RETRACTED: Kinase inhibitors and airway inflammation

European Journal of Pharmacology 533 (2006) 118 – 132 www.elsevier.com/locate/ejphar

Review

Kinase inhibitors and airway inflammation Ian M. Adcock ⁎, K. Fan Chung, Gaetano Caramori, Kazuhiro Ito

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Cell and Molecular Biology Group, Airways Disease Section, National Heart and Lung Institute, Imperial College London, Dovehouse Street, London, SW3, 6LY, United Kingdom Accepted 13 December 2005 Available online 8 February 2006

Abstract

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Kinases are believed to play a crucial role in the expression and activation of inflammatory mediators in the airway, in T-cell function and airway remodelling. Important kinases such as Inhibitor of κB kinase (IKK)2, mitogen activated protein (MAP) kinases and phsopho-inositol (PI) 3 kinase regulate inflammation either through activation of pro-inflammatory transcription factors such as activating protein-1 (AP-1) and nuclear factor κB (NF-κB), which are activated in airway disease, or through regulation of mRNA half-life. Selective kinase inhibitors have been developed which reduce inflammation and some characteristics of disease in animal models. Targeting specific kinases that are overexpressed or over active in disease should allow for selective treatment of respiratory diseases. Interest in this area has intensified due to the success of the specific Abelson murine leukaemia viral oncogene (Abl) kinase inhibitor imatinib mesylate (Gleevec) in the treatment of chronic myelogenous leukaemia. Encouraging data from animal models and primary cells and early Phase I and II studies in other diseases suggest that inhibitors of p38 MAP kinase and IKK2 may prove to be useful novel therapies in the treatment of severe asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis and other inflammatory airway diseases. © 2006 Elsevier B.V. All rights reserved.

Introduction . . . . . . . . . . . . . . . . . . . . . Inflammatory component of respiratory disease . . . The role of protein kinases in respiratory disease . . Kinase inhibitors. . . . . . . . . . . . . . . . . . . 4.1. MAP kinase inhibitors . . . . . . . . . . . . 4.1.1. p38 MAPK inhibitors . . . . . . . . 4.1.2. JNKs . . . . . . . . . . . . . . . . 4.2. NF-κB . . . . . . . . . . . . . . . . . . . . 4.3. Protein kinase C (PKC) . . . . . . . . . . . 4.4. Inhibitors of other protein kinases . . . . . . 4.5. Tyrosine kinases . . . . . . . . . . . . . . . 4.5.1. Non-receptor tyrosine kinases . . . . 4.5.2. Receptor associated tyrosine kinases 4.6. Phosphoinositide 3-kinases (PI-3Ks) . . . . . 5. Conclusions . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . .

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1. 2. 3. 4.

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Contents

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Keywords: Asthma; COPD; Inhibitor; MAP kinase; IKK; Gene expression

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⁎ Corresponding author. Tel.: +44 207 351 8183; fax: +44 207 351 8126. E-mail address: [email protected] (I.M. Adcock). 0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2005.12.054

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Between 1980 and 1996, the number of Americans diagnosed with asthma more than doubled to almost 15 million. Interestingly, the highest rate of increase was reported in children under 5 years old. This represents a profound public health problem responsible for over 9 million visits to primary health care providers, just under 2 million emergency room visits and over 460,000 hospitalisations per year in the United States of America (USA) alone. In 1990, the annual cost of asthma to the U.S. economy was estimated to be $6.2 billion, with the majority of the expense attributed to medical care. Even if rates were to stabilize, asthma would continue to be a profound public health problem. In addition to placing a considerable burden in terms of direct medical costs, asthma also has enormous indirect costs and is one of the leading causes of work or school absenteeism (Barnes et al., 1996). Most patients with asthma respond well to current therapies, however, a small percentage (10%) fail to respond well and these account for N 50% of the total asthma health care costs. Importantly, it appears as if enhanced kinase activity may be responsible, at least in part, for a failure of glucocorticoid action in these patients (Leung and Bloom, 2003; Adcock and Lane, 2003). Chronic obstructive pulmonary disease (COPD), a chronic inflammatory disease of the lower airways and lung, is one of the commonest causes of morbidity and mortality in the world, and is increasing in prevalence. COPD is predicted to become the third most common cause of death and the fifth most common cause of disability in the world by 2020 (Lopez and Murray, 1998). Cystic fibrosis is also recognised as an inflammatory disease with increased neutrophilia, inflammatory gene expression and enhanced activation of several transcription factors under the control of Toll-like receptor activation (Koehler et al., 2004). Exacerbation of disease induced by viral and/or bacterial infection also leads to kinase activation as a prequel to increased inflammation (Mejias et al., 2005).

“acute-on-chronic” inflammation may be observed during exacerbations, with an increase in eosinophils and sometimes neutrophils (Busse and Lemanske, 2001). Over one hundred mediators have now been implicated in inflammatory diseases of the airway and lung, including multiple cytokines, chemokines and growth factors (Barnes et al., 1998). Blocking a single mediator is therefore unlikely to be very effective in this complex disease and mediator antagonists have so far not proved to be very effective compared with drugs that have a broad spectrum of anti-inflammatory effects, such as glucocorticoids (Leckie et al., 2000). Inflammatory mediators are not released solely from classical infiltrating inflammatory cells and resident/structural cells also produce inflammatory mediators. Thus airway epithelial cells, endothelial cells, fibroblasts and airway smooth muscle cells show an altered phenotype in asthma and express multiple inflammatory mediators, including cytokines, chemokines and peptides. Indeed, these cells then become important targets for asthma therapy and there is increasing evidence that airway epithelial cells are major targets for inhaled glucocorticoids (Schwiebert et al., 1996). Chronic inflammation may also lead to structural changes in the airway, including sub-epithelial fibrosis, increased thickness of airway smooth muscle, increased numbers of blood vessels (angiogenesis) and increased number of mucus secreting cells (Beckett and Howarth, 2003). These changes, referred to as airway remodelling, may not be fully reversible with current treatments and explain why there is an irreversible component of airway obstruction in some patients with moderate to severe asthma and why there is an accelerated decline in lung function [forced expiratory volume in 1 s (FEV1)] in non-smoking asthmatic patients (Lange et al., 1998). Why some patients develop these irreversible changes whereas others do not may be determined by so far unknown genetic factors modulating the inflammatory/anti-inflammatory responses. COPD is a chronic inflammatory disease of the lower airways and lung, which is enhanced during exacerbations (Barnes, 2000). The pathological hallmarks of COPD are destruction of the lung parenchyma (pulmonary emphysema), inflammation of the peripheral airways (respiratory bronchiolitis) and central airways along with parenchymal inflammation (Hogg et al., 2004; Saetta et al., 2001). Most patients with COPD have all three pathologic conditions (chronic obstructive bronchitis, emphysema, and mucus plugging), but the relative extent of emphysema and obstructive bronchitis within individual patients can vary widely (Boschetto et al., 2003). There is a marked increase in macrophages and neutrophils in bronchoalveolarlavage fluid and induced sputum (Hogg et al., 2004; Saetta et al., 2001). Patients with COPD have infiltration of T-cells (with an increased ratio between CD8+ and CD4+ T-cells), macrophages and an increased number of neutrophils within the airways' mucosa and lung parenchyma (Hogg et al., 2004; Saetta et al., 2001). This was particularly marked with respect to B cells and CD8 cells in GOLD stage 3 and 4 (Hogg et al., 2004). Severity of COPD is also strongly associated with airway wall volume and luminal mucous exudate of the small airways (Hogg et al., 2004).

