression in the bronchoalveolar lavage fluid. In smokers, increased expression of adhesion mole-cules results in sequestration of neutrophils in the pulmonary microcirculation, which further exacerbate COPD [14,15].

A number of signs of inflammation, such as MCP-1 overexpression, have also been observed in smokers with normal lung function, but to a lesser extent than in COPD patients.

Recent data from animal studies have implicated other signaling molecules in airway remodeling. Activated mem-

ory T cells home into the lung and act as effector cells. Tc1 cell differentiation into memory cells is facilitated by Th1 cells. INF- secreted from Th1 and Tc1 cells activates macrophages to secrete MMP-9 and MMP-12, leading to elastolysis and emphysema. Similarly, Tc1 cells release cytotoxic perforins and granzyme B that cause alveolar cell death and apoptosis, which is also a feature of emphysema. Lung secretions contain only a small percentage of T cells because most T-lymphocytes reside in the subepithelial and smooth muscle region of the tissue. During COPD within the tissue, there is an increase in either the CD8+/CD4+ ratio of T cells or the total number of both CD8+ and CD4+ T cells [16,17].

In bronchial asthma, an allergen, infection or pollutant in predisposing individuals activates the submucosal mast cells in the lower airways, leading to bronchoconstriction within seconds. The disease progression in bronchial asthma has been carefully characterized; it has been reported that den-dritic cells (DCs), eosinophils and Th2 cells initiate bron-chial asthma, while IL-4, 5, 9 and 13 promote progression and GM-CSF expression leads to persistence of the condition [18].

Airway remodeling is a prominent structural change of severe bronchial asthma. Airway remodeling is characterized by epithelial damage, airway smooth muscle remodeling, sub-epithelial fibrosis, mucus production, goblet cell meta-plasia and angiogenesis. Different growth factors, such as TGF- and fibroblast growth factor, orchestrate the above-listed structural changes, which contribute to both airway hyperresponsiveness and a permanent loss of lung function with apparently irreversible airflow limitation [19].

SPECIFIC INHIBITION OF NF- B ACTIVATION - NEW PHARMACEUTICAL TARGETS

NF- B is a TF that governs the expression of genes in-volved in the immune response, embryo and cell lineage development, cell apoptosis, cell cycle progression, inflam-mation and oncogenesis. During the past few years, consid-erable attention has been given to the upstream signaling pathways that lead to NF- B activation. Many of these sig-naling molecules can serve as potential pharmaceutical tar-gets to specifically inhibit NF- B activation, leading to inter-ruption of the disease processes. However, how these mole-cules interact with each other is still a debatable issue. Since many of the signal molecules in this pathway relay more than one of the upstream signals to downstream targets, it has been suggested that activation of NF- B requires the transmission of a network of signals, rather than a linear sequence. Thus, a detailed characterization of all of the up-stream signaling molecules that activate NF- B will be re-quired before specific pharmaceutical inhibitors of NF- B activation can be developed. Such inhibitors are predicted to have potent anti-inflammatory and/or anti-carcinogenic ef-fects [20,21].

ANTIOXIDANT THERAPEUTIC TARGETS

Inflammatory lung diseases, such as asthma and COPD, are characterized by systemic and local chronic inflammation and oxidative stress. The sources of the increased oxidative

Bronchial Asthma, Chronic Obstructive Pulmonary Disease Current Medicinal Chemistry, 2009 Vol. 16, No. 7 869

stress in patients with asthma and COPD derive from the increased burden of inhaled smoking oxidants and from the increased amounts of ROS and RNS generated by the nu-merous inflammatory, immune and structural cells of the airways [22].

Increased levels of airway produced ROS is reflected by increased markers of oxidative stress in the airspaces, spu-tum, breath, lungs and blood of patients with asthma and COPD. ROS, either directly or via the formation of lipid peroxidation products [23], such as acrolein, 4-hydroxy-2-nonenal and F2-isoprostanes, may play a role in enhancing the inflammation through the activation of the stress kinases, JNK, MAPK, p38 and phosphoinositide 3 (PI-3)-kinase/PI-3K-activated serine-threonine kinase Akt, and redox-sensitive transcription factors, such as NF- B and AP-1. Recent data have also indicated that oxidative stress and pro-inflammatory mediators can alter nuclear histone acetyla-tion/deacetylation, allowing for the access of TF DNA bind-ing that leads to enhanced pro-inflammatory gene expression in various lung cells. Since a variety of oxidants, free radi-cals and aldehydes are implicated in the pathogenesis of asthma and COPD, therapeutic administration of multiple antioxidants might provide effective treatment of these dis-eases [24].

An overview of the antioxidant therapeutic targets is shown in Fig. (1).

Antioxidant and/or anti-inflammatory agents, including the thiol molecules ((glutathione and the mucolytic drugs (N-acetyl-L-cysteine and N-acystelyn)), dietary polyphenol (curcumin-diferuloylmethane, a principal component of turmeric), resveratrol (a flavanoid found in red wine), theo-phylline and epigallocatechin-3- gallate (found in green tea), ergothioneine (xanthine and peroxynitrite inhibitor), quer-cetin, erdosteine and carbocysteine lysine salt, are reported to control NF- B activation and regulate glutathione biosyn-thesis genes, chromatin remodeling and inflammatory gene expression. Specific spin traps, such as a catalytic antioxi-dant (ECSOD mimetic), manganese (III) meso-tetrakis (N,N'-diethyl-1,3-imidazolium-2-yl) porphyrin (AEOL 10150 and AEOL 10113) and a SOD mimetic (M40419) have also been reported to inhibit cigarette smoke-induced inflammatory responses in vivo [25].

A reducing agent, L-2-oxothiazolidine-4-carboxylic acid (OTC), which is a prodrug of cysteine, reduces bronchial inflammation and airway hyper-responsiveness. ROS gen-eration in the bronchoalveolar lavage fluids was increased by ovalbumin (OVA) inhalation, but this increase diminished

Fig. (1). Schematic summary of reactive oxygen species (ROS) and reactive nitrogen species (RNS) -mediated NF- B activation in bronchial asthma and COPD. Efficacy of antioxidants in redox-sensitive transcription factors (NF- B pathway) and mediators.

MARK, mitogen activated protein kinase; JNK, Jun NH2-terminal kinase; ERK, extracellular signal-regulated kinase; SIRT, silencing infor-mation regulator; TRx, thioredoxin; GSH, glutathione; GSSG, S-glutathione.

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with administration of OTC. In lungs after OVA inhalation, IL-4, IL-5, IL-13 and eosinophil cationic protein levels were significantly reduced by OTC administration. In addition, the increased expression of ICAM-1, VCAM-1, RANTES and eotaxin in the lungs after OVA inhalation was significantly reduced by OTC administration. It was also reported that the increased NF- B levels in the nuclear protein extracts of lung tissues at 72 h after OVA inhalation were decreased by OTC administration. These findings suggest that OTC may reduce airway inflammation and hyperresponsiveness through regulation of NF- B activity [26].

