Selective PDE inhibitors as novel treatments for respiratory diseases

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Selective PDE inhibitors as novel treatments for respiratory diseases Clive P Page and Domenico Spina Phosphodiesterases (PDEs) are a family of enzymes which catalyse the metabolism of the intracellular cyclic nucleotides, c-AMP and c-GMP that are expressed in a variety of cell types and in the context of respiratory diseases, It is now recognised that the use of PDE3, PDE4 and mixed PDE3/4 inhibitors can provide clinical benefit to patients with asthma or chronic obstructive pulmonary disease (COPD). The orally active PDE4 inhibitor Roflumilast-n-oxide has been approved for treatment of severe exacerbations of COPD as add-on therapy to standard drugs. This review discusses the involvement of PDEs in airway diseases and various strategies that are currently being pursued to improve efficacy and reduce sideeffects of PDE4 inhibitors, including delivery via the inhaled route, mixed PDE inhibitors and/or antisense biologicals targeted towards PDE4. Address Sackler Institute of Pulmonary Pharmacology, Institute of Pharmaceutical Science, Franklin Wilkins Building, King’s College London, London SE1 9NH, UK Corresponding author: Page, Clive P ([email protected])

concomitant glucocorticosteroids [5,6]. However, xanthines have a very narrow therapeutic window (exhibiting a wide range of side effects and many drug/drug interactions) which has been ascribed to the fact that xanthines are non-selective phosphodiesterase (PDE) inhibitors, as well as having other pharmacological actions such as adenosine receptor antagonism. In an attempt to find new drugs for the treatment of respiratory diseases that have a wider therapeutic window than theophylline, a considerable body of work has been undertaken to look for more selective PDE inhibitors. Theophylline has been show to inhibit the activity of a cyclic 30 , 50 nucleotide PDE with a Ki of 100 mM [7] and this activity has been suggested to contribute to its ability to promote suppressor cell activity in lymphocytes [8] and its beneficial anti-inflammatory actions in patients with asthma [9] and COPD [10]. This research has in part, promoted an interest in the role of the various PDEs in regulating cyclic AMP levels within relevant cells involved in asthma and COPD and in the potential for modulating the function of such cells following inhibition of this enzyme with highly potent and selective inhibitors.

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Introduction

There are presently 11 known families of PDE and at least 21 isoforms with numerous splice variants that are characterised by differences in structure, substrate specificity, inhibitor selectivity, tissue and cell distribution, regulation by kinases, protein–protein interaction and subcellular distribution [11]. However, targeting PDE 3 and/or 4, the enzymes responsible for metabolising cyclic AMP in airway and pulmonary smooth muscle, and inflammatory cells respectively has been the focus for the development of drugs that could prove beneficial in the treatment of respiratory diseases such as asthma and COPD [12].

Xanthines such as theophylline have been used in the treatment of respiratory diseases such as asthma and chronic obstructive pulmonary disease (COPD) since the 1930s although their popularity has declined due to the introduction of other classes of drugs, particularly the inhaled long acting b2-adrenoceptor agonists and glucocorticosteroids. Nonetheless, xanthines are recognised as being clinically effective in both asthma and COPD patients, showing benefit that is complementary to existing drug classes. There is now good evidence in the literature that xanthines can exhibit both bronchodilator and anti-inflammatory actions in patients with asthma [1,2] or COPD [3,4] and several studies have described worsening of asthma and COPD when xanthines are withdrawn, even in patients taking

It is therefore of interest that plasma levels achieved with a dose of theophylline that demonstrated significant anti-inflammatory activity in patients with asthma [9] were well below the Ki for PDE inhibition and suggested that PDE4 inhibition alone does not completely explain the clinical effectiveness of this drug. Nevertheless, highly potent and selective PDE4 inhibitors have been developed as new anti-inflammatory drugs for use in patients with asthma or COPD. Targeting PDEs is not unique to inflammatory diseases as exemplified with the development and clinical success of a number of PDE5 inhibitors for the treatment of erectile dysfunction as exemplified by sildenafil [13] and indeed PDE5 inhibitors are now being investigated as

This review comes from a themed issue on Respiratory Edited by M Gabriella Matera and Mario Cazzola Available online 11th April 2012 1471-4892/$ – see front matter # 2012 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coph.2012.02.016

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treatments for pulmonary hypertension [14–16] and as bronchodilators [17].