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1. Introduction

2. Inflammatory component of respiratory disease

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Inflammation is a central feature of many respiratory diseases and the specific characteristics of the inflammatory response in each disease and the site of inflammation differ but all involve the recruitment and activation of inflammatory cells and changes in the structural cells of the lung. These conditions are characterized by an increased expression of components of the inflammatory cascade including cytokines, chemokines, growth factors, enzymes, receptors and adhesion molecules (Busse and Lemanske, 2001). Inflammation in asthma is associated with increased airway hyperresponsiveness leading to recurrent episodes of wheezing, breathlessness, chest tightness, and coughing, particularly at night or in the early morning. These episodes are usually associated with widespread but variable airflow obstruction that is often reversible either spontaneously or with treatment (Busse and Lemanske, 2001). This inflammation is present even in those with very mild asthma and is unique in that the airway wall is infiltrated by T lymphocytes of the T-helper (Th) type 2 phenotype, eosinophils, macrophages/monocytes and mast cells. In addition, an

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The bronchioles are obstructed by fibrosis and infiltrated with macrophages and T lymphocytes. In contrast to the situation with asthma, eosinophils are not prominent except in patients with concomitant asthma or in some patients during exacerbations. The relationship between inflammation and clinical symptoms of asthma and COPD is not clear. There is evidence that the degree of inflammation is loosely related to airway hyperresponsiveness, as measured by histamine or methacholine challenge but other factors, such as structural changes in the airway wall, are important (Barnes, 2003). 3. The role of protein kinases in respiratory disease

4. Kinase inhibitors

Protein kinases are classified as either protein tyrosine (Tyr) kinases or serine/threonine (Ser/Thr) kinases although some dual kinases exist. The human genome consists of 518 kinases along with a smaller number of phosphatases (Manning et al., 2002). All kinases transfer the γ-phosphate of ATP to hydroxyl acceptor groups of tyrosine, serine and threonine residues of target proteins. The ATP-binding pocket is between the two lobes of the kinase fold. This site, together with less conserved surrounding pockets, has been the focus of inhibitor design that has exploited differences in kinase structure to achieve selectivity and most small inhibitor molecules of protein kinases target the active ATP-binding site and act as competitive inhibitors. However, the high degree of structural similarity in the ATP-binding pocket of protein kinases presents a major challenge to the development of potent, selective kinase inhibitors. Nonetheless, Gleevec and Tarceva, for example, have shown effectiveness in treating a range of cancers (Noble et al., 2004) (Table 1). Analysis of inhibitor kinase complex structures has indicated potential strategies for targeting the unphosphorylated inactive or active form of the kinase, for targeting the ATP-binding site globally or at or less conserved additional pockets or single residues, or alternatively into targeting noncatalytic domains. The EGF receptor tyrosine kinase inhibitors Iressa (AstraZeneca, Loughborough, UK) and Tarceva (Oncogene Sciences, Uniondale, USA) developed for non-small cell lung cancer and pancreatic cancer bind competitively to the ATP-binding domain. Recently more effective inhibitors that bind irreversibly to the ATPbinding domain through covalent bond formation with a cysteine have been developed (CI-1033, Pfizer, New York; EKB569, Wyeth, Collegeville, USA) and their clinical efficacy is being evaluated. Kinases, such as Gleevec, are targeted towards a unique inactive configuration. The crystal structure of the Abl kinase domain in complex with Gleevec shows that the pyrimidine and the pyridine rings of the drug overlap with the ATP-binding site and are surrounded by a hydrophobic cage (Noble et al., 2004). The rest of the molecule is wedged between the activation

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Many external inflammatory signals important in the inflammatory response seen in asthma and COPD (such as viral and bacterial infection, allergen, cytokines and growth factors) can activate intracellular kinases following binding to transmembrane receptors on responsive cells (Lee and Goodbourn, 2001). The rapid amplification of the initiating signal is due to the number of enzymes involved in each kinase cascade. In addition to mitogen activated protein (MAP) kinases (MAPKs), other kinase pathways are essential for the inflammatory response, e.g. Inhibitor of κB kinase (IKK)2, phospho-inositol-3 (P-I3) kinase and more signal specific Janus kinases (JAKs)/signal transducer and activator of transcription (STATs) pathways. Although each pathway can activate specific downstream transcription factors there is considerable cross-talk between kinase pathways both at the membrane proximal and the transcription factor proximal ends of each pathway which allows signal integration. The importance of these kinase pathways has been shown by the ability of selective inhibitors of these pathways to modulate the expression of numerous inflammatory mediators and adhesion molecules, control granulocyte apoptosis and chemotaxis and T-cell, macrophage and epithelial cell function (Leckie et al., 2000; Kumar et al., 2003). Furthermore, kinase inhibitors have been reported to regulate ASM proliferation and various other factors involved in airway remodelling (Eynott et al., 2003). In addition, mice either overexpressing or lacking some of these kinases, often have altered lung function after allergen challenge (Pernis and Rothman, 2002). An important recent paper from the group of Donna Davies has reported increased tyrosine phosphorylation in the airway epithelium of patients with severe, but not mild, asthma (Hamilton et al., 2005). Although specific substrates were not identified the authors postulated the involvement of epidermal growth factor (EGF) receptor kinase and extracellular regulated kinase (ERK). Changes in phosphorylation status of cells has also been reported in severe asthma where an association with reduced glucocorticoid responsiveness has been proposed (Adcock and Lane, 2003). Thus, enhanced activation of ERK, c-Jun N-terminal kinase (JNK) and p38 MAPKs, and STATs have all been proposed to play a role in steroid-insensitive asthma (Tsitoura and Rothman, 2004; Sousa et al., 1999; Irusen et al., 2002; Goleva et al., 2002; Li et al., 2004). Furthermore, since many inflammatory proteins are not expressed in the resting noninflamed tissue or cell they must be induced following activation of transcription factors and resultant gene transcription (Car-

amori and Adcock, 2003). Many of the important transcription factors involved in regulating gene expression in asthmatic and COPD inflammation such as NF-κB and AP-1 for example are regulated by kinases. Phosphorylation of transcription factors can affect their intracellular localisation, DNA binding and protein–protein interactions with coactivator/repressor factors as well as protein degradation. Therefore, phosphorylation of transcription factors acts as a switch to rapidly translate extracellular inflammatory signals into alterations in gene transcription. STAT, nuclear factor of activated T-cells (NF-AT) and GATA3 proteins are key downstream regulators of Th2 cytokine function and are phosphorylated/dephosphorylated in the asthmatic airway (Pernis and Rothman, 2002). In addition, downstream components of the JNK activation pathway have also been reported to be up-regulated in asthma (Demoly et al., 1995) along with enhanced NF-κB activation in both asthma and COPD (Hart et al., 1998; Di Stefano et al., 2002).