OTC also increases intracellular glutathione (GSH). Re-duced GSH is one of the most important reducing agents against oxidant free radicals (ROS) in asthma. GSH deple-tion leads to sustained activation of NF- B, while GSHEE (GSH ethyl ester) prevents it. Furthermore, inhibition of NF-

B activation restored myogenesis, despite the GSH deple-tion. GSH contributes to the formation of myotubes from satellite myoblasts by ensuring inactivation of NF- B; hence, maintaining optimal GSH levels may be beneficial in restoring muscle mass in patients with chronic inflammatory disorders [27].

A recent study [28] that investigated the apoptotic behav-iour of human blood eosinophils when incubated with the antioxidant, N-acetyl-L-cysteine (NAC), showed that con-centrations above 5mM increased both the GSH levels of eosinophils and D-NAC, which is the inactive form of NAC. It is of interest that the levels of NF- B activity were mark-edly decreased in NAC-treated eosinophils, and this was accompanied by an acceleration of apoptotic events in the eosinophils. The above findings are of great importance because the eosinophils were preincubated with either GM-CSF or TNF- , which are both known to increase their lifespan.

VEGF increases vascular permeability and leads to air-way inflammation. In addition, VEGF has been shown to enhance the expression of receptor activator of NF- B (RANK) in endothelial cells. OTC and VEGF receptor in-hibitors, which both inhibit upregulation of VEGF expres-sion, also modulate RANK expression that may be associ-ated with the regulation of vascular permeability, suggesting that VEGF regulates RANK expression. These findings provide a crucial molecular mechanism for the potential use of antioxidants to prevent and/or treat asthma and other air-way inflammatory disorders [29].

In the respiratory tract, NO is produced by residential and inflammatory cells. NO is generated via oxidation of L-arginine that is catalysed by the enzyme NO synthase (NOS). NOS exists in three distinct isoforms: neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS). NO, derived from the constitutive isoforms of NOS (nNOS and eNOS), and other NO-adduct molecules (nitroso-thiols) are able to modulate bronchomotor tone. NO, derived from iNOS, upregulates different cytokines via an NF- B-dependent pathway and seems to be a proinflammatory me-diator with immunomodulatory effects. The production of NO under oxidative stress conditions secondarily generates strong oxidizing agents (reactive nitrogen species) that may amplify the inflammatory response in asthma and COPD. Moreover, NO can be exhaled, and its levels are abnormal, in

stable atopic asthma and during exacerbations in both asthma and COPD. Therefore, exhaled NO levels might provide a non-invasive tool to monitor the underlying inflammatory process. It is suggested that NOS regulation provides a novel target in the prevention and treatment of chronic inflamma-tory diseases of the airways such as asthma and COPD. Ex-ogenous addition of TNF- and lipopolysaccharide (LPS) to the media of in vitro grown rat pulmonary microvascular endothelial cells, induces iNOS gene expression at the tran-scriptional or posttranscriptional level. The induction of iNOS gene expression by TNF- and LPS is dependent on the activation of NF- B. Antioxidants may inhibit the induc-tion of iNOS gene through the inhibition of NF- B activa-tion [30].

Cigarette smoke-induced oxidative stress [31-33] also initiates the activation of signal transduction systems, such as the redox-sensitive transcription factors NF- B and AP-1 [31,34], which have a central role in regulating many proin-flammatory genes [20,23,32,35]. In human lung tissue, a smoking-related increase occurs in NF- B nuclear transloca-tion, associated with degradation of the inhibitor of factor kB (IkB ) and an imbalance between histone deacetylation and acetylation in favor of acetylation [36]. These changes may contribute to the enhanced inflammation observed in smokers who are susceptible to the development of COPD [36]. The importance of oxidant–antioxidant imbalance in COPD pathogenesis has been further underscored in another study, that showed similarities with aging using a knockout mouse model of senescence [37]. Moreover, Henderson and colleagues [38] examined the effect of a small molecule, MOL 294 (methyl (4R/S)-4-hydroxy-4-[((5S,8S)/(5R,8R))-8-methyl-1,2-dioxo-2-phenyl-2,3,5,8-tetrahydro-1H-[1, 2, 4] triazolo[1,2-a]pyridazin-5-yl]-2-butynoate), on airway in-flammation and airway hypereactivity (AHR) in a mouse model of asthma. MOL 294, which is a selective inhibitor of the oxidoreductase, Trx [39], has a redox-active disul-fide/dithiol with a conserved Cys-Gly-Pro-Cys sequence that functionally blocks NF- B and AP-1 activation. Treatment of OVA-sensitized/challenged mice with this molecule re-sulted in a net decrease in airway eosinophilia, mucus hyper secretion, IL-13 and eotaxin production, and AHR.

The lung injury that is caused by oxidative stress and ex-cessive inflammatory responses may be counteracted by naturally occurring anti-oxidants such as vitamin E ( -tocopherol). Hammarström and colleagues [40] investigated the action of vitamin E in human alveolar type II and bron-chial epithelial cells that were stimulated with TNF- . They observed that treatment with vitamin E reduced the DNA binding of nuclear NF- B within the nucleus in A549 cells, with the most pronounced effect observed with 50 M, fol-lowed by decreased levels of IL-8 and ICAM-1. This sup-ports the idea that the observed suppression of the TF re-sulted in reduced gene transcription. In addition, they inves-tigated if the specific I B inhibitor, BAY11-7082, could abolish the secretion of IL-8 and expression of ICAM-1 in lung epithelial cells. They found that the I B inhibitor al-most completely inhibited TNF- -induced secretion of IL-8 and expression of ICAM-1. These results indicate that the induction of these inflammatory mediators is dependent on the NF- B activation pathway; they also implicate vitamin E

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as an anti-inflammatory agent that, in addition to its anti-oxidant properties, inhibits the NF- B pathway.

THE I B INHIBITORY MOLECULES

The activity of NF- B is tightly regulated by its interac-tion with the I B proteins. In most resting cells, NF- B is sequestered in the cytoplasm in an inactive form associated with the inhibitory molecules I B , I B , I B , p105 or p100. This interaction blocks the ability of NF- B to bind to DNA and results in an NF- B complex that is primarily localized to the cytoplasm. Following exposure to inflamma-tory cytokines, UV light, ROS or bacterial and viral toxins, the NF- B signalling cascade is activated, leading to the complete degradation of I B. This allows the translocation of free NF- B to the nucleus, where it binds to the promoter of target genes and regulates their transcription. In the nu-cleus, acetylation of NF- B determines its active or inactive state. p300 and CBP acetyltransferases play a major role in the acetylation of RelA(p65), principally targeting Lys for modification. Acetylated NF- B is active and resistant to the inhibitory effects of I B. However, when histone deacetylase 3 (HDAC3) deacetylates NF- B, I B readily binds to NF- B and causes its translocation into the cytoplasm. In this way, HDAC3 serves as an intranuclear molecular switch that turns off the biological processes triggered by NF- B. One of the target genes activated by NF- B is I B . Newly synthesized I B can enter the nucleus, remove NF- B from DNA and export the complex back to the cytoplasm to restore its origi-

nal latent state. As mentioned above, the activation of NF- B by extracellular inducers depends on the phosphorylation and subsequent degradation of I B proteins. Activation of NF-

B is achieved through the action of a family of ser-ine/threonine kinases (IKK). The IKK contains two catalytic subunits (IKK and IKK ) and a regulatory/adapter protein NEMO (also known as IKK ). IKK and IKK phosphory-late I B proteins and the members of the NF- B family. All I B proteins contain two conserved serine residues within their N-terminal area, which are phosphorylated by IKK. IKK and IKK share about 50% sequence homology and can interchangeably phosphorylate the Ser residue of both I B and I B . These phosphorylation events lead to the immediate polyubiquitination of I B proteins and rapid degradation by the 26S proteasome [7].