Phosphodiesterase 4 (PDE4) PDE4 catalyses the hydrolysis of cyclic AMP which terminates the downstream signalling of this second messenger. There are 4 gene families (A, B, C, D), although there is added complexity with over 20 splice variants [11]. Whilst hydrolysis of cyclic AMP is a common feature of this family, it is clear that these isoforms can be targeted to different domains within the extracellular compartment and their activity differentially regulated by kinases, suggesting that these isoforms have specific functions in the control of cellular activity. X-ray crystallography has resolved the catalytic domain of these enzymes which comprises 3 important domains, consisting of a bivalent metal binding pocket (Zn2+, Mg2+) which forms a complex with the phosphate moiety of cyclic AMP, a pocket containing glutamine (Q pocket) which forms hydrogen bonds with the nucleotide (purine) moiety of cyclic AMP and a solvent pocket. PDE4 inhibitors occupy this active site through a number of important interactions and prevent cyclic AMP metabolism. These include indirect binding to the metal ions via the formation of hydrogen bonding to water, whilst hydrophobic interactions between the planar ring structure of these inhibitors and hydrophobic amino acid residues like

phenylalanine and isoleucine serve to ‘clamp’ the inhibitor within the active site and hydrogen bond interaction between the aromatic ring structure of these inhibitors and the invariant glutamine residue in the Q pocket, the site which is normally occupied by the nucleotide moiety of cyclic AMP [18–20]. There are considerable challenges to the synthesis of subtype selective inhibitors due to the high degree of sequence and structural homology within the catalytic domains of the PDE4 subtypes [18–20]. One possibility might be to exploit subtle differences between the interaction of these inhibitors to the catalytic active site, or alternatively, via non-active site inhibition by targeting the N terminal region of the enzyme which contains phosphorylation sites and/or protein binding sequences and so indirectly interfere with PDE4 activity [19,20], or by promoting binding to a recently described novel site involving C-terminus residues [21]. PDE4 is expressed in a number of cell types that are considered important in the pathogenesis of inflammatory airway disease (Table 1) and thus it might reasonably be argued, that targeting PDE4 could potentially suppress the function of numerous inflammatory cell types. However, it is well known that other PDE enzymes are also expressed in inflammatory cells and the contribution of

Table 1 PDE distribution within human cells of interest for the treatment of respiratory diseases like asthma and COPD. Cell type

PDE4 subtype c

Other PDE’s

T lymphocytes CD4 CD8 Th1, Th2 Th17

A, B, Da

3, 7

Inhibition of proliferation and cytokine release

[26,52,94,107–109]

B cells Eosinophils Neutrophils Monocyte Macrophages Dendritic cells Osteoblast Chondrocytes Mast cells

A, A, A, A, A, A, A, A,

7 7 7 7 1,3,7 1,3 3 1

Increased proliferation Inhibition of superoxide anion generation; delayed apoptosis Inhibition of superoxide anion and neutrophil elastase release Inhibition of TNFa release Inhibition of TNFa releaseb Inhibition of TNFa release Stimulates RANKL-induced osteoclast formation Inhibition of IL-1b stimulated production of nitric oxide Little if any mast cell stabilisation

[52,110] [52,94,95] [52,94,111] [52,94,111,112] [50–52,94] [94,112] [113] [114] [115,116]

Airway epithelial cells

1–3,4, 5, 7,8

Increased production of PGE2; Inhibition of IL-6 production

[117,118]

Endothelial cells

2,3,4,5

Inhibition of adhesion molecule expression

[111,119]

B, B, B, B, B, B, B, B,

D D D D D D D D

Biological consequence of PDE4 inhibition

Reference

Fibroblasts

A, B, D

1,4,5,7

Inhibition of fibroblast chemotaxis; inhibition of pro-MMP1,2 release Differentiation into myofibroblasts

[52,120–123]

Sensory nerves d

D

1,3

Inhibition of neuropeptide release

[124]

a b c d

PDE4D absent in Th1 cells. In the presence of a PDE3 inhibitor. PDE4 subtype mRNA expression illustrating relative abundance in cells. Guinea-pig sensory nerves.

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other PDEs to the regulation of inflammatory cell function (e.g. PDE3, PDE7) is also being explored [22]. Whilst it would seem prudent to develop subtype selective PDE4 inhibitors in attempts to maximise therapeutic benefit at the expense of adverse effects, there is also the possibility that mixed PDE inhibitors targeting more than one PDE enzyme subtype may offer a better approach in targeting multiple target cells in the disease process. This approach may be seen as analogous to the success of clozapine as a better anti-psychotic than newer generation atypical anti-psychotics because clozapine targets numerous receptors, and as such, has been described as a ‘magic shotgun’, for the treatment of Schizophrenia [23].