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Table 1 Summary of human and animal studies with kinase inhibitors Target type

Compound names

Developer

Clinical indication

Study phase

References

Outcomes

p38 MAPK

GSK

Manning and Davis (2003) Fijen et al. (2002)

Awaited

p38 MAPK

VX-745

Vertex

RA, Crohn's, psoriais, COPD Crohn's, RA, psoriasis, rhinitis RA

I

p38 MAPK

GSK-681323 GSK-856553 RWJ-67657

II

p38 MAPK p38 MAPK p38 MAPK

BIRB-796 Ro-320-1195 Scio-469

Boehringer Hoffman–LaRoche Scios

Asthma, allergy, RA RA RA

II II II

Discontinued — crosses blood brain barrier Awaited Awaited Awaited

JNK

CC-401

Celgene

RA

JNK/p38

CNI-1493

Crohn's

JNK/MLK

CEP-1347

Cytokine Pharma Sciences Cephalon

Alzheimer's, Parkinson's

II

PKCβ LY333531 EGF receptor Iressa kinase

Eli–Lilly AstraZeneca

Retinopathy, macular oedema Cancer

III

Stelmach et al. (2003) Kuma et al. (2005) Cohen (2002) Nikas and Drosos (2004) Manning and Davis (2003), Eynott et al. (2003) Hommes et al. (2002) Manning and Davis (2003), Bozyczko-Coyne et al. (2002) Aiello (2002) Traxler (2003)

Abl kinase

Gleevec

Novartis

CML

IKK

AS206868 SC-514 BMS345541 TPCA-1 Antisense BAY 61-3606

Celgene/Serono, Pharmacia, Bristol Myers Squib, GSK University of Alberta Bayer

RA, asthma

Rat Mouse

Asthma

Rat

Abbott

Autoimmune and inflammatory diseases Asthma

Mouse

Lyn

pyrrolo[2,3-d] pyrimidines Lipopeptide

Berlin and Lukacs (2005) Frelin et al. (2003), Burke et al. (2003), Luedde et al. (2005), Podolin et al. (2005) Stenton et al. (2002), Yamamoto et al. (2003) Burchat et al. (2002)

Mouse

Adachi et al. (1999)

JAK3

WHI-P131

Immune disease

I

Inflammation and proliferation

Rat

Manning and Davis (2003), Cetkovic-Cvrlje et al. (2003) Finan and Thomas (2004)

PI3K

LY294002

Parker–Hughes Institute

segment and the C helix, which locks the kinase in an inactive conformation. In contrast, Tarceva binds to the active form of their target kinase. PD173955 (Parke–Davis, USA), a pyrido[2,3]pyrimidine-based compound, is a more potent inhibitor of Abl than Gleevec, and the structure (Nagar et al., 2002) has suggested that its greater potency may be because it can bind to multiple conformations of Abl (active or inactive), whereas Gleevec requires a specific inactive conformation. Pyrimidine imidazoles, quinazolinones, and pyridol-pyrimidines are inhibitors that derive considerable selectivity for p38α from probing a small hydrophobic pocket at the back of the ATPbinding site that ATP does not exploit (McInnes and Fischer, 2005). In the majority of protein kinase structures, entry to this pocket is blocked by bulky side chains (e.g. Phe, Asp or Glu residues) but in p38α this so-called “gatekeeper” residue

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Awaited

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University of Texas

Lilly

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Lck

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Syk

J&J

Well tolerated entering Phase II Phase II

Lacked effectiveness

Licensed in Japan, USA for lung cancer. No effect on survival Licensed Reduces swelling and bone erosion/destruction. Prophylactic Inhibits allergen-induced BAL and tissue inflammation and BHR Inhibits IL-2 release in vivo Blocked airway eosinophillia Awaited

Inhibits cancer cell proliferation, lacks specificity

is a much smaller threonine residue as in Abl kinase. 3,4Dihydropyridol[3,2-d]pyrimidines and 2(1H)-quinazolinones have been reported as two new classes of p38α inhibitor that have greater selectivity for p38 than for c-Jun N-terminal kinases (JNKs) and extracellular signal-regulated kinases (ERKs), which could not be rationalized on the basis of predicted p38α-inhibitor interactions (Fitzgerald et al., 2003). Structure determination revealed that the enzyme undergoes a rather subtle conformational change in the hinge sequence to accommodate an inhibitor hydrogen bond-accepting group. This conformational change is calculated to be more energetically favourable in p38α, β and γ, where the hinge region sequence is Met109– Gly110 than in other JNKs and ERKs where Gly110 is replaced with a bulkier side chain. The diaryl ureas, exemplified by the p38 MAPK inhibitor BIRB 796, exploits novel binding sites

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scription factors. The resulting changes in gene expression affect fundamental cellular processes such as proliferation, differentiation, survival and chronic inflammation (Karin, 1998; Johnson and Lapadat, 2002). ERK kinases are widely expressed and are involved in the regulation of meiosis, mitosis and postmitotic functions in differentiated cells. ERKs are activated by oncogenic ras in tumour cells and inhibitors of this pathway are directed more towards cancer (Johnson and Lapadat, 2002). Inhibiting MAPK pathways may not necessarily require direct inhibition of the MAPK themselves since these pathways are involved in processes other than inflammation. If inhibition is to be effective and safe, normal physiological functions must be retained, possibly suggesting that intervention at downstream kinases may have a safer safety profile.

4.1.1. p38 MAPK inhibitors p38 MAPK kinase is a Ser/Thr kinase involved in many processes thought to be important in lower airways' inflammatory responses and tissue remodeling. Several of these events are hallmarks of asthma and COPD. There is, however, a paucity of reports specifically addressing the role of p38 MAP kinase in asthma and COPD but recent in vitro and in vivo results suggest a therapeutic opportunity in this area (Lee et al., 2000). There are four members of the p38 MAP kinase family (α, β, γ and δ). All isoenzymes are activated by the same upstream MAPK kinases (MKK3 or MKK6) in response to a number inflammatory signals but they differ in their tissue distribution, regulation of kinase activation and subsequent phosphorylation of downstream substrates (Saklatvala, 2004). They also differ in terms of their sensitivities toward the p38 MAP kinase inhibitors. In general, p38 MAPKs are activated by many stimuli, including cytokines, hormones, ligands for G protein-coupled receptors, and stresses such as osmotic shock and heat shock and elevated levels of these cytokines are associated with several chronic inflammatory diseases, such as severe asthma and COPD (Pargellis et al., 2002). The synthesis of many inflammatory mediators such as TNFα, IL-4, IL-5, IL-8, RANTES (Regulated on Activation, Normal T Expressed and Secreted) and eotaxins, thought to be important in asthma and COPD pathogenesis is regulated through activation of p38 MAPK (Kyriakis and Avruch, 2001). The p38 MAP kinase pathway is also important for the production of T-cell cytokines such as IL-12 and interferon (IFN)γ and induction of CD54/Intercellular adhesion molecule (ICAM)-1 expression (Craxton et al., 1998). In addition, p38 MAPK appears to be involved in glucocorticoid-resistance in asthma (Irusen et al., 2002). The α and β isoforms are very effective at activating kinases downstream of p38 MAPK such as MAPK activated protein (MAPKAP)2/3 and mitogen- and stress-activated kinase (MSK) 1/2 and have some ability to activate transcription factors. In addition, the α isoenzyme is able to regulate mRNA half-life. However, the relative importance of the β isoensyme is unclear as it is expressed at much lower levels than the α isoenzyme except in endothelial cells (Saklatvala, 2004). In contrast, the γ and δ isoenzymes are less effective at activating MAPKAP2 and other downstream kinases and better at targeting transcription factors such as activating transcription factor (ATF)2, Ets(E26)-