An overview of the therapeutic targets of IKK inhibitors is shown in Fig. (2).

Broide and colleagues [42], published a paper describing the critical role played by NF- B protein in the development of airway remodeling. Their results suggest that blocking the activity of this protein, perhaps by using an inhaled inhibitor, may be an effective strategy for treating asthma and other lung diseases. To investigate whether NF- B within epithe-lial cells of the airways is involved in allergen-induced air-way remodeling, Broide and colleagues used a mouse model of asthma. When these mice were repeatedly exposed to the allergen ovalbumin, they responded by becoming sensitive to ovalbumin and then developed chronic asthma. The airway

Fig. (2). Network of cellular mechanisms that lead to NF- B activation and are implicated in the pathogenesis of bronchial asthma: Com-pounds that inhibit IKKB.

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remodeling that was observed appears to be similar to human airway remodeling. The researchers genetically manipulated a subset of these mice to selectively knock-out or delete the gene that encodes IKK in the epithelial cells of the airways. Given that IKK regulates NF- B activity and in its absence NF- B cannot be activated, with the elimination of IKK the researchers effectively prevented NF- B activation in the airway epithelial cells in the IKK -ablated mice. In epithelial cells lacking this gene, the levels of activated NF- B were very low. The researchers then compared what happened when the IKK -deficient mice and normal mice (with a functional IKK gene) were sensitized to ovalbumin and then repeatedly exposed to ovalbumin. The results showed that the mice deficient in activated NF- B had dramatically reduced airway modelling that is usually observed in ex-perimental asthma, less mucus production and lower levels of both eosinophils in the lungs and peribronchial Th2 cells. Decreased inflammation and remodeling in these mice were also accompanied by lower levels of soluble mediators that exacerbate asthma. Perhaps an inhaled NF- B antagonist or an IKK inhibitor could provide effective therapy for chronic respiratory diseases, such as asthma and COPD, in human patients. As mentioned above, the IKK kinase com-plex is a major activator of NF- B but it has been shown that IKK is 20-fold more active than IKK-1 in the phosphoryla-tion of I B [43-45]. Kishore and colleagues [46] described a selective IKK inhibitor, called SC-514, which they used to study NF- B activation in rheumatoid arthritis-derived synovial fibroblasts (RASF cells) after stimulation with IL-1 . They observed that SC-514 inhibited all forms of recom-binant human IKK-2, including the rhIKK homodimer, rhIKK-1/rhIKK-2 heterodimer and the constitutively active form of rhIKK (Ser177-Glu, Ser181-Glu), with comparable IC50 values in the 3-12 μM range. Furthermore, they ob-served that SC-514 specifically inhibited the phosphorylation of p65 by IKK-2 only on the serine 536 residue. Finally, SC-514 was also efficacious in vivo in a rat LPS-induced TNF- model of acute inflammation. On the minus side, SC-514 does not have good oral bioavailability (2%), it has a very short half-life (0.2 h), and its efficacy was more prominently demonstrated with intraperitoneal administration.

Extending these results, Birrel and colleagues [47] exam-ined the impact of another IKK inhibitor, TPCA-1 (2-[(aminocarbonyl)amino]-5-[4-fluorophenyl]-3-thiophenecar-boxamide), on stimulated cultured human airway smooth muscle (HASM) cells and in an in vivo model of antigen-driven airway inflammation. TPCA-1 was demonstrated as a potent and selective IKK inhibitor with an IC50 of 7.74±0.18 μM on isolated IKK and had a 22-fold selectiv-ity over IKK and >550-fold selectivity over other kinases and enzymes. Furthermore, in vivo experiments revealed that TPCA-1 inhibited IL-1 , IL-4, IL-5, IL-13, eotaxin and TNF- gene expression in the ovalbumin asthma model. Similar findings were observed with the glucocorticoid com-parator, budesonide, which also significantly inhibited anti-gen-induced cytokine gene expression. To confirm that TPCA-1 had prevented NF- B nuclear translocation in this asthma model, an EMSA analysis was perfomed using nu-clear extracts from lung tissue. The autoradiograph revealed that TPCA-1, but not budesonide, had blocked the antigen-induced NF- B nuclear translocation. Another interesting

finding was that the oral bioavailability of the compound, TPCA-1 was approximately linear, ensuring long systemic exposure (>4h).

In a recent study, Newton and colleagues [48] demon-strated that the IKKB selective inhibitors PS-1145 [N-(6-chloro-9H- -carbolin-8-ly) nicotinamide] and ML120B [N-(6-chloro-7-methoxy-9H- -carbolin-8-yl)-2-methyl-nicotina-mide], were able to dose-dependently inhibit NF- B-dependent transcription of IL-6, IL-8, IL-1 , TNF- , RAN-TES, GM-CSF and MCP-1 genes in both human A549 pul-monary cells and primary human bronchial epithelial (HBE) cells. Parallel effects on ICAM-1 expression and a signifi-cant repression of IL-8 release were also observed. In con-trast, the corticosteroid dexamethasone, had no effect on NF-

B-dependent transcription or on the expression of ICAM-1. By using mouse embryo fibroblasts (MEFs) lacking both the

and subunits of IKK, it was found that these proteins are required for the induction of a major subset of IFN- -stimulated genes and that this requirement is independent of NF- B activation. Furthermore, there is no defect in either the IFN- -stimulated signal transducer and Stat1 activation or function in the IKK / -null MEFs. Therefore, although activated Stat1 dimers are necessary for the activation of these genes in response to IFN- , they are not sufficient. These results reveal an important additional pathway for IFN- -stimulated gene expression, in which an NF- B-independent function of IKK is required. It has been previ-ously suggested that IKK might contribute to IFN- -dependent gene expression. It was proposed that INFs can activate NF- B directly as a component of IFN-stimulated gene expression. IFN- appears to be able to activate NF- B by phosphorylating a portion of p65 NF- B that is constitu-tively free in the nucleus in some cell lines. However, studies in several cell types fail to reveal direct and complete activa-tion of NF- B by IFN- . There is ample evidence of an im-portant cross talk between the activation of the IKK pathway by TNF- or IL-1 and IFN-dependent signalling, and, there-fore, the combination of TNF- and IFN- leads to synergis-tic induction of inflammatory genes. Keslacy and colleagues [49] showed that exogenously added TNF- with either exogenous or endogenous INF- in airway smooth muscle (ASM) cells, act synergically by promoting the production of

various pro-inflammatory genes regulated through activation of RANTES, which is expressed and secreted by normal T-cells. It is known that ASM cells can act as effector cells in

the initiation and/or perpetuation of airway inflammation in

asthma by producing various inflammatory chemokines or cytokines.