Asthma and PDE4 The underlying pathophysiological features of asthma are a consequence of the contribution of numerous cells including the epithelium, dendritic cells, T lymphocytes, eosinophils, mast cells and airway smooth muscle, involving a complex web of interconnecting interactions between different cell types and inflammatory mediators, and whilst glucocorticosteroids remain the mainstay for the treatment of this disease, there still remains a group of asthmatics with poor symptom control despite treatment with these drugs [24]. It is therefore of interest that many of the inflammatory cell types illustrated in Table 1 express PDE4 and inhibition of this enzyme can suppress the function of these cells. A number of studies have investigated whether the pathogenesis of asthma might be a consequence of increased PDE4 protein expression and activity. This hypothesis had some currency in view of studies in the dermatitis field which purported to demonstrate the expression of a novel and distinct monocyte derived cyclic AMP-PDE obtained from individuals with atopic dermatitis. Monocytes from these individuals had increased functionality and this corresponded with the presence of protein with increased PDE activity. This was thought to be the underlying basis for the pathology associated with atopic dermatitis [25]. However, soluble PDE4 activity was not increased in a range of peripheral blood leukocytes from atopic subjects of either mild or severe severity [26]. Similarly, whilst increased total PDE catalytic activity was observed in peripheral blood monocytes from individuals with mild asthma, this was associated, paradoxically, with reduced PDE4 activity [27] and the expression of PDE4A-D was not increased in peripheral blood CD4 positive T lymphocytes in patients with mild asthma [28]. Together these studies indicate that the underlying pathogenesis of mild asthma cannot be attributed to enhanced PDE4 expression or activity and whilst a genome wide association search has demonstrated single nucleotide polymorphisms on chromosome 5q12 for PDE4D [29], it remains to be established as to the significance of these findings given that this isoform www.sciencedirect.com

has been linked to emesis, and drug targeting of PDE4A,B has been suggested as a rational approach to developing non-emetic anti-inflammatory PDE4 inhibitors [21,30]. However, the challenge remains to develop a PDE4 subtype selective inhibitor that differs by at least 3 orders of magnitude and this has yet to be achieved after more than a decade of work in this field. Numerous preclinical studies in models of allergic pulmonary inflammation have repeatedly documented the ability of PDE4 inhibitors to inhibit two important characteristic features of asthma, namely, the recruitment of eosinophils to the airways and bronchial hyperresponsiveness (BHR). One disadvantage of these studies is the inability to ascertain the role of particular PDE4 isoforms because of the non-selective nature of the PDE4 inhibitors currently under development. However, the use of genetically modified mice has revealed some interesting findings. Airway inflammation characterised by recruitment of eosinophils to the airways of mice deficient in PDE4D was no different to wild type controls [31]. This indicated that other PDE4 subtypes contributed to the metabolism of intracellular cyclic AMP in these inflammatory cells, since cell recruitment to the airways was inhibited when animals were treated with non-selective PDE4 inhibitors [32,33]. However, airway obstruction caused by methacholine was enhanced in wild type allergic mice, but was abolished in PDE4D gene deficient mice. These mice were hyporesponsive to methacholine, even in the absence of allergic sensitisation and this feature appeared to be related to an increase in bronchodilator prostaglandin production in the airways of these PDE4D gene deficient mice [31,34]. However, this effect was specific for methacholine because the enhanced airway obstruction in response to another spasmogen, serotonin, was unaffected by the deletion of PDE4D [31]. Similar experiments have shown that unlike PDE4A and PDE4D, PDE4B knockout mice do not develop many of the constellation of events associated with allergic inflammation, namely Th2 cytokine production, eosinophil recruitment and BHR [35] and suggests the potential for the development of PDE4B subtype selective inhibitors for the treatment of respiratory diseases. One could argue that PDE4B is important for the sensitisation of T cells to an allergic phenotype and we must await the results from conditional knockout studies to ascertain whether PDE4B is critical for Th2 effector function. These studies highlighted the potential complimentary role of PDE4 isoforms in regulating allergic airway inflammation, and the need to target more than one PDE4 isoform in order to counter all of the changes seen in allergic airways, since the inflammatory response, BHR and airway remodelling in allergic wild type mice is inhibited by non-isoform selective PDE4 inhibitors such as rolipram or roflumilast [32,33,36]. The anti-inflammatory potential of PDE4 inhibitors documented in models of allergic inflammation and in Current Opinion in Pharmacology 2012, 12:275–286