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within the ATP-binding pocket, one of which is created by inducing a conformational change in the enzyme that ultimately yields a structure incompatible with ATP binding. The structure of the EGF receptor 2 (also known as HER2) in complex with Herceptin shows that Herceptin binds EGF receptor 2 on the C-terminal portion of domain IV at a site that encompasses the binding pocket for other domains in the closed inactive forms of EGF receptor 1 and EGF receptor 3. This binding close to the juxtamembrane region of the receptor may allow engagement with the endocytotic machinery while avoiding kinase activation, which would explain the inhibitory effects of Herceptin on receptor signalling (Noble et al., 2004). In vitro studies have shown an important role for several kinases in the regulation of inflammatory gene expression and are the focus of many reviews (Kumar et al., 2003; Manning et al., 2002). We will discuss some of the potential kinases implicated in playing a critical role in chronic inflammatory lower airway diseases, specifically asthma and COPD, and the progress made in developing selective small molecule inhibitors to these targets. Some of the protein kinases are also thought to play a major role in other chronic inflammatory diseases such as rheumatoid arthritis (RA) and in oncoogenesis. Clinical studies are generally more advanced in these later diseases. The selectivity profile for a specific kinase inhibitor may differ depending on the therapeutic needs of the disease and the intended use of the drug. Thus for chronic inflammatory diseases, in which life-long therapy may be required, a much greater degree of selectivity may be needed to achieve an acceptable therapeutic window. In asthma and COPD, for example, it will be necessary inhibit the target kinase whilst not inhibiting potentially debilitating off-target kinases that may cause adverse side effects. In acute disease, however, a broader selectivity profile may be tolerated and, potentially, desirable. These multiplex kinase inhibitors, such as PTK787 and SU11248 used to inhibit angiogenesis, have reached Phase III clinical studies for cancer (Noble et al., 2004). Inhibition of MAPK and NF-κB appear to be the most obvious targets for asthma and COPD and the review has to an extent focussed on these. In addition, non-receptor tyrosine kinases are intimately involved in immune cell receptor signalling and T-cells, eosinophils, and mast cells all use these kinases. Inhibiting these kinases and JAK-STAT kinases regulated by Th2 cytokines may modulate the immune response in asthma.

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4.1. MAP kinase inhibitors

MAPKs are a family of serine/threonine kinases that transduce extracellular signals to the nucleus. In mammalian cells, three major groups of MAPK that differ in their substrate specificity have been characterized: ERK, JNK and p38 MAP kinase. Activation of MAPK requires dual phosphorylation on threonine and tyrosine by upstream kinases and occurs in response to diverse stimuli, such as environmental stress (hyperosmotic shock, heat shock, UV irradiation), endotoxins, mitogenic stimuli and proinflammatory cytokines such as interleukin (IL)-1β and tumour necrosis factor (TNF)α. Once activated, MAPKs phosphorylate selected intracellular proteins including tran-

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activation and disease severity in animal models. For example SB203580 was shown to attenuate BAL TNFα production in an ovalbumin challenged rat model of asthma (Escott et al., 2000) and SB2439063 reduced neutrophilia and mediator expression in rat COPD models (Underwood et al., 2000). A respirable p38α antisense oligonucleotide (ISIS 101757) has recently been reported to significantly inhibit ovalbumin-induced pulmonary eosinophilia, mucus hypersecretion, and airway hyperresponsiveness in a murine model of asthma (Duan et al., 2005). Interestingly, a novel p38α and β MAPK inhibitor (CGH2466) with additional adenosine A1, A2B and A3 receptor antagonist and the phosphodiesterase 4D (PDE4D) inhibitory properties isoenzyme inhibited the ovalbumin- or lipopolysaccharide-induced airway inflammation in mice more potently than the single receptor antagonists or enzyme inhibitors used alone (Trifilieff et al., 2005). BIRB796 is also effective in animal models of asthma and in Phase I clinical studies is well tolerated and shows good pharmacokinetic and pharmacodynamic properties (Regan et al., 2002). It has also shown anti-inflammatory effects (decreased TNFα and IL-6 expression) in a trial with 24 human endotoxemia patients (Branger et al., 2002). In Phase II studies BIRB796 show a decrease in TNFα and a drop in C-reactive protein (Regan et al., 2002). Several structurally diverse p38 MAPK inhibitors have now been evaluated in Phase I (and early Phase II) studies (Kuma et al., 2005). For example, in endotoxin injected human volunteers, RWJ67657 reduced the febrile response and cytokine production to only 10% of that of placebo treated subjects and prevented neutrophil and endothelial activation and was effective against several disease biomarkers (Fijen et al., 2002).

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like kinase (Elk)-1 and serum response factor (SRF) accessory protein (SAP)-1 (Fig. 1) (Saklatvala, 2004). The pyridinylimidazole compounds, exemplified by SB 203580, were originally prepared as inflammatory cytokine synthesis inhibitors that subsequently were found to be selective inhibitors of p38α and β MAP kinase. SB 203580 inhibits the catalytic activity of p38 MAP kinase by competitive binding in the ATP pocket. These drugs inhibit many inflammatory cytokines, chemokines and inflammatory enzymes and interestingly, have a preferential inhibitory effect on synthesis of Th2 compared to Th1 cytokines (Schafer et al., 1999). Furthermore, p38 MAPK inhibitors have also been shown to decrease eosinophil survival by activating apoptotic pathways (Kankaanranta et al., 1999). Interestingly, unlike most protein-kinase inhibitors, which use the ATP-binding pocket to compete directly with ATP binding, the p38 MAPK selective inhibitor BIRB796 (Boehringer Ingelheim) indirectly competes with ATP binding by stabilizing a conformation of the kinase that is incompatible with ATP binding (Pargellis et al., 2002) and is able to inhibit α, β and γ isoenzymes at much lower concentrations than those that affect JNK (Kuma et al., 2005). Recently, Stelmach et al. have reported (in a rat model) potent, orally bioavailable dihydroquinazolinone hybrid p38α inhibitors based on a pyridinyl imidazole p38α inhibitor developed by Merck and on the Vertex compound VX-745 (Stelmach et al., 2003). p38 MAPK inhibitors have been shown to be efficacious in several disease models, including those for asthma and COPD (Adams et al., 2001). In all cases, p38 activation in key cell types correlated with disease initiation and progression. Treatment with p38 MAP kinase inhibitors generally attenuated both p38

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Fig. 1. Mitogen activated protein kinase (MAPK) signalling pathways and selective inhibitors. The modules shown represent pathway connections for the respective MAPK phosphorelay systems. There are multiple MAPK kinase kinase (MKKK), MAPK kinase (MKK) and MAPKs for each system. The connections between the MAPK systems are often cell dependent and are not yet completely elucidated. Inhibitors of particular kinases are also shown. ALK: anaplastic lymphoma kinase, ATF-2: activating transcription factor-2, C/EBP: CCAAT enhancer binding protein, ELK: ETS-like kinase, ERK: extracellular regulated kinase, JNK: c-Jun N-terminal kinase, MAPK: mitogen activated kinase, MEF: myocyte-specific enhancer factor, MLK: mixed lineage kinase, MNK: MAP kinase-interacting kinase, MOS: mouse sarcoma, RSK: ribosomal S6 kinase, TAK: Tat-associated kinase, TCFs: transcription factors, TPL: tumour progression locus.