Recently, the same group [50] reported that IFN- , added at concentrations of 1000 U/ml, markedly inhibited TNF- -induced expression of IL-6, IL-8 and eotaxin. These genes were also found to be NF- B-dependent in that TNF- -induced expression of IL-6, IL-8 and eotaxin was dose-dependently inhibited by the selective IKK inhibitor, 4-(2'-aminoethyl)amino-1,8-dimethylimidazo[1,2-a]quinoxaline

(BMS-345541) (1-30 M). Moreover, IFN- , added at con-centrations of 10-1000 U/ml, failed to inhibit NF- B-dependent gene transcription. In addition, IFN- (at 1000 U/ml) decreased TNF- -induced histone acetyl transferase (HAT) and increased histone deacetylase (HDAC) activities. Taken together, these data indicate that IFN- is a potent

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inhibitor of specific TNF- -inducible inflammatory genes by acting on NF- B trans-activation via the modulation of

HDAC function. In addition, IL-1 and TNF- enhance the phosphorylation of the serine-727 residue of Stat1, which

occurs incompletely in response to IFN- alone and, thus, can potentiate IFN- -mediated, Stat1-driven gene expression. Moreover, INF- and increase the trans-activation function of constitutive basal NF- B already free in the nucleus in several cell types by stimulating phosphorylation of the p65 subunit of NF- B. IKKs role in IFN- -dependent signaling is critical for many IFN- -stimulated genes [50]. Mast cells (MC) are known to be major effector cells for allergic dis-eases. The cross-linking of allergen-specific IgE bound to its high-affinity receptor, Fc RI, results in a series of molecular events leading to nuclear factor NF- B activation [51,52]. Peng and colleagues [53] demonstrated that a cell-permeable peptide targeting IKK and four individual chemical IKK inhibitors, 15 deoxyprostaglandin J2 (15dPGJ2), BMS-345541, SC-514, and sulindac, significantly inhibited Ag-IgE-induced TNF production by MC. IKK is rapidly phos-phorylated in MC after Ag-IgE stimulation. Inhibition of IgE-dependent IKK phosphorylation by sulindac correlates with decreased NF- B activation and TNF- production. Thus, IKK may be a therapeutic target that can stabilize the MC in allergic diseases. Moreover, human bronchial biopsies from people with asthma contain higher levels of one of the NF- B protein subunits, compared with lung tissue biopsies taken from people without asthma. Together, these data suggest that epithelial cell-specific NF- B participates indi-rectly in airway remodeling by regulating inflammatory responses that contribute to mucus production, fibrosis and airway wall thickening in experimental asthma.

During lung inflammation, the induction of apoptosis by IKK inhibitors could aid in the clearance of activated in-flammatory cells from the lung and, therefore, lead to re-duced inflammation of the airways. However, a common feature of asthmatic airways is a shedding of the epithelium, which itself correlates with airway hyperresponsiveness [54,55]. Thus, IKK inhibitors could promote epithelial damage and lead to increased airway hyperresponsiveness [56]. In the context of COPD, however, apoptosis may prove beneficial, by preventing, for example, goblet cell hyperpla-sia. Finally, it may be possible to use IKK inhibitors to reduce NF- B activity rather than completely eliminating it. This may prevent the induction of apoptosis and reduce problems of associated tissue damage. Although both IKK and I B can be ubiquitinated and degraded by the 26S pro-teasome complex, proteasome inhibition is commonly asso-ciated with suppression of NF- B activity, as observed in cancer cell lines [57,58].

CORTICOSTEROIDS

Corticosteroids are very effective in treating chronic in-flammatory diseases. Corticosteroids exert their anti-inflammatory effect by switching off the expression of mul-tiple genes, including cytokines, chemokines, adhesion molecules, receptors and proinflammatory enzymes. Corticosteroids enter cells and bind to glucocorticoid receptors (GRs) in the cytoplasm. Occupied GRs rapidly translocate to the nucleus, where they may bind to CREB-binding protein (CBP) or other coactivators, such as p300.

(CBP) or other coactivators, such as p300. p300/CBP-associated factors inhibit HAT activity, histone acetylation and chromatin restructure. Although this leads to the tran-scription of anti-inflammatory genes, this effect does not adequately explain how corticosteroids switch off multiple inflammatory genes. Thus, alternate explanations, including corticosteroid effects on transcription factors and histone deacetylases that regulate gene transcription, have been pro-posed. Transcription factors that are important in asthma include NF- B and AP-1, both of which are activated in the airways of asthmatics. One mechanism by which glucocorti-coids regulate NF- B expression is by regulating the expres-sion of I B. Another explanation is that NF- B is regulated when it interacts with other coactivators, such as CBP, p300 or p300/CBP, which are known to interact with NF- B to promote transcription [59].

The endocrine and metabolic effects of steroids that are responsible for the systemic side effects of corticosteroids are likely to be predominantly mediated via DNA binding through an interaction of GRs with negative GREs (cis-repression). This has led to a search for novel corticosteroids that selectively trans-repress without significant trans-activation or cis-repression, thus reducing the potential risk of systemic side effects. Since corticosteroids bind to the same GR, this seems, at first, to be an unlikely possibility. Nevertheless, separation of trans-activation and trans-repression has been demonstrated using reporter gene con-structs with selective mutations for the GR in transfected cells. In addition, in mice with GRs that do not dimerise, there is no trans-activation, but trans-repression appears to be normal. Furthermore, some steroids, such as the antagonist RU486, exhibit greater trans-repression than trans-activation effects. Indeed, the topical steroids used in asthma therapy today, such as fluticasone propionate and budesonide, appear to show more potent trans-repression than trans-activation effects, which may account for their selection as potent anti-inflammatory agents. Recently, a novel class of dissociated steroids has been described, in which there is potent trans-repression with relatively little trans-activation. These ster-oids, including RU24858 and RU40066, have anti-inflammatory effects in vitro, although there is little separa-tion of the anti-inflammatory and systemic side effects in vivo. Other dissociated corticosteroids appear to show disso-ciation in vivo. Several dissociated corticosteroids are now in clinical development and may lead to inhaled steroids with greater safety or even to oral steroids, which are less likely to produce significant adverse effects. The recent resolution of the crystal structure of the ligand-binding domain of GRs may help in achieving a better design for dissociated steroids [60].

