Development status of second generation PDE4 inhibitors for asthma and COPD: the story so far

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1 Monaldi Arch Chest Dis 2002; 57: 1, STATE OF ART Development status of second generation PDE4 inhibitors for asthma and COPD: the story so far M.A. Giembycz ABSTRACT: Development status of second generation PDE4 inhibitors for asthma and COPD: the story so far. M.A. Giembycz. The beginning of the 1990s saw the synthesis and evaluation of orally-active, second generation phosphodiesterase (PDE) inhibitors that have high specificity for the PDE4 subtype. In vitro and in vivo studies established that this class of compounds suppresses the activity of many pro-inflammatory and immune cells indicating that they may be effective in the treatment of airway inflammatory diseases. In this article we review the development status of the most advanced and extensively studied PDE4 inhibitors for asthma and chronic obstructive pulmonary disease. Monaldi Arch Chest Dis 2002; 57: 1, Keywords: Cilomilast, roflumilast, asthma, COPD, phosphodiesterase 4, cyclic AMP, airways inflammation. Thoracic Medicine, National Heart & Lung Institute, Faculty of Medicine, Imperial College of Science, Technology and Medicine; London, United Kingdom. Correspondence: Mark A. Giembycz; Thoracic Medicine; National Heart & Lung Institute; Faculty of Medicine; Imperial College of Science, Technology and Medicine; Dovehouse Street; London SW3 6LY; United Kingdom; Received: January 25, 2002; accepted: February 6, Definition of asthma and COPD Asthma is a specialized chronic inflammatory disease of the airways characterised, clinically, by reversible airways obstruction. The inflammation in asthma is multifaceted, often allergic, involving both infiltrating leukocytes and structural cells. The current dogma is that CD4 + T-lymphocytes play a pivotal role in the pathogenesis of asthma by orchestrating pulmonary eosinophil recruitment, which is a hallmark of this disease, although the role of the eosinophil was recently questioned [1 3]. Asthma is a disease with a low morbidity and, in all but the most severely afflicted (about 2% of sufferers), is rarely fatal. Chronic obstructive pulmonary disease (COPD) is a generic term that embraces several debilitating inflammatory pathologies that often co-exist, and is characterised by a slowly progressive and largely irreversible (<15%) decrement in lung function [4, 5]. In particular, persistent airflow limitation, usually associated with airway collapse, edema and fibrosis are present to a greater or lesser extent and account for the wide spectrum of disease. Thus, individuals with COPD might present with chronic bronchitis as a dominant feature or with emphysema due to destruction and collapse of terminal alveoli. COPD is a disease of the middle aged and elderly that is caused, almost always, by chronic cigarette smoking. It has a long latency period (35 years) before it manifests itself and afflicts at least 15% of all smokers. Current dogma holds that COPD is a neutrophilic inflammatory disorder of the lung that is governed, in large part, by resident macrophages; it is distinct from asthmatic inflammation. Size of the problem Current trends indicate that asthma is set to become the most common chronic disease in industrialised countries within 30 years with a much higher incidence in children than in adults [6]. In 1997, the global prevalence of asthma was 30 million, being approximately the same in males and females, and is estimated to rise to 35 million by 2005 [7, 8]. In all industrialised nations the mean prevalence of asthma is about 5% of the population although notable differences are apparent between countries. In rural parts of Europe including Greece, Portugal and Southern Italy the incidence is lower than the average whereas in the UK and Scandinavia the prevalence is 6 to 7% and is higher still (~10%) in New Zealand [7]. The increase in incidence of asthma is reflected by a huge and sustained growth in the global market for asthma therapies. In 1997 the US and European markets were valued at $2.95 and $2.54 billion respectively, totalling 63% of the total global market [7]. While approximately 50% of the total cost of asthma ($5.8 billion in 1994) in the US is attributable to the drugs bill, medical care costs, including emergency admissions to hospital when severe asthma is not well controlled, and indirect