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human cells in vitro has to some degree been corroborated in clinical trials in patients with asthma. Twice daily treatment for 9.5 days with the PDE4 inhibitor CDP840 inhibited the development of the late phase response in the lungs of patients with allergic asthma (long considered a clinical model associated with airways inflammation) by around 30% [37]. A similar degree of inhibition of the late phase response was observed following once daily treatment for 7–10 day with roflumilast [38], whilst 2 week treatment with MK-0359 improved baseline FEV1 and reduced symptom scores in chronic asthmatic subjects [39]. Whilst the effect of roflumilast on the development of the late response was modest, there was a 30–50% reduction in sputum eosinophil number and levels of sputum eosinophil cationic protein was reduced following 2 week treatment with roflumilast which was the first study to demonstrate anti-inflammatory activity in asthma [40]. There is also limited clinical information demonstrating an anti-inflammatory action of inhaled PDE4 inhibitors. For example, 7 days of treatment with GSK256066 produced modest attenuation of both the early and late asthmatic response to antigen challenge [41]. However, in all these clinical studies, the effect of drug treatment on the acute allergen-induced bronchoconstriction was weak and is consistent with the lack of demonstrable action of PDE4 inhibition on both mast cell and airway smooth muscle function. These findings highlight the role of other PDE enzymes, particularly PDE3 in the regulation of the function of these other important cell types in the airways of patients with inflammatory airway diseases (Table 1). This could be exploited in the development of mixed PDE3/4 inhibitors which possess both bronchodilator and anti-inflammatory activity residing in the same molecule [42]. The effect of roflumilast against BHR has also been evaluated and with modest protection against allergeninduced BHR [43]. The degree of protection afforded by roflumilast was in the order of 1 doubling dilution [40]. This might suggest that PDE4 is not a good target to modify BHR, or that higher doses are required to provide complementary and persistent inhibition of the enzyme in order to attenuate this phenomenon, as certainly there are a number of preclinical studies that have shown inhibition of BHR with PDE4 inhibitors [44]. It is of interest that Roflumilast has a plasma half-life of 16 hours following a single oral administration to man, and is metabolised by CYP3A4 to the active N-oxide metabolite which has considerably greater bioavailability with a half life of 20 hours that would favour prolonged enzyme exposure [45]. This favourable pharmacokinetic profile would be anticipated to produce longer periods of PDE4 inhibition. However, at the doses of roflumilast that can be safely be administered to man, whilst there was a Current Opinion in Pharmacology 2012, 12:275–286

significant reduction in the activity of circulating monocytes in subjects maintained on Roflumilast for 4 weeks, the magnitude of this change was small, resulting in only an approximately 1.3 fold reduction in TNFalpha production by monocytes in response to endotoxin challenge ex vivo [46]. One could argue that sufficient PDE4 inhibition was probably not achieved in relevant inflammatory cells within the airway tissue compartment at the doses that can safely be employed in clinical studies. Unfortunately, the doses achievable clinically with most orally active PDE4 inhibitors are limited by side effects, the most commonly reported being headache, nausea and diarrhoea of a mild to moderate severity [38,39]. The availability of other non-emetic PDE4 inhibitors such as CDP840 [37] and tetomilast [47] might provide hope that PDE4 inhibitors can be developed for the treatment of asthma having a wider therapeutic index, a worthy goal to pursue in light of at least one clinical study reporting comparable clinical efficacy between Roflumilast and beclomethasone diproprionate in persistent asthma [48].

COPD and PDE4 Unlike asthma, COPD is primarily caused by cigarette smoking, although, in developing countries smoke derived from burning biomass fuels is also a predisposing factor [49]. COPD is also an inflammatory disease but the nature of the inflammatory response is distinct from asthma and is characterised by the activation of macrophages and airway epithelial cells, which in turn, secrete a range of chemokines and lipid mediators resulting in the recruitment of neutrophils and CD8+ T lymphocytes to the lung. The secretion of a range of proteases from neutrophils (elastase, MMP9, cathepsins) and macrophages (MMP12) is thought to contribute towards airway fibrosis of the small airways, increased mucus secretion and destruction of the alveolar wall [24]. These pathological changes give rise to the symptoms of cough, mucus secretion, difficult breathing and emphysema. Many of the cell types implicated in COPD express PDE4 (Table 1). The expression of PDE4A–D in peripheral blood neutrophils and CD8T cells is not altered in subjects with mild COPD [28]. However, the expression of PDE4A4 and total cyclic AMP PDE activity was significantly increased in macrophages purified from bronchoalveolar lavage fluid from subjects with mild to moderate COPD compared with healthy subjects or smokers who did not present with COPD [50]. Of the 12 PDE4 variants analysed, only the activity of PDE4A4 was increased and suggested that local events/processes within the lung of subjects with COPD specifically upregulated this variant [50]. However, the functional consequence of this change remains to be established in light of findings showing that PDE4 inhibition has only a modest effect in suppressing TNFa production from human macrophages derived from cultured monocytes and the contribution of PDE3 and PDE7 www.sciencedirect.com