The glucocorticoids formoterol and budesonide, when administered to atopic asthmatics, were shown to signifi-cantly decrease (1) the number of submucosal cells staining for (total) NF- B, GM-CSF, and TNF- , (2) the number of mucosal eosinophils and (3) the expression of VCAM-1 in the endothelium and IL-8 in the epithelium. In the case of formoterol treatment, these changes were not accompanied by reduced immunoreactivity for adhesion molecules or cytokines. Thus, at least some of the therapeutic efficacy of inhaled corticosteroids is mediated through the inhibition of NF- B-regulated gene expression, whereas the reduction in

874 Current Medicinal Chemistry, 2009 Vol. 16, No. 7 Charokopos et al.

airway eosinophilia by long-acting agonists probably oper-ates through alternative pathways [61]. Recently, TOPIGEN Pharmaceuticals, Inc., announced a phase II study investigat-ing a novel, inhaled, NO-donating derivative of budesonide (TPI1020) in patients with asthma who are smoking (see company web site).

The 2-agonist, salmeterol, has direct anti-contractile/ anti-inflammatory effects on human lung myofibroblasts that are amplified in vitro by the combined use of low concentra-tions of the glucocorticoid fluticasone propionate. Sal-meterol±fluticasone inhibits the constitutive and TGF- -induced expression of alpha-SMA. In addition, the two drugs block the TNF- -induced nuclear translocation of NF- B. Finally, salmeterol decreases TNF- -induced production of IL-6. The complementary anti-inflammatory/anti-contractile effects displayed by salmeterol and fluticasone on lung myo-fibroblasts in vitro may be related to the improvement in lung function and symptom control obtained in vivo by the early use of low doses of glucocorticoids in combination with long-acting 2-agonists [62].

LEUKOTRIENE INHIBITORS

The leukotrienes are a family of polyunsaturated lipoxy-genated eicosatetranoic acids that are derived from arachi-donic acid and exhibit a wide range of pharmacological and physiological actions. In biological systems, their actions are limited by their relative rates of synthesis and degradation. Of the three enzymes exclusively involved in the formation of the leukotrienes, namely, ALOX5, LTC4 synthase and LTA4 epoxide hydrolase, ALOX5 is the enzyme required for the production of the cysteinyl leukotrienes LTC4, LTD4, LTE4 and LTB4. Over the past 10 years, it has been shown that pharmacological inhibition of ALOX5 action or antago-nism of the cysteinyl leukotrienes action at their receptor is associated with an amelioration of asthma. Inhibition of ALOX5 activity or blockade of the CysLT1 receptor by cLI is associated with clinically significant improvements in asthma outcome. The regulation of ALOX5 is multifaceted; known mechanisms include regulation of action of the ma-ture protein and regulation of ALOX5 gene transcription and translation. The ALOX5 gene promoter contains numerous consensus-binding sites for many known transcription fac-tors, including Sp1, Sp3, Egr-1, Egr-2, GATA, Myb and NF-

B. It may be possible to genotype asthmatics at the ALOX-5 locus and to predict which patients will improve with leu-kotriene inhibitors; however, asthmatics with mutant alleles at the ALOX-5 should probably avoid cLI because they are unlikely to derive clinical benefit [63].

ACTIVATED PROTEIN C

Activated protein C (APC) is a serine protease with po-tent anti-inflammatory effects. In an in vivo asthma model, BALB/c mice inhaled APC and were subsequently exposed to their allergen, OVA. Inhalation of APC significantly in-hibited the expression of Th2 cytokines, IgE secretion, eosi-nophilic inflammation and hyperresponsiveness in OVA-sensitized/challenged animals. In addition, it was shown that the function of STAT6 and NF- B isolated from lung tissue was significantly reduced in mice that inhaled APC [64].

LYSOPHOSPHATIDIC ACID (LPA)

LPA is a potent bioactive phospholipid that elicits di-verse cellular responses through activation of the G-protein-coupled receptors, LPA1-LPA4. LPA-mediated signalling is regulated by lipid phosphate phosphatases (LPP)-1, -2 and -3, which belong to the phosphatase superfamily. Overexpres-sion of LPP-1 attenuates LPA-induced increases in the intra-cellular Ca2+ concentration, phosphorylation of I B and translocation of NF- B to the nucleus, and almost com-pletely prevents IL-8 secretion. Thus, LPPs could represent potential targets in regulating leucocyte infiltration and air-way inflammation [65].

MOLECULAR THERAPY, NF- B DECOY AND HIS-

TONE DEACETYLASE (HDAC) INHIBITORS

Modern therapeutic methods for manipulation of gene expression in allergic diseases have been receiving increased attention in the emerging era of functional genomics. With the growing application of gene silencing technologies, pharmacological modulation of translation represents a great advance in molecular therapy for allergy. Several strategies for sequence-specific post-transcriptional inhibition of gene expression are being developed. They include antisense oligonucleotides (AS-ONs), ribozymes (RZs), DNA en-zymes (DNAzymes) and RNA interference (RNAi) triggered by small interfering RNAs (siRNAs). Potential anti-mRNA drugs in asthma and other allergic disorders may target tran-scription factors involved in Th2 differentiation and allergic inflammation, including STAT-6, GATA-3 and NF- B. New-generation respirable AS-ONs, external guide sequence ribozymes and RNA interference-based therapies have the potential to satisfy unmet needs in allergy treatment, acting at a more proximal level to a key etiopathogenetic molecular process that results in abnormal expression of genes. Moreo-ver, antisense and siRNA technologies imply a more rational design of new drugs for allergy [66]. Research focuses on a serine/threonine kinase involved in glycogen metabolism called glycogen synthase kinase-3B (GSK- 3B), which pro-duces an enzyme that is critical in relaying genetic informa-tion or cellular signals that control early embryonic devel-opment and cell growth in adult animals. GSK-3B is also responsible for the phosphorylation of the transcription fac-tors c-Jun, c-Myc and CCAAT/enhancer-binding protein, translation eukaryotic initiation factor 2 and cytoskeletal proteins. Stimulation with mitogens or growth factors leads to the inactivation of GSK-3B by phosphorylation of the regulatory serine residue at position 9. GSK-3B influences the activity of NF- B. Recent experiments showed that GSK-3B regulates phosphorylation of p65 at the Ser-468 residue and thereby controls the basal activity of NF- B and also regulates the stability of NF- B p105 subunit through phosphorylation. Recently, researchers examined whether the selective GSK-3B inhibitor 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8) could ameliorate airway inflammation in BALB/c mice that were sensitized and chal-lenged with ovalbumin [67]. They showed that TDZD-8 substantially reduced airway eosinophilia and serum levels of OVA-specific IgE. Moreover, TDZD-8 significantly re-duced OVA-induced airway hyperresponsiveness. This find-

Bronchial Asthma, Chronic Obstructive Pulmonary Disease Current Medicinal Chemistry, 2009 Vol. 16, No. 7 875

ing could lead to the design of the next generation of anti-inflammatory drugs.