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there is increased expression of the components of the proinflammatory transcription factor activator protein 1 (AP-1) and enhanced JNK activity (Sousa et al., 1999). JNK consist of three isoforms, encoded by three different genes, of which the JNK1 and 2 isoforms are widely distributed, while JNK3 is mainly located in neuronal tissue (Manning and Davis, 2003; Martin et al., 1996). Gene disruption studies in mice demonstrate that JNK is essential for TNFα-stimulated c-Jun phosphorylation and activity (Manning and Davis, 2003), and is also required for some forms of stress-induced apoptosis and the regulation of inflammatory genes including cytokines, growth factors, cell surface receptors, cell adhesion molecules and proteases such as matrix metalloprotease 1 (MMP-1) (Manning and Davis, 2003). JNKs enhance the transcriptional activity of AP-1 by phosphorylation of the AP-1 component c-Jun and thereby increasing AP-1 association with the basal transcriptional complex (Hibi et al., 1993). JNKs may also enhance the activity of other transcription factors such as ATF-2, Elk-1 and SAP-1a (Manning and Davis, 2003). JNKs are phosphorylated and activated by the dual specificity MAPK kinases MKK4 and MKK7, which are activated by multiple upstream MAPKK kinases including the mixed lineage kinases (MLKs). The STKE JNK Pathway (http://stke.sciencemag.org/cgi/cm/ stkecm;CMP_10827) Connections Map indicates that there are 13 MKKKs that regulate the JNKs. This diversity of MKKKs allows a wide range of stimuli to target this MAPK pathway (Manning and Davis, 2003). JNK activation may also be important in the regulation of the immune response e.g. JNK polarizes the differentiation of CD4+ T-cells to a Th1-type immune response (Fig. 2) (Dong et al., 1998). Signal Pharmaceuticals/Celgene have reported the discovery of SP600125, a JNK inhibitor with at least a 20-fold selectivity against other protein kinases (Bennett et al., 2001), however, this selectivity has recently been questioned (Bain et al., 2003). Despite this, SP600125 has been a useful tool in assessing the role of JNK in various disease models. SP-600125 inhibited

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Despite early initial hopes with these first-generation inhibitors, it soon became clear that liver toxicity would limit their effectiveness (Cohen, 2002). In addition, the first-generation p38α/β/γ inhibitor VX-745 (Haddad, 2001), despite showing some success in a Phase II trial for rheumatoid arthritis, was able to cross into the CNS and further development of this compound has ceased. Unlike the earlier compounds that suffer from undesirable liver side effects, the second-generation p38 MAPK inhibitors have much reduced effects on cytochrome P450 (Adams et al., 1998). Several of these newer generation inhibitors, e.g. VX-702 (Vertex Pharmaceuticals) and HEP 689 have been evaluated in clinical trials for stroke and psoriasis, respectively and at present, at least two large placebo-controlled, dose-ranging Phase II studies, investigating the efficacy of MAPK inhibitors in chronic inflammatory disorders, are under way and results are expected this year (Waetzig et al., 2003). The second-generation p38α inhibitor VX-702 went into Phase II late in 2003 and the results are expected in 2004. CNI-1493, an inhibitor of upstream kinases of JNKs and p38 kinases arthritis (Bianchi et al., 1995), is being tested in patients with Crohn's disease and rheumatoid arthritis (Hommes et al., 2002), and the Boehringer–Ingelheim p38α inhibitor BIRB796 is being evaluated in Crohn's disease, rheumatoid arthritis and psoriasis. Preclinical data in rats with carrageen arthritis demonstrated that oral SCIO-469 and SCIO-323, both potent p38 MAPK inhibitors, lead to a suppression of induced TNFα levels over a wide range of doses. Currently, Scios are nearing completion of a Phase II clinical trial evaluating SCIO-469 for the potential treatment of rheumatoid arthritis (Nikas and Drosos, 2004). In addition, Scios also has begun a Phase I, double-blind, placebo-controlled, dose escalation study of SCIO-323, its second-generation oral p38 MAP kinase inhibitor. Whether this new class of anti-inflammatory drugs will be safe in long-term studies remains to be established. For the successful use of MAPK inhibitors in clinical trial on patients with asthma and COPD, these compounds must be very specific to reduce the side-effects of the plethora of physiological MAPK functions. The limiting side-effects of p38 MAPK inhibition may also possibly be avoided by targeting of the downstream substrates such as MAPKAPK2 since, in contrast to p38 knockout mice, MAPKAPK2 knockouts are viable and exhibit an antiinflammatory phenotype (Kotlyarov et al., 1999). However, in the treatment of asthma and COPD, inhalation may be a feasible alternative therapeutic approach to oral administration.

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4.1.2. JNKs The c-Jun NH2-terminal kinases (JNKs) phosphorylate and activate members of the activator protein-1 (AP-1) transcription factor family and other cellular factors implicated in regulating altered gene expression, cellular survival (apoptosis), differentiation and proliferation in response to cytokines, growth factors and oxidative stress and cancerogenesis. Since many of these are common events associated with the pathogenesis of asthma and COPD, the potential of JNK inhibitors as therapeutics has attracted considerable interest (Manning and Davis, 2003). Furthermore, in patients with severe glucocorticoid-resistant asthma

Fig. 2. The role of mitogen activated protein (MAP) kinase in CD4+ Th1/Th2 differentiation and CD8+ T-cell activation and death. c-Jun amino terminal kinase (JNK)2 and p38 MAP kinases are required for interferon (IFN)γ production by Th1 cells and interleukin (IL)-4 and -5 cytokine release from Th2 cells. Extracellular response kinase (ERK) is required for Th2 differentiation.

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USA.) reduced RANTES production in bronchial epithelial cells (Kujime et al., 2000). These inhibitors are in Phase II studies in Parkinson's and Alzheimer's disease for example where JNK regulates neuronal apoptosis (Manning and Davis, 2003; Bozyczko-Coyne et al., 2002). Several other companies (Aventis, Eisai, Hoffman–LaRoche, Merck, Serono, Takeda and Vertex) have also reported specific JNK inhibitors with potential therapeutic utility in asthma and COPD. Few, however, have reported any additional pharmacological data. Due to the cross-talk between protein kinases recent interest has focussed on the regulation of kinases upstream of JNK and p38 MAPK such as MKK3, MKK6 and MKK4. Celgene, amongst others, has recently announced a number of specific inhibitors of these kinases (Manning and Davis, 2003). Due to the extensive cross-talk within the JNK signalling cascade it is difficult to predict potential adverse events that might arise from pathway inhibition. However, the fact that JNK inhibitors are progressing in clinical trials indicates that the utility of targeting this pathway for therapeutic benefit in asthma and COPD will probably be determined within the near future. 4.2. NF-κB

NF-κB is a major family of transcription factors activated during the inflammatory response in asthma and COPD. NF-κB normally resides in the cytoplasm held in an inactive state by its inhibitor chaperone, IκBα. Phosphorylation of IκBα results in ubiquitination and proteolysis of IκBα, which then releases NF-κB to promote gene transcription (Fig. 3). The multi-subunit IκB kinase responsible for this phosphorylation contains two catalytic subunits, termed IκB kinase (IKK)-1 and IKK-2 (Ghosh and Karin, 2002). NF-κB is a key transcriptional regulator of multiple pro-inflammatory mediators such as TNFα, interleukins, ICAMs, vascular cell adhesion molecules (VCAMs), MMPs and

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TNFα and IL-2 production in human monocytes and Jurkat cells respectively and attenuated TNFα- and IL-1β-induced granulocyte–macrophage colony stimulating factor (GM-CSF), RANTES and IL-8 production in primary human airway smooth muscle cells (Oltmanns et al., 2003). We have recently shown that in acute and chronic animal models of asthma SP-600125 (30 mg/kg s) reduces bronchoalveolar lavage accumulation of eosinophils and lymphocytes, cytokine release, serum IgE production and smooth muscle proliferation after repeated allergen exposure (Eynott et al., 2003; Nath et al., 2005). Similar results were seen with the dual AP-1/nuclear factor κB (NF-κB) inhibitor SP100030 (Huang et al., 2001). Furthermore, SP600125 was able to block JNK activation, MMP-3 expression, and joint destruction in a rat adjuvant arthritis model (Han et al., 2001). SP600125 has also recently been reported to be effective in bleomycin-induced pulmonary fibrosis in mice (Blease et al., 2003). These data indicate that JNK inhibitors may be effective in chronic airway inflammatory diseases such as asthma and COPD. A more selective second-generation JNK-selective inhibitor (CC-401, Celgene) has successfully completed a Phase I, doubleblind, placebo-controlled, ascending single intravenous dose study in healthy human volunteers (Manning and Davis, 2003). Future studies will examine whether CC-401 will be glucocorticoid sparing (lacking many of the steroid side effects) in humans. CNI-1493, a dual inhibitor of JNK and p38 MAPK, has proved effective in a small study in 12 patients with Crohn's disease. Over 58% of patients showed clinical improvement at 8 weeks with 42% of patients having complete remission. Importantly, CNI-1493 was also steroid-sparing in 11 / 12 subjects and recruitment for a Phase II study is currently underway (Hommes et al., 2002). Furthermore, mixed lineage kinase (MLK) family inhibitors such as CEP-1347 and CEP-11004/KT-8138 (Cephalon Inc.,