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in regulating function in this cell type cannot be ignored [51,52]. Genotyping studies have also revealed the presence of single nucleotide polymorphisms within the PDE4D gene in COPD subjects compared with smokers within the Japanese population that was in linkage disequilibrium with the IL-13 gene, although the significance of these findings remains to be established since this relationship was absent in an Egyptian sample [53].

reduced the number of neutrophils and lymphocytes recruited to the airways and the levels of two biochemical markers of this disease, namely IL-8 and neutrophil elastase. The magnitude of the change in the number of these inflammatory cells and concentration of mediators was between 30 and 50% which might contribute to the clinical benefit these drugs exhibit in phase III clinical trials.

There are a limited number of preclinical in vivo models of COPD. However, the recruitment of neutrophils to the airways can be readily induced using the bacterial wall component, endotoxin, although it is widely appreciated that this stimulus can only model one aspect of COPD, namely neutrophil recruitment to the airways. The recruitment of these cells to the airways of wild type mice was inhibited by around 50% in PDE4B and PDE4D deficient mice and a greater degree of inhibition was observed when wild type mice were treated with the PDE4 inhibitor rolipram [54]. Such research has once again highlighted the complimentary roles of PDE4 isoforms in regulating neutrophil recruitment to the airways. Similarly, smoking induced neutrophil recruitment to the airways, release of chemokines and emphysematous changes to the lung were attenuated by PDE4 inhibitors [55,56]. Together, these studies highlight the potential value of inhibiting PDE4 in various cell types implicated in this disease.

Increasingly, lipopolysaccharide and ozone are being used to evaluate the efficacy of novel therapeutic agents for the treatment of COPD. Healthy subjects or smokers without COPD are exposed to these stimuli and the ensuing inflammatory response, namely recruitment of neutrophils and levels of various biological proteins (e.g. cytokines, matrix metalloproteinase) are used as a biomarker of disease. The acute treatment with glucocorticosteroids has been reported to have either no effect [64,65] or reduced neutrophil recruitment to the airways by 30– 50% [66–68] and whether these differences are explained by patient selection remains unclear. However, set against this background selective CXCR2 antagonists were shown to inhibit the neutrophil recruitment induced by ozone by 50–80% [68,69]. Using a similar design it has been shown that roflumilast caused a 40% inhibition of neutrophil recruitment to the airways in response to lipopolysaccharide and none of the inflammatory markers (e.g. IL-8 levels in lavage fluid) was inhibited [70].

A number of phase III clinical trials have assessed the potential utility of PDE4 inhibitors in the treatment of COPD [57–59]. All three studies report modest but significant improvements in spirometry over placebo, quality of life scores and reduction in the number of exacerbations in the severest group of COPD subjects. However, more recent clinical trials evaluating the effect of Roflumilast in symptomatic moderate to severe patients with COPD have reported more promising findings [60,61]. Roflumilast caused a significant 48 ml improvement in FEV1 compared with placebo and reduced exacerbation rates of moderate to severe intensity by 17% over a 52-week period [61]. Similarly, Roflumilast consistently improved baseline FEV1 in moderate to severe COPD patients already taking either a long-acting beta2agonist (salmeterol) or a long-acting muscarinic antagonist (tiotropium bromide) by 49 and 80 ml, respectively during 24 weeks of treatment [60].

These inflammatory biomarker studies highlight a recurring theme that complete inhibition of PDE4 is difficult to achieve because of dose-limiting side-effects. As discussed for asthma, it also remains plausible that other PDE isoforms (e.g. PDE3, 7 see Table 1) may require targeting to obtain a full anti-inflammatory action to be revealed in this disease [22].

The mechanism of the improvement in spirometry is unlikely to be due to relaxation of airway smooth muscle because this drug class has weak bronchodilator activity. It is possible that this improvement is due to an antiinflammatory action of the drugs (Table 1), although no biomarker of inflammation was measured in these studies. However, separate studies have addressed whether PDE4 inhibitors are anti-inflammatory in patients with COPD. Both Roflumilast [62] and Cilomilast [63] www.sciencedirect.com