Recently, Corgentech Inc. announced that the company has begun treating patients in a multi-center phase 1/2 clini-cal trial of its NF- B decoy drug (NF- B Decoy). NF- B Decoy is a candidate drug for the treatment of atopic derma-titis, a chronic skin disease also known as eczema, that af-fects about 15 million adults in the United States. Another similar trial was conducted in Australia and Switzerland in approximately 120 patients in the mid-2005. NF- B Decoy is a highly selective and potent inhibitor of the TF NF- B, and, if successful in eczema, it will also be tested in other inflammatory diseases such as inflammatory bowel disease and asthma [68].

Histone deacetylase (HDAC) inhibitors (Fig. (3)) induce cell cycle arrest and differentiation in cancer cells and have been in phase 1/2 clinical trials for the treatment of various solid or hematological malignancies. In recent years, HDAC inhibitors have been used as anti-inflammatory drugs in rheumatoid arthritis or lupus erythematosus.

The molecular mode of action of HDAC inhibitors is still controversial, but it seems to rely on a reduced inflammatory mediator production, such as NO or cytokines, which also implies inhibition of the TF NF- B. It is possible, therefore, to use HDAC inhibitors in the future for asthma [69].

PEROXISOME PROLIFERATOR-ACTIVATED RE-

CEPTORS (PPARs)

COPD, although characterized by a specific pattern of in-flammation in the airways and lung parenchyma, possesses significant extra pulmonary effects that may also contribute to morbidity and mortality in individual patients [70]. COPD involves several systemic features, particularly in patients with severe disease, and these features have a major impact on the survival of patients with comorbid diseases [71-73]. Cachexia is commonly seen in patients with severe COPD. There may be a loss of skeletal muscle mass and weakness as a result of increased apoptosis and/or muscle disuse. The above clinical manifestations are probably the end result of the oxidative stress, which is present locally and systemically in the skeletal muscle of COPD patients. The generation of ROS and RNS have shown to increase muscle proteolysis, inhibit muscle-specific protein expression, increase muscle cell apoptosis and increase the concentrations of inflamma-tory mediators, including TNF- , IL-6 and ROS, which may potentate some of these effects [74,75]. Furthermore, the generation of ROS leads to a reduction in the histone deace-tylase activity in lung tissue from COPD patients, which may lead to enhanced expression of inflammatory genes and also to a reduction in the antiinflammatory action of glucocorti-costeroids [76]. Interestingly, NF- B activation has been shown in skeletal muscle of severely underweight COPD

Fig. (3). Schematic representation of histone acetylation and deacetylation through NF- B pathway and histone deacetylases (HDAC) com-pounds inhibitors in bronchial asthma and COPD. Histone acetyltranferase can activate histone acetylation through the complex p50-p65-co-activators or directly through the p50-p65 complex acetylation.

876 Current Medicinal Chemistry, 2009 Vol. 16, No. 7 Charokopos et al.

patients (decreased content of IkB and increased DNA binding of NF- B) [77], and NF- B activation per se, is sufficient for the induction of muscle atrophy [78].

The peroxisome proliferator-activated receptors (PPARs) belong to the larger superfamily of steroid nuclear receptors. To date, three different PPAR isoforms have been isolated, -

, - / and - (also referred to as NUC-1, PPAR or FAAR). All three exhibit tissue-specific expression, ligand-specific activation and the ability to heterodimerize with retinoid X receptors. Moreover, PPARs have also been shown to pos-sess important anti-inflammatory properties by modulation of inflammatory signaling through the NF- B pathway [79].

An overview of the PPARs’ therapeutic targets is shown in Fig. (4).

PPAR- is expressed in many skeletal muscles, including airway smooth muscle. The primary role of PPAR- in skeletal muscle is regulation of fatty acid homeostasis and transcriptional control of lipid regulatory genes [80]. How-ever, recent data have demonstrated that it also exhibits anti-inflammatory effects [81]. It was demonstrated that induc-tion of chronic airway inflammation by LPS administration in homozygous PPAR- knock-out mice resulted in a huge recruitment of inflammatory cells into the airways, mainly neutrophils and macrophages, associated with an increase in bronchoalveolar lavage of TNF- , the CXC chemokines MIP-2 and KC, and an increase in the CC chemokine, MCP-1, compared to PPAR- +/+ animals. In contrast, the admini-stration of fenofibrate reduced the inflammatory infiltration,

cytokines and chemokines in the PPAR- +/+ animals, but not in the PPAR- -/- animals. These results are very interesting because it is known that PPAR- activators inhibit the NF-

B pathway and re-establish control over pro-inflammatory cytokine (IL-6, TNF- ) production in mouse tissues. Moreo-ver, activation of PPAR- was found to induce expression of I B in primary smooth muscle cells [82,83].

PPAR- is also activated by a number of naturally occur-ring lipid-derived molecules, including long-chain fatty acids, eicosanoids and leukotriene B4, while the fibrate class of hypolipidaemic drugs, including fenofibrate and gemfi-brozil, serve as synthetic PPAR- ligands. Several studies demonstrated that activation of PPAR- in vivo also causes an upregulation in a number of antioxidant enzymes, includ-ing catalase, copper(II) and zinc(II) superoxide dismutase (SOD) and mediators of the glutathione pathway [83-85].

Although most abundant in adipose tissue, PPAR- is also expressed at low levels in the skeletal muscle and in a number of hematopoietic cells, including DCs, eosinophils, macrophages and T cells. From a pulmonary perspective, PPAR- has been shown to inhibit the release of proinflamat-tory cytokines from activated macrophages and airway epithelial cells and to inhibit vascular airway smooth cell proliferation and induce apoptosis in endothelial cells, T-lymphocytes and macrophages. PPAR- is also expressed in the human lung. Like PPAR- , PPAR- attenuates the proin-flammatory cytokine influx by blocking NF- B activation [86]. Anti-inflammatory effects of PPAR- activation in the

Fig. (4). Compounds that activate PPAR- and inhibit the NF- B pathway. The activated PPAR- -compound complex inhibits simultane-ously the nuclear corepressors NCoR/HDA3 and/or SMRT/HDA3 and the formation of the nuclear P50-P65 complex resulting, thus, in the inhibition of the transcription of inflammatory genes.

TZD, thiazolimediones; NCoR, nuclear receptor corepressor complex; SMRT, silencing mediator for retinoid and thyroid hormone receptor; RXR, 9-cis retinoid acid receptor.

Bronchial Asthma, Chronic Obstructive Pulmonary Disease Current Medicinal Chemistry, 2009 Vol. 16, No. 7 877

lung have been shown consistently in experimental models of asthma and other airway diseases, including COPD [87]. PPAR- is the main target of the thiazolidinedione (TZD) class of insulin-sensitising drugs, which are currently used to treat type 2 diabetes mellitus [88-90]. Besides these synthetic ligands, PPAR- is activated by several naturally occurring compounds, such as prostaglandin J2 derivates and polyun-saturated fatty acids (PUFAs) [80]. Moreover PUFAs are common components of fatty fish and olive oil and are known PPAR activators. Broekhuizen and colleagues [91] observed that nutritional supplementation with PUFAs, as an adjunct to exercise training in COPD patients, markedly enhanced exercise capacity, compared with a placebo-treated group.