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Fig. 3. Activation of nuclear factor κB (NF-κB) by TNFα. NF-κB heterodimers (p50/p65) are sequestered in the cytoplasm by IκB inhibitory proteins (IκBα). Stimulation by stress-inducing agents, or exposure to inflammatory cytokines (TNFα), mitogens or a diverse array of bacterial and viral pathogens leads to the activation of signaling cascades converging on the IκB kinase (IKK) complex. Phosphorylation of IκBα by activated IKK is a signal for its ubiquitylation and proteasome-dependent degradation. Freed NF-κB dimers translocate to the nucleus where they bind to κB elements and activate the transcription of a variety of genes involved in the control of cell proliferation/survival and in the inflammatory and immune response. Inhibition of NF-κB activation is achieved by proteasomal

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of airway inflammation (Ziegelbauer et al., 2005). This was associated with a reduction in NF-κB transactivation, chemokine-, cytokine-, and adhesion molecule expression, and T- and B-cell proliferation. There are concerns that inhibition of NF-κB may cause side effects such as increased susceptibility to infections, which as been observed in gene disruption studies when components of NF-κB are inhibited (Barnes and Karin, 1997; Orlowski and Baldwin Jr., 2002). In addition, IKK2 knockout mice do not survive due to liver apoptosis. Whether this is a clinical concern will need to be tested further. 4.3. Protein kinase C (PKC)

Protein kinase C (PKC) is a family of serine/threonine kinases that play a ubiquitous role in intracellular signal transduction (Goekjian and Jirousek, 2001). PKCs are activated by diverse stimuli and participates in cellular processes such as growth, differentiation, and apoptosis. The PKCs comprise twelve different isoforms (isozymes) that are grouped based on structure into three subfamilies: The classical PKCs (α, β1, β2 and γ) that are activated by diacylglycerol (DAG) and calcium, the novel PKCs (δ, ε, η, and θ) that are activated by DAG, and the atypical PKCs (ζ and λ/ι) that do not respond to either DAG or calcium (Goekjian and Jirousek, 2001). PKC isozymes appear to play distinct, and in some cases opposing roles in the transduction of intracellular signals and are often overexpressed in disease states. Individual isozymes have been implicated in many cellular responses important in both normal lung function and the pathogenesis of asthma and COPD. These responses include permeability, contraction, migration, hypertrophy, proliferation, apoptosis, inflammation and secretion (Dempsey et al., 2000). Understanding how individual isozymes contribute to the pathogenesis of asthma and COPD will enable isoform specific agonists and antagonists to be used as viable therapeutic targets. At present little work has been performed in human lung tissues and animal models of asthma and COPD with specific inhibitors of PKC isoforms. Allergen challenge in man resulted in increased expression and activation of the PKCξ isoform in sputum eosinophils (Evans et al., 1999). In addition, Vachier and colleagues have reported abnomal regulation of PKC activity in alveolar macrophages of asthmatic patients (Vachier et al., 1997). Furthermore, activation of PKCδ in 16-HBE airway epithelial cells enhanced IL-8 expression via an effect on NF-κB suggesting that PKCδ plays a key role in the regulation of NF-κB-dependent gene expression in these cells (Page et al., 2003). Both isoform selective and non-selective PKC inhibitors have been tested in animal models and current data suggests that non-selective PKC agents will be less effective than selective agonists or inhibitors (Dempsey et al., 2000). Some isoform specific PKC inhibitors e.g LY333531 (Lilly, USA) are in Phase III studies for diabetic retinopathy and diabetic macular oedema which suggests that the compounds are safe but their potential utility in asthma and COPD models is awaited (Aiello, 2002).

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cyclooxygenases (COXs)(Ghosh and Karin, 2002) and enhanced activation of NF-κB has been implicated in asthma (Hart et al., 1998) and COPD (Di Stefano et al., 2002). NF-κB also plays a major role in the regulation of cell apoptosis/survival (Ghosh and Karin, 2002). IKK2 is the important kinase for the control of these inflammatory genes and conditional IKK2 knockout mice fail to mount an effective antibody response to T-cell-dependent and independent antigens (Li et al., 2003). A large number of commonly used anti-inflammatory drugs have been shown in vitro to be capable of inhibiting NF-κB including non-steroidal antiinflammatories such as sulindac, cyclopentenone prostaglandins, arsenic trioxide, thalidomide, a variety of anti-oxidants, natural products such as parthenolide and resveratrol and importantly glucocorticoids (Barnes and Karin, 1997). Recently a peptide that blocks the interactions of IKKγ with the catalytic core of IKK and pharmaceutically developed ATP-competitive synthetic small-molecule inhibitors of the IKK complex have become available (Kapahi et al., 2000; Orlowski and Baldwin Jr., 2002). As an example of these compounds, AS602868/SPC839 (Celgene/Serono) showed a concentration-dependent inhibition of ICAM-1 and VCAM-1 in human umbilical vein endothelial cells (HUVEC) cells (Frelin et al., 2003). PS-1145, an IKK inhibitor developed by Millenium, blocks TNFα-induced NF-κB activation and ICAM-1 expression in multiple myeloma cells and interestingly this effect was enhanced by dexamethasone (Hideshima et al., 2002). Bristol Myers Squib has developed an oral small molecule inhibitor of IKK2 (BMS345541) that reduces the incidence and severity of arthritis in a mouse model after daily dosing but was less potent than dexamethasone (Burke et al., 2003). In contrast, AS602868 is able to reduce swelling and bone erosion/destruction in a rat arthritis model when dosed at 30 mg/kg/day. AS602868 was reported to be as effective as 1 mg/kg of dexamethasone in reducing TNFα but was not as potent as indomethacin (Grimshaw et al., 2001). In addition, AS602868 (10 mg/kg) was effective in preventing murine liver ischaemia-reperfusion injury (Luedde et al., 2005). No animal toxicity has been seen to date. GlaxoSmithKline have also developed an oral small molecule inhibitor of IKK2 (TPCA-1) that produces a dose dependent reduction in paw edema and cytokine production in a mouse arthritis model (Podolin et al., 2005). In these studies, 20 mg/kg twice daily produces similar results to etanercept. SC-514 (Pharmacia) is another selective ATP-competitive inhibitor of IKK2. SC-514 inhibits transcription of NF-κB-dependent genes such as COX-2, IL-6 and IL-8 in synovial fibroblasts. In addition, SC-514 (50 mg/kg i.p.) was able to inhibit LPS-induced serum TNFα production in a rat model of inflammation to a similar extent to dexamethasone (1 mg/kg p.o.). Oral gavage of SC-514 was less effective due to its short half-life and low bioavailability (Kishore et al., 2003). A recent study performed by Bayer (Kyoto, Japan) indicated that a bioavailable selective ATP-competitive IKK2 inhibitor inhibited cockroach allergen-induced airway inflammation and hyperreactivity and efficiently abrogated leukocyte trafficking induced by carrageenan in mice or by ovalbumin in a rat model