2 PHOSPHODIESTERASE 4 IN ASTHMA AND COPD costs due to impaired quality of life, account for the remainder [7, 9]. It has been estimated that 14% of prescribing costs in the UK is due to asthma drugs. However, much of this expenditure may also be due to inappropriate or poor control of asthma as well as poor patient compliance, leading to increased consultations and hospital admissions [10]. COPD is the most common of all respiratory disorders in the world with a global prevalence of diagnosed disease predicted to reach 38 million by 2006 [7, 11]. It is likely that this figure is significantly understated as COPD is heavily under-diagnosed, perhaps by as much as 50%. Unlike asthma, COPD has a high morbidity and currently is thought to account for 3 million deaths per year, 33% of which are in industrialised nations [12]. In the UK and US the current annual death rate is approximately 30,000 and 100,000 respectively. The Global Burden of Diseases Study ranked COPD as the sixth most common cause of death in 1990 [13] and the twelfth most common cause of disability as assessed by disability-adjusted life years [14]. Currently, COPD is the third and fourth leading cause of death in the UK and US respectively [15]. The World Health Organisation predicts that because of the increased prevalence and poor treatment of COPD, it will become the third most common cause of death worldwide by 2020 accounting for 8.4 million lives [13]. Unmet needs of currently available therapies Although asthma, for the most part, is well treated with existing bronchodilator and anti-inflammatory therapies, there are still concerns over the use of glucocorticosteroids, which are prone to cause adverse effects especially in children. Furthermore, in severe asthmatic subjects, in whom oral glucocorticosteroids are the only effective treatment, there is a need to develop new anti-inflammatory drugs that can be administered systemically without the side-effects associated with glucocorticosteroids. One such group of compounds are cyclic adenosine monophosphate (AMP) phosphodiesterase (PDE) inhibitors and some of these are now in late clinical development (table 1) including cilomilast and roflumilast. In contrast to asthma, there are no currently effective treatments for COPD and the efficacy of glucocorticosteroids is controversial [16]. Thus, there are clear unmet needs for patients with COPD including effective anti-inflammatory therapy and mucolytics allied with disease-modifying drugs that can repair the anatomical and functional consequences of years of chronic inflammation. The possibility that PDE4 inhibitors could have application in COPD as well as asthma was recently appreciated given their predicted ability to alleviate the neutrophilic inflammation that is characteristic of this generic disorder. PDE Inhibitors: an introduction Cyclic nucleotide PDEs are a family of enzymes that catalyse the degradation of cyclic purine (camp, cgmp) nucleotides to their corresponding 5 -nucleotide monophosphates. Eleven PDE families are currently defined that differ in primary sequence, substrate specificity, co-factor requirements and sensitivity to inhibitors [17]. Of these, perhaps the most extensively studied in recent years is PDE4. This subfamily comprises a group of immunologically and pharmacologically distinct enzymes [18, 19] that, in humans and other species, are encoded by at least four distinct genes (PDEA, PDEB PDEC and PDED), have absolute specificity for camp [17] and may represent a potential therapeutic targets for the treatment inflammatory diseases, including asthma and COPD, with small molecule inhibitors [18, 20, 21]. The rationale for developing compounds that attenuate PDE4 activity is based on several critical findings: i) PDE4 is abundant and the major regulator of camp metabolism in almost every pro-inflammatory and immune cell (table 2); ii) PDE4 inhibitors, of varied structural classes, suppress a myriad of responses such as cytokine generation, NADPH oxidase activity, degranulation, im- Table 1. PDE4 inhibitors known to be, or have been, in clinical development for asthma and/or COPD PDE Inhibitor Company Indication Status Cilomilast GlaxoSmithKline COPD Phase III GlaxoSmithKline Asthma Phase II* Roflumilast Byk-Gulden Asthma & COPD Phase III V-11294A Napp Asthma Phase II* BAY Bayer COPD Phase III** Bayer Asthma Phase II* Pumafentrine Byk-Gulden Asthma Phase II SCH Schering-Plough Asthma Phase I YM-976 Yamanouchi Asthma Phase I* * Probably discontinued for asthma. ** Phase III studies delayed. PDE = phosphodiesterase. COPD = chronic obstructive pulmonary disease. 49

3 M.A. GIEMBYCZ Table 2. Expression of PDE isoenzymes in human immune and pro-inflammatory cells Immune/ Pro-inflammatory Cells PDE Isoform(s) Expressed Airways Smooth Muscle 1, 2, 3, 4, 5, 7 Cholinergic Nerve 1, 3, 4 Sensory Nerve 1, 3, 4 Mast Cell 4, 7 Macrophage 1, 3, 4, 5, 7 T-Lymphocyte 3, 4, 7 Eosinophil 4, 7 Basophil 3, 4, 5, 7 Neutrophil 4, 7 Monocyte 1, 3, 4, 7 Platelet 1, 2, 3, 5 Epithelial Cell 1, 2, 3, 4, 5, 7, 8 Endothelial Cell 2, 3, 4, 5 munoglobulin E (IgE) production, proliferation, lipid mediator and histamine generation, and chemotaxis; and iii) PDE4 inhibitors are efficacious in animal models of pulmonary inflammation [18]. If these observations hold in humans then, conceptually, PDE4 inhibitors should show a pleiotropic profile of activity on those cell types involved in airways inflammation and so differ from classical mediator antagonists whose importance in disease progression might vary between asthma/copd sufferers. A further prediction is that inhibition of PDE4 should potentiate the effects of endogenous anti-inflammatory agents that stimulate adenylyl cyclase through Gs-coupled receptors such as catecholamines, prostaglandin E 2 and prostacyclin [22]. Taken together, the pre-clinical pharmacology of these compounds provides an exciting rational basis for the development of novel anti-inflammatory pharmaceuticals that may display steroid-like activity without the associated side-effects. A major obstacle that became clear in the development of PDE4 inhibitors is their propensity for non-steroid-like side effects of which nausea and vomiting are the most common and worrisome. Side-effects represent an extension of the pharmacology of these compounds and are typical of first generation PDE4 inhibitors, such as rolipram. To reduce the low therapeutic ratio of these drugs, several strategies have been considered [18, 21]. With respect to the most clinically advanced compounds it is important to understand that PDE4 may adopt at least two non-interconvertible, or slowly interconvertible, conformations, PDE4