The most common side effect reported with Roflumilast included diarrhoea (9%), headache (5%) and nausea (5%) [57,59–61] which was of the same order of magnitude as that reported with cilomilast, although abdominal pain and vomiting were also reported for this drug [58]. The adverse effects appeared to disappear with continued use but were a major reason why patients discontinued with the study during the first 3–4 weeks of treatment. No cardiovascular liabilities were noted. One unanticipated side-effect noted following prolonged treatment with Roflumilast was a significant reduction in body weight in subjects that could be attributed to gastrointestinal discomfort in susceptible patients, although it was interesting to note that weight loss was also reported in patients not complaining of gastrointestinal side-effects [60,61]. One might anticipate that the risk/benefit ratio would be improved by inhaled delivery of a PDE4 inhibitor directly to the lung. Unfortunately, to date only one study evaluating the effect of an inhaled PDE4 nonselective subtype inhibitor in moderate to severe COPD Current Opinion in Pharmacology 2012, 12:275–286

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subjects used FEV1 as a primary outcome measure and this was negative [71]. As no inflammatory biomarkers were measured it is unclear whether sustained PDE4 inhibition was achieved, although the high dose was associated with adverse events like headache and improvements in lung function were found at 2 weeks, but not following 6 weeks of treatment.

Unwanted side-effects Nausea and other gastrointestinal side effects are commonly reported side-effects associated with most PDE4 inhibitors which remain a major drawback for the wider therapeutic use of these drugs. The mechanism(s) responsible for these side-effects have been investigated in an attempt to discover non-emetic PDE4 inhibitors. The direct recording of neuronal activity within the area postrema conclusively demonstrated that substances known to cause nausea (e.g. apomorphine) caused the excitation of neurones within the area postrema of dogs [72]. Neuronal activity within the area postrema was also increased following the systemic administration of 8bromo cyclic AMP or following elevation of endogenous levels of cyclic AMP within neurones by forskolin, an activator of adenylyl cyclase [72]. Elevated levels of cyclic AMP within the area postrema enhanced the emetogenic response. Dogs treated with the non-selective PDE inhibitor theophylline and the PDE4 selective inhibitor, 4-(3-butoxy-4-methoxyphenyl)methyl-2-imidazolidone (Ro 20-1724) reduced the emetic threshold of the D2 agonist, apomorphine [72]. One consequence of elevated levels of cyclic AMP is the transcriptional activation of the early response gene c-fos, following upstream activation of the transcription factor, cyclic AMP response element binding (CREB) by protein kinase A. This method was employed to demonstrate increased c-fos immunoreactivity in neurones within area postrema and nucleus tractus solitarius following systemic administration of a PDE4 inhibitor and providing conclusive proof that this drug class can lead to the activation of neurones within the emetic centres of the CNS [73]. The intracerebroventricular administration of highly potent PDE4 inhibitors which can be used to limit systemic bioavailability of drug and provide a means of directly activating neurones within the CNS, also induced emesis in the ferret [74]. Furthermore, the emetic response to systemically administered PDE4 inhibitors is reduced by anti-emetic agents including the 5HT3antagonist, ondansetron, and the NK1 antagonist, (+)(2S,3S)-3-(2-[11C] methoxybenzylamino)-2-phenylpiperidine (CP-99,994) [74,75]. Similarly, the increased expression of c-fos within the emetic centres of the brain was also reduced following treatment with the NK1 antagonist, RP67580, and implicating substance P in this response [73]. Current Opinion in Pharmacology 2012, 12:275–286

Many studies have documented the expression of PDE4D within the area postrema, nucleus tractus solitaris and nodose ganglion neurones in various species including man and implicated this isoform in nausea and vomiting [76–79]. However, it should also be recognised that detectable transcript for PDE4B was also found within the nucleus tractus solitaris and area postrema in humans and rodents, respectively, and could be involved in the emetic response [79,80]. Since rodents do not possess an emetic reflex, it is not possible to directly investigate the role of different isoforms of PDE4 in emesis. However, a surrogate biological response which measures the reversal of anaesthesia induced by alpha2-adrenoceptor agonists has been used to study the role of PDE4 subtypes in emesis [75,81]. It was previously shown that the ability of PDE4 inhibitors to induce emesis in the ferret was inhibited by the alpha2selective agonist, clonidine [75] and suggested that raising cyclic AMP within central noradrenergic terminals by PDE4 inhibitors promoted emesis, and this could be attenuated via alpha2-adrenoceptor mediated inhibition of adenylyl cyclase. The hypnotic action of xylazine was reversed in rodents treated with PDE4 inhibitors [75] and therefore used as a surrogate for emesis. Deletion of PDE4D and not PDE4B reduced the duration of anaesthesia induced by xylazine, compared with wild type mice, and secondly, the ability of PDE4 inhibitors to shorten xylazine-induced anaesthesia was impaired in PDE4D, but not PDE4B knockout mice [82]. Together these studies suggested that PDE4 inhibitors with low affinity for PDE4D should have reduced emetic potential. However, it is not entirely clear whether PDE4D inhibition alone is the sole basis of emesis since there are examples of PDE4 inhibitors that document in vivo antiinflammatory activity but that are not emetogenic [83– 85]. Furthermore, the emetic profile of various PDE4 inhibitors (PMNPQ > R-rolipram > CT-2450) could not be explained by a preferred selectivity for PDE4D over PDE4A or PDE4B [74,82]. One possible explanation might be that some PDE4 inhibitors preferentially partition within the CNS and hence the potential to inhibit PDE4D inhibition in area postrema neurones might explain the differences in the emetic potential of these drugs. Indeed, brain penetrance by PMNPQ was 46-fold greater than CT-2450, although the concentration of the ‘low emetic’ PDE4 inhibitor, CT-2450 within the CNS was still 475-fold greater than the PDE4D inhibitory potency for CT-2450, and presumably still in sufficient concentrations to inhibit this enzyme [81]. In contrast, brain penetrant allosteric inhibitors of PDE4D were shown to be non-emetic despite producing functional inhibition of PDE4D in the CNS [86]. Finally, it should be recognised that the area postrema is not completely behind the blood–brain barrier and therefore accessible to free drug within the circulation www.sciencedirect.com