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Tyrosine kinases are involved in many signalling pathways crucial for basic cellular processes, such as cellular proliferation (Fig. 4)(Corry, 2002). Although the intracellular compartment might be less therapeutically accessible compared with the cell surface, the feasibility of tyrosine-kinase blockade has recently been shown for Gleevec in AML and Iressa in non-small cell lung cancer (NSCLC) (Druker et al., 2001). 4.5.1. Non-receptor tyrosine kinases

4.5.1.1. Spleen tyrosine kinase (Syk) inhibitors. Syk (p72Syk) kinase is a protein tyrosine kinase that plays a pivotal role in signalling of the high affinity IgE receptor (FcεRI) in mast cells and in syk-deficient mice, mast cell degranulation is inhibited (Costello et al., 1996). Syk is also involved in antigen receptor signalling of B and T lymphocytes and in eosinophil survival in response to IL-5 and GM-CSF suggesting that this might be an important potential target for the development of new antiasthma drugs (Yousefi et al., 1996). BAY 61-3606, a potent and selective inhibitor of Syk kinase inhibited lipid mediator release, cytokine synthesis and mast cell degranulation and had inhibitory effects on atopic human basophils, eosinophils and monocytes (Yamamoto et al., 2003). In addition, oral administration of BAY 61-3606 significantly suppressed antigen-induced passive

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The first examples of therapies targeting kinase enzymes that play a critical role in disease are Herceptin (trastuzumab, Genetech) for the treatment of metastatic breast carcinoma and Gleevec (imatinib mesylate) for the therapy of patients with Philadelphia chromosome-positive (Ph+) chronic myelogenous leukaemia (CML). The reason for this efficacy lies in the critical role the target kinases play in these diseases (Cohen, 2002). For example, the HER2/neu receptor tyrosine kinase is an oncogene expressed at up to 100 times the level seen in normal cells in neoplastic cells of about 30% of patients with breast carcinoma and a significant clinical correlation was shown between HER2/ neu overexpression and several clinical parameters of malignancy (Chazin et al., 1992; Traxler, 2003). Intriguingly, Gleevec has also been reported to show efficacy in an allergic model of asthma (Berlin and Lukacs, 2005) and hypereosinophilic syndromes (Cortes et al., 2003). Many other growth factor related tyrosine kinases being proposed as anti-cancer therapies and small molecule inhibitors directed against different kinases such as EGF receptor kinase, vascular endothelial growth factor (VEGF) receptor kinase, platelet-derived growth factor (PDGF) receptor kinase, Kit and Fms-like tyrosine kinase [Flt] are in the pipeline of several pharmaceutical companies (Traxler, 2003). Since several of these kinases may also play a role in the pathogenesis of asthma and COPD it is possible that these compounds may prove effective in respiratory disease. Furthermore, as T-cells are important in the pathogenesis of asthma and COPD, interfering with the

Fig. 4. Inter relationship between various kinase pathways in the activation, proliferation and synthesis of inflammatory mediators. Stimulation of cell surface cytokine and/or growth factor receptors results in activation of parallel kinase pathways including MAPK kinase (ERK, p38 and JNK), JAK and IKK/NF-κB. These can crosstalk with each other and modify cell proliferation and mediator release through changes in gene transcription controlled by transcription factors such as NF-κB, AP-1 (Fos–Jun), STATs, ATF2 and Elk. Receptor activated PI3K also leads to Akt activation and cross-talk with the NF-κB pathway and other cytosolic targets that regulate protein expression. In addition, T- and B-cell activation by receptor cross-linking results in stimulation of Lyn and other tyrosine kinases including Syk with subsequent activation of PKC, PI3K, ERK and other pathways. Abbreviations used: Akt — protein kinase Akt2; ATF2 — activating transcription factor 2; BCR — B-cell receptor; Elk — E26 like; ERK — extracellular signal regulated kinase; IKK— IκB kinase; JAK — Janus kinase; JNK — Jun N-terminal kinase; MEKK5 — MAPK/ ERK kinase kinase 5; NF-κB — nuclear factor κB; P38 — p38 MAPK; PAK — p21-activated kinase; PI3K — phosphatidylinositol 3-kinase; PKC — Protein kinase C; PLC — phospholipase C; Rac— ras-related C3 botulinum toxin substrate; S6K— ribosomal protein S6 kinase; SOS — son of sevenless; STAT — signal transducers and activators of transcription; TCR — T-cell receptor; TNFα— tumour necrosis factor α; TRAF— TNFα receptor associated factor; ZAP70 — zeta chain associated protein 70; Lyn, Lck and Syk are all non-receptor tyrosine kinases.

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cutaneous anaphylactic reaction, bronchoconstriction, and bronchial oedema at 3 mg/kg in the rat. Furthermore, BAY 613606 attenuated ovalbumin-induced airway inflammation in rats (Yamamoto et al., 2003). Further indications that this may be a target for new drug development is indicated by recent evidence that aerosolised Syk antisense oligodeoxynucleotide inhibits allergen-induced BAL and tissue inflammation in a rat model of allergic asthma and also suppressed allergen-mediated contraction of the rat trachea (Stenton et al., 2002). The Syk inhibitor, R-112 (Rigel Pharmaceuticals), is currently undergoing Phase II clinical trials for allergic rhinitis having recently successfully completed a Phase II Park study where it showed effectiveness against symptoms with a rapid onset (Meltzer et al., 2005). A separate compound, R-406/788 has entered a Phase II study for rheumatoid arthritis. In addition, the acute bronchoconstrictor response to ovalbumin in rats was blocked by the Syk tyrosine Kinase/ZAP70 (zeta chain associated protein kinase 70 kDa) inhibitor piceatennol (Novartis AG) (Wong and Leong, 2004) although future development of this compound has ceased due to toxicological problems.

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4.5.1.2. Src kinases. The src family of tyrosine kinases is a closely related group of non-receptor kinases involved in signaling pathways that, in response to the activation of cell-surface receptors by growth factors, cytokines, or cell-surface ligands, control the growth and differentiation of cells (Thomas and Brugge, 1997). There are at least nine members of this family: c-src, fyn, lck, hck, lyn, fgr, blk, yes, and yrk. C-src is expressed in a wide range of tissues and is implicated in a variety of processes; other members of the family, such as hematopoietic cell kinase (hck) and lymphoid cell kinase (lck) are more limited in their expression. Two c-Src inhibitors (CGP77675 and CGP 76030) reduced MMP-9 expression levels in a PC3 cancer cell line and may have similar effects in models of COPD (Recchia et al., 2003). Lyn kinase is an upstream kinase of Syk and an inhibitor of Lyn kinase, PP1, has an inhibitory effect on inflammatory cells and mast cell activation (Amoui et al., 1997). Lyn is also involved in eosinophil activation and IL-5 signalling (Adachi et al., 1999; Lynch et al., 2000) and a Lyn blocking peptide inhibits eosinophilic inflammation in a murine model in vivo (Adachi et al., 1999). However, as Lyn and Syk are widely distributed in the immune system, there are doubts about the long-term safety of selective inhibitors. Lck is expressed primarily in T lymphocytes and plays an essential role in the immune response. Lck (−/−) mice have a SCID-like phenotype implicating lck as an immunosuppressive target. Both Abbott laboratories and AstraZeneca have recently reported orally available lck inhibitors in animal models of T-cell inhibition. A selective inhibitor of lck should inhibit T-cell activation and have broad application for the treatment of autoimmune and inflammatory diseases (Burchat et al., 2002).