There is now a large body of evidence indicating that al-lergic asthma is the result of a predominant Th2 type immune

response, and targeting the production of Th2 cytokines has been proposed as a way to regulate disease. PPAR- ligands can affect T cell cytokine production, and PPAR- expres-sion is increased in the bronchial submucosa, airway epithe-lium and smooth muscle cells from asthmatic patients [79,92,93]. In a mouse model of bronchial asthma, it was shown that activation of PPAR- alters the maturation proc-ess of DCs [94]. It was also shown that ciglitazone, a selec-tive PPAR- agonist, abolished the movement of DCs in regional lymph nodes after antigenic stimulation, and this was accompanied by an increase in IL-10 production. It was speculated that the above observations were probably a result of PPAR- agonists disrupting the NF- B pathway by block-ing the influx of TNF- and IL-1 cytokines, which are involved in the movement of DCs at the regional lymph nodes. This speculation was proven correct by another study with mice [95] that showed that the PPAR- agonists, rosiglitazone, pioglitazone and AdPPAR, had an effect on TDI-induced bronchial inflammation and remodeling, which are prominent features of bronchial asthma. Furthermore, it was shown that inhibition of NF- B activation decreased Th2 cytokine levels, adhesion molecules, chemokines and TGF- 1 after TDI inhalation.

Because PPAR- ligands can affect T cell cytokine pro-duction

in vitro, August and colleagues [96] examined if these ligands affect symptoms in a murine model of allergic asthma. The data revealed that ciglitazone and GW1929, two agonistic ligands for PPAR- , significantly inhibited airway inflammation during allergic asthma induction. Oral treat-ment with ciglitazone and GW1929 inhibited airway in-flammation, and, to a lower extend, AHR. By contrast, intra-nasal exposure to GW1929 significantly reduced AHR fol-lowing exposure to allergen, while GW9662, a PPAR- an-tagonist, had no effect. In vitro, T cells from ciglitazone-treated mice secrete significantly less IL-4 and IFN- in re-sponse to restimulation. These data suggest that PPAR- agonists may be useful for the treatment of allergic asthma.

Respiratory viruses have also been detected in a high proportion of the more severe exacerbations requiring hospi-talization in bronchial asthma and COPD subjects. Viruses have also been implicated in the pathogenesis of asthma exacerbations in adults [97,98]. Although the mechanisms of virus-induced asthma exacerbations differ from the mecha-nisms of virus-induction in COPD exacerbations [99], the

syndrome following the respiratory virus infection is both a consequence of the direct harmful effects of the virus itself and of the immunopathology resulting from the host immune response. This has led to the concept that rhinovirus and RSV infection are, at least in part, immune-mediated dis-eases with proinflammatory cytokines, chemokines and inflammatory cell products producing a pathology that is, perhaps, more than a direct cytotoxic effect of the virus. Arnold and colleagues [101] investigated the role of the PPAR- agonists 15d-PGJ(2), ciglitazone and troglitazone on the synthesis of RSV-induced cytokine release from RSV-infected human lung epithelial cells. They observed that all PPAR- ligands inhibited in a dose-dependent manner the release of TNF- , GM-CSF, IL-1 , IL-6. IL-8 and RANTES from RSV-infected epithelial cells. Concomitantly, the PPAR- ligands diminished the cellular amount of mRNA encoding for IL-6, IL-8 and RANTES, and the RSV-induced binding activity of the transcription factors NF- B, AP-1. The above data indicate a potential application of PPAR ligands in the anti-inflammatory treatment of RSV infection in bronchial asthma and COPD exacerbations.

PPARs are essential regulators of fatty acid utilization and energy homeostasis in several tissues, including the heart and skeletal muscle [102]. Recently, Remels and colleagues [103] showed that PPAR- protein content is reduced in the skeletal muscle of COPD patients, compared with healthy controls. In addition, mRNA levels of the PPAR coactivator 1 (PGC-1 ), which also regulates mitochondrial biogene-sis, were lower in COPD patients, compared with controls, and mRNA levels of PPAR- were significantly lower in cachectic patients, compared with non-cachectic patients. These results show a strong overlap between PPAR- and PPAR- to control fatty acid oxidation by regulating the genes involved in fatty acid transport, -oxidation and mito-chondrial respiration.

Given both the strong involvement of the PPARs and PGC-1 (1) in the regulation of the skeletal muscle oxidative phenotype, (2) in blocking the NF- B pathway and (3) the observation of reduced PPAR expression levels in skeletal muscle of COPD patients, it is tempting to suggest a possible therapeutic role for PPAR activators in muscle metabolism in chronic obstructive pulmonary disease.

ARYL HYDROCARBON RECEPTOR (AHR)

The TF aryl-hydrocarbon-receptor (AhR) plays an impor-tant role in the response to environmental pollutants. How-ever, its role in normal physiology is unclear. To investigate the role of AhR in acute lung inflammation, a group [104] studied the effect of exposure to inhaled cigarette smoke or bacterial endotoxin control on AhR knock-out mice. Smoke-induced lung inflammation was 2-3 fold more severe in AhR-

/- mice than in controls. Interestingly, levels of TNF- and IL-6 in the bronchoalveolar lavage of air-exposed AhR-/- mice were equal to the levels of smoke-exposed controls, suggesting that AhR-deficient mice are inflammation prone. AhR-/- mice when challenged with inhaled endotoxin, which does not contain AhR ligands, also developed greater lung neutrophilia than controls, and their bronchoalveolar lavage cells produced elevated levels of TNF- and IL-6 when treated with endotoxin in vitro. NF- B DNA-binding activity

878 Current Medicinal Chemistry, 2009 Vol. 16, No. 7 Charokopos et al.

Table 1. Drugs for Asthma and COPD: (a) Currently in Use, (b) Experimental Drugs, and (c) Drugs that Failed in Clinical Trials

Treatment Condition Ref.