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[87]. Whether differential partitioning of these inhibitors within the area postrema accounts for why some PDE4 inhibitors have a reduced emetic profile remains to be established. A number of preclinical studies have highlighted a number of other potential disadvantages to targeting PDE4 and these include the development of mesenteric vasculitis, immunosuppression [88], heart failure and arrhythmia [89]. However, none of these events appear to be realised in phase II and phase III clinical trials undertaken to date, at least with cilomilast and Roflumilast. Similarly, slow release theophylline has been used for decades in the treatment of asthma and COPD and has not been associated with a number of these potentially adverse events, despite being shown to cause mesenteric vasculitis preclinically [90,91]. It has also been suggested that PDE4 inhibitors may have pro-inflammatory properties and is based on the finding that at very high doses Roflumilast (100 mg/kg) promoted the recruitment of neutrophils to the airways and this correlated with the release of IL-8 from cultured endothelial cells in vitro [92]. This mechanism might explain why animals chronically treated with high doses of PDE4 inhibitors results in a vasculitis. However, the concentrations required to achieve these untoward effects are at least 1000 times greater than the ED50 and EC50 values reported for Roflumilast against several in vivo biomarkers of inflammation and cell function in vitro, respectively [93,94]. However, it is unlikely that the plasma concentrations required to produce this purported pro-inflammatory effect could be achieved even with chronic dosing. Similarly, another study has shown that PDE4 inhibitors, at concentrations that are pharmacologically relevant, delay apoptosis of neutrophils and eosinophils, an effect that increased when combined with beta2-adrenoceptor agonists [95]. Furthermore, cyclic AMP elevating drugs are purported to increase the expression of chemokines selective for CXCR2 and CCR2 during the differentiation of human monocytes to macrophage. Whilst PDE4 inhibitors alone were ineffective, their combination with prostaglandin E2 or forskolin caused a greater increase in chemokine protein expression and might suggest that PDE4 inhibition could be a proinflammatory signal in monocytes/macrophages, particularly if cyclic AMP elevating agents (e.g. prostaglandins, beta-agonists) are also present [96]. However, to what extent these findings translate into the clinic is unclear, particularly as the beneficial effect of Roflumilast in moderate to severe COPD subjects was not compromised in patients taking long acting beta-agonists [60]. Overall, the clinical evidence suggests that PDE4 inhibitors suppress rather than exacerbate inflammation in the airways [62,63]. Furthermore, there was no evidence of an increased incidence of pneumonia in patients with COPD clinically treated with Roflumilast which might be anticipated if PDE4 inhibitors interfere with host defence [60,61]. Interestingly, weight loss was an unexpected www.sciencedirect.com

effect in subjects taking roflumilast independently of any reports of gastrointestinal discomfort [60,61] which could be a result of the well recognised effect of elevating cyclic AMP in adipocytes, promoting lipolysis, since it has been previously reported that beta2-agonists have a well known lipolytic action. Mice deficient in PDE4D, but not PDE4B, have abnormal growth development as demonstrated by reduced fertility and litter size by female mice, reduced growth rates and body weight in both sexes, the latter a consequence of a significant reduction in the weight of bone, muscle and body organs [97,98]. Although the effect of these gene deficiencies was not studied on lipolysis per se, both PDE4B and PDE4D are present in rat adipocytes and inhibition of these enzymes promotes basal lipolysis [99].