to receptor-associated Janus kinases (JAKs) following cytokine receptor stimulation. The activated STAT dimerises, translocates to the nucleus and enhances inflammation-related gene transcription. For example, IL-4 and IL-12 drive Th2 and Th1 cells differentiation respectively through activation of STAT6 and STAT4 (Heim, 1999). This suggests that targeting JAKs (and STATs) could be an effective therapeutic strategy for asthma. JAK3 is required for IL-2Rγ signalling (Russell et al., 1994) and its genetic absence results in T-cell depletion and severe immunodeficiency in humans and mice. JAK3 also plays a pivotal role in IgE receptor/FcepsilonRI-mediated mast cell responses, and targeting JAK3 with a specific inhibitor, such as WHI-P131, may provide the basis for new and effective antiasthma drugs. 18 small molecule inhibitors of JAKs have been reported in the literature. WHI-P131 (Parker–Hughes Institute), a JAK3 specific inhibitor, without effect on JAK1 and 2, inhibits degranulation and cytokine release from lgE receptor/FcepsilonRI cross-linked mast cells and can prevent cutaneous as well as systemic fatal anaphylaxis in mice following i.v. administration (Malaviya et al., 1999) at nontoxic dose levels. Furthermore, WHI-P131 prevented the development of insulitis and diabetes in NOD-scid/scid females after adoptive transfer of splenocytes from diabetic NOD females. Thus WHI-P131 shows immunosuppressive activity in an animal model of autoimmune disease (Cetkovic-Cvrlje et al., 2003). Unfortunately, JAK3 and JAK2, are so widely used in other signalling pathways that they are probably impractical targets (Corry, 2002). Of the many STAT proteins, only STAT6 is unique to the asthma-related cytokines IL-4 and IL-13. Again, the other STAT factors are so widely used in other cytokine and hormonesignalling pathways that any systemic targeting approach is likely to induce significant toxicity. Th2 cell differentation is coordinated through a pathway that obligatorily includes IL-2-inducible tyrosine kinase (ITK) (Fowell et al., 1999). In the absence of ITK, mouse Th2 cells do not develop in vivo, without affecting Th1 function, indicating that ITK might represent a suitable intracellular target for blockade in asthma (Fowell et al., 1999). These problems may be overcome by the use of inhaled drugs.

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4.5.1.3. Janus Kinases (JAKs). STATS are the primary signal specific mediators of cytokine regulated gene expression. For example, phospho-STAT1 controls IFNγ-induced ICAM-1 production. STATs are phosphorylated and activated by recruitment

4.5.2. Receptor associated tyrosine kinases EGF receptors may play a critical role in the regulation of mucus secretion from asthmatic airways in response to multiple stimuli (Takeyama et al., 1999). An orally active small molecule inhibitor of EGF receptor tyrosine kinase (Gifitinib/Iressa, Astra Zeneca) has now been developed for the treatment of epidermal cancers, but may also suppress airways mucus secretion (Wakeling, 2002). Furthermore, epithelial damage and airway remodelling are consistent features of bronchial asthma and are correlated with disease chronicity, severity, and bronchial hyperreactivity. EGF receptor expression in the bronchial epithelium is enhanced with increasing asthma severity and is insensitive to glucocorticoid therapy (Puddicombe et al., 2000). Iressa has gained clinical approval on studies showing a success rate of 10%. This was not seen in 2 further Phase 3 studies.

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5. Conclusions Kinase pathways have become recognised as key cellular signal transducers and several protein kinase inhibitors are in development for the treatment of asthma and COPD with a major focus on MAPK inhibitors and those targeting NF-κB. To obtain similar success to that achieved with Gleevec in CML and Iressa in NSCLC it will be important to validate the role of each protein kinase in the disease relevant cells of patients. Although some hints as to inhibitor potential of specific kinases may be achieved with conditional knockout mice and in vivo pharmacology indications for drug use will be defined by kinase activity profiles in specific diseases. The design of specific inhibitors, either orally active (the patients preference) or inhaled to minimise possible side-effects, will be dependent upon a combination of an accurate 3D structure of the active site pocket, good molecular modelling and excellent medicinal chemists. It is most likely that kinase inhibitors will be more efficacious in more severe steroid-insensitive asthma and in COPD where glucocorticoids are of limited effectiveness. Finally, it is likely that specific kinase inhibitors will be more efficient in augmenting current therapies, particularly corticosteroids, rather than as monotherapy.

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involved in the generation of these lipid mediators, may become important targets for development. The lack of selective inhibitors has limited research in this area however (Baumruker et al., 2005).

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The likely explanation for this is that patients were not selected for those whose disease progression and tumour proliferation was associated with EGF receptor kinase activity. This was not the case in those patients with Gleevec however (Dancey and Freidlin, 2003) and it would be disappointing if Iressa did not reach its potential due to a failure to treat patients with abnormal EGF receptor kinase expression. Vascular endothelial growth factor A (VEGF-A) is also highly expressed in asthmatic lungs and may lead to increased airway vascular permeability resulting in increased thickening of the airway mucosa and narrowing of the airway lumen (Wagner, 2003). In asthma, the lower the FEV1 the higher the VEGF-A expression suggesting that VEGF-A may play an important role in the pathogenesis of asthma (Wagner, 2003). In contrast, in pulmonary emphysema low VEGF-A expression correlates with reduced FEV1. This suggests that use of VEGFR kinase inhibitors such as PTK787/ZK 222584 directed against VEGF receptor 1 and VEGF receptor 2 receptor tyrosine kinases that decreased VEGF-A activity over a long period may lead to COPD (Wagner, 2003).

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Phosphoinositide 3-kinases (PI-3Ks) are important enzymes that generate lipid second messengers that regulate a number of cellular responses including cell growth/division, cell apoptosis/survival and activation (Condliffe et al., 2000; Finan and Thomas, 2004). Class 1A PI-3Ks are involved in signalling by cytokines, antigens and co-stimulatory molecules and are activated by receptor associated tyrosine kinases. PI-3Ks may also contribute to the pathogenesis of asthma by affecting airway smooth muscle proliferation and eosinophil recruitment (Condliffe et al., 2000; Finan and Thomas, 2004). The results of studies using selective PI-3K inhibitors (Wortmanin and Ly294002) raise the possibility that inhibition of these kinases may prove efficacious. However, the lack of isoform specificity of these compounds has prevented useful information being obtained in vivo on the role of specific enzymes in this family. Gene knockout experiments are only just beginning to define the role of some of these family members in cellular function and we await information on isoform specific inhibitors in animal models of disease (Finan and Thomas, 2004; Koyasu, 2003). Akt plays a pivotal role in cell survival, proliferation and inflammation through a number of downstream effectors. Recently, novel allosteric inhibitors have been reported which exhibit some Akt isozyme selectivity e.g. the indazole-pyridine A-443654 (Abbott Labs, IL, USA). These compounds show some efficacy in murine cancer models but have a narrow therapeutic window and are associated with abnormalities in glucose metabolism which will limit there clinical development (Barnett et al., 2005). Sphingosine and ceramide 1-phosphates (S1P and CIP) are known to act either as extracellular mediators or as intracellular second messengers while C1P currently is only known for its intracellular actions and play important roles in mast cell activation and degranulation, monocyte activation and neutrophil priming. Potentially, SIP and CIP kinases, the enzymes

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