(a) Dugs currently available in clinical practice

Short acting 2 agonists asthma, COPD [70, 117]

Long acting 2 agonists asthma, COPD [70, 114, 118]

Inhaled steroids asthma, COPD [70, 118, 122, 123]

Oral steroids asthma [70]

Cysteinyl-leucotriene receptor antagonists asthma [124]

Theophylline COPD [70, 125]

Short-acting anticholinergics COPD [70, 118]

Long-acting anticholinergics COPD [70, 118]

mAb to human IgE asthma [126]

(b) Potential drug targets

mAb to TNF- asthma [118]

IKK-2 inhibitors asthma, COPD [46-48]

INF- asthma [49]

Selective HDAC2 inhibitors COPD [69]

PPAR- activators asthma, COPD [81-91, 101]

Proteasome inhibitors asthma, COPD [57, 58]

Antioxidants COPD [28, 37-40]

Toll-like receptor 9 -Th1- stimulators asthma [109]

Type IV PDE inhibitors COPD [128, 129]

5-lipoxygenase enzyme inhibitor asthma, COPD [130, 145]

CC chemokine receptor 3 antagonist asthma [131, 132]

GSK-3B inhibitors asthma [67]

human recombinant (hr) IL-12 asthma [133]

hr IL-10 asthma [134]

P-selectin antagonists asthma [135]

PGD2/DP2 antagonist asthma [144]

Statins COPD [150]

MARK inhibitors asthma [106]

JNK inhibitors asthma [107]

p72SYK inhibitors asthma [108]

vitamin D3 COPD [113]

EGFR inhibitor COPD [115]

(c) Drugs that failed in clinical trials

Neuropeptide-PAF antagonists asthma [136]

Bradykinin antagonists asthma [137]

mAb to IL-5 asthma [138]

mAb to IL-4 asthma [139, 140]

mAb to TNF- COPD [141]

Allergen specific VLA-4 antagonist asthma [142]

Antihistamines asthma [143]

Mast cell stabilizers asthma [41]

5-Lipoxygenase activating protein (FLAP) inhibitors asthma [146]

Leucotriene B4-BLT1 antagonist asthma [147]

IL-8 inhibitors COPD [148]

Hyaluronic acid asthma [149]

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was elevated in smoke-exposed AhR-/- mice, compared with controls, and it was associated with a rapid loss of RelB. It seems that AhR is a previously unrecognized regulator of inflammation that interacts with NF- B so that, in the ab-sence of AhR, RelB is prematurely degraded, resulting in heightened inflammatory responses to multiple proinflamma-tory stimuli [104].

CONCLUDING REMARKS

Table 1 summarizes currently available drugs, therapeu-tic potential drug targets and drugs that have been tried and failed in asthma and COPD.

Inflammatory airway diseases impose a significant health care problem worldwide. Conventional therapy consists of

2-adrenoceptor agonists in combination with glucocorti-coids and/or anticholinergics. Nevertheless, there is an ur-gent need to focus on multiple target strategies to treat bron-chial asthma, such as inhaled glucocorticoids with lower systemic side-effects, that prevent the activation of gene transcription through DNA-glucorticoid receptor binding. In COPD patients, more potent antioxidants could help to re-store steroid sensitivity and phosphodiesterase type 4 (PDE4) inhibitors with isozyme selectivity [105]. Research into the molecular pathways and, in particular, the transcription fac-tors governing the pathophysiology of bronchial asthma and COPD is of critical importance to identify human-relevant targets.

NF- B regulates the transcription of a wide array of gene products that are involved in the molecular pathobiology of the lung. Three lung cell types, epithelial cells, macrophages and neutrophils, have been shown to be involved in the gen-eration of lung inflammation through signaling mechanisms that are dependent on the activation of the NF- B pathway. The basic molecular biology of the NF- B activation path-way is well described, and approaches to modify this axis have involved inhibition of various components of the clas-sical activation pathway, including ubiquitination and pro-teosomal degradation of I B. Recently, there have been detailed characterizations of molecular mechanisms that involve reversible post-translational modification of RelA, including phosphorylation and acetylation, that may be therapeutically targeted.

DNA decoy, antisense and siRNA technologies that in-terfere with NF- B expression and binding have been tested in experimental settings, but it is not yet clear whether these approaches can be practically or effectively applied in pa-tients. A promising approach is inhibition of IKKs since they appear to be highly specific for the NF- B activation path-way and are affected by conventional small molecule phar-maceutics [5]. Apart from IKKs, several kinase inhibitors are in phase I trials; these include p38 MARK inhibitors [106], JNK inhibitors [107] and p72SYK inhibitors [108].

Airways display robust NF- B activation and represent targets for anti-inflammatory asthma therapies, but the func-tional importance of NF- B activation in airway epithelium remains enigmatic. Transgenic mice in which NF- B activa-tion is repressed specifically in the airways demonstrate significantly ameliorated inflammation in response to in-haled Ag, reduced levels of chemokines, T cell cytokines,

mucus cell metaplasia and circulating IgE, compared with littermate controls, but not reduced Ag-driven airways’ hy-perresponsiveness. It can be therefore deduced that airway epithelial NF- B activation orchestrates Ag-induced inflam-mation and subsequent adaptive immune responses, but does

not contribute to airways’ hyperresponsiveness [109].

For many years, researchers have attempted to down-modulate the immune mechanisms that initiate and maintain inflammation. Today, the only widely used disease-modifying form of an allergy treatment is the specific immu-notherapy with allergen extracts. More recently, the use of anti-IgE was approved for patients with allergic asthma.

Other immunomodulatory methods under investigation in-clude the administration of microbial adjuvant that inhibit Th2 reactivity, and the design of molecules that interrupt the activity of key allergic cytokines, chemokines or other Th2 effectors mediators [110,111]. In addition, the administration of the antioxidant diferulomethane (curcumin) and sulforap-hane, which occurs naturally in broccoli [112], may also be an attractive approach toward novel bronchial asthma and COPD treatment.

Alternatively, molecular therapies targeting the genera-tion of Tregs via NF- B blockade of DC maturation may be a novel approach to modulate asthmatic inflammation in early stages. It is of interest that Tregs from the lungs in contrast to those isolated from the spleen produced INF- in addition to TGF- and IL-10. Encouraging evidence about Treg manipulation comes from a recent report [113] that demosntrated that the administration of Vitamin D3 in a small group of patients with glucocorticoid-resistant bron-chial asthma promote responsiveness to glucocorticoids and increase IL-10 production by Tregs.

In COPD neutrophils infiltrate the airways. Epidermal growth factor receptor (EGFR) plays a key role in signalling pathway which lead to mucin production (MUC5AC) and neutrophil accummulation via IL-8 production. EGFR ex-pression correlates also with disease severity and neutrophil infiltration in asthmatic airways. Acute exacerbations of asthma and COPD are also associated with steroid refractory neutrophilic inflammation. Upregulation of EGFR ligands induces an increase of IL-8 cytokine and ICAM-1 expression both of which are under NF- B control [114]. It was recently reported that the EGFR inhibitor AG1478 completely abol-ished the expression of IL-8 and ICAM-1 from infected epithelial cells with rhinovirus infection commonly seen in COPD patients; therefore, inhibition of EGFR may also suppress the activation of NF- B and the transcription of inflammatory genes [115]. In addition, Bergin et al. [116] showed that meprin alpha, an oligomeric metalloendopepti-dase, also induced nuclear localisation and activation of NF-

B in a MyD88-dependent manner. It is of interest that in-flamed epithelial cells express meprin alpha in excess and, in response to neutrophic elastase, can generate EGFR ligands through EGFR and TLR4 to NF- B; therefore, blocking EGFR ligands may also result in NF- B inactivation.

In conclusion, results from experimental and clinical studies suggest that inhibitors of NF- B that are successful in other diseases, may prove to be useful novel therapies in the treatment of asthma and chronic obstructive pulmonary disease.

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Received: November 22, 2008 Revised: January 28, 2009 Accepted: January 30, 2009