Conclusions Strategies aimed at improving the risk/benefit ratio for PDE4 inhibition will be important if this drug class is to be widely used in the treatment of respiratory diseases such as asthma and COPD. The therapeutic window between the anti-inflammatory action of these drugs and side-effects such as nausea and emesis is probably not wide enough for cilomilast, and may limit the wider use of Roflumilast. It is therefore of interest that there are newer PDE4 inhibitors currently in development which appear to lack significant emetic action (e.g. oglemilast) and IPL512602 [100], although the molecular basis for this lack of emesis has not yet been elucidated. Many PDE4 inhibitors under development are designed for oral administration, but because of the recognised side effect limitations of this class of drug to date, other approaches are being tried to improve the therapeutic window. A number of drugs are now in development as inhaled PDE4 inhibitors which can directly target cells within the lung and thereby minimise systemic absorption and thus minimise adverse effects. This is of course a widely accepted route of administration in the pulmonary field as a way of minimising systemic side-effects with other drug classes. Both AWD 12-281 (N-(3,5-dichloropyrid-4-yl)-[1-(4-fluorobenzyl)-5-hydroxy-indole-3-yl]glyoxylic acid amide [101] and UK-500,001 [71,102] and GSK25066 are examples of inhaled PDE4 inhibitors. Interestingly, 7 days of treatment with GSK256066 produced modest attenuation of both the early and late asthmatic response to antigen challenge [41]. Clearly it should be possible to obtain good anti-inflammatory activity by direct delivery of a PDE inhibitor directly to the lung, although this does not always mean a complete loss of emesis and nausea, as exemplified by the inhaled mixed PDE3/4 inhibitor zardaverine which induced bronchodilation clinically, but only at doses that also induced nausea and vomiting [103]. Another promising approach might be the use of antisense oligodeoxynucleotides targeting PDE4 which Current Opinion in Pharmacology 2012, 12:275–286

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could be delivered by the inhaled route, particularly in view of the positive results obtained in the successful targeting of the adenosine A1 receptor in a rabbit model of allergic inflammation [104], illustrating the potential of this approach. A preclinical study has demonstrated that antisense oligonucleotides directed against mRNA for PDE4B/4D and PDE7 delivered topically to the lung suppressed inflammation following 2 weeks cigarette smoke exposure in mice [105]. The advantage of this technique is that side-effects of nausea and emesis could be avoided. Alternatively, the discovery of allosteric modulators of PDE4 that are proposed to confer lower emetic potential because they only partially inhibit PDE4 enzyme activity, but nonetheless retain sufficient activity to fully inhibit inflammatory cell activity [86] is another approach that could be exploited in the discovery of nonemetic PDE4 inhibitors for the treatment of inflammatory airway diseases. As discussed above, it is also possible that the reason why targeting PDE4 alone may not fully inhibit airway inflammation is the fact that other PDE types exist in both structural and inflammatory cells in the lung which play an important role in modulating cellular function (Table 1). Therefore, it may be that targeting multiple PDE enzymes is required for optimal anti-inflammatory action and of course the non-selective inhibition of PDEs has long been proposed as a major mechanism explaining the benefit of xanthenes in the treatment of airway diseases. For example, the macrophage is viewed as a critical cell type in the pathogenesis of COPD [24]. However, the activity of these cells is only inhibited to a small degree by PDE4 inhibitors [94] and the potential functional involvement of PDE3 and PDE7 in these cells cannot be completely ignored. The inhibitory action of PDE4 inhibitors on the cellular activity of CD8+ T lymphocytes and macrophages was also significantly increased in the presence of PDE7 selective inhibitors [52]. More recently the combination of a selective PDE3 and a selective PDE 4 inhibitor was shown to provide greater inhibitory effects on the function of alveolar macrophages [106]. Another approach to inhibiting more than one enzyme is the design of mixed PDE inhibitors and recently a mixed PDE3/4 inhibitor RPL 554 has been described which preclinical has both bronchodilator and anti-inflammatory action [42]. Several clinical studies have now described the bronchodilator activity of this drug in patients with asthma or COPD (www.veronapharma.com) and the antiinflammatory effects of this drug in the clinic are eagerly awaited.

Conflict of interest The authors are both Consultants for Veronapharma plc who are developing RPL554 as a novel inhaled PDE3/4 inhibitor for the treatment of inflammatory airways disease. CP is also a co-founder and has equity in Veronapharma plc. Current Opinion in Pharmacology 2012, 12:275–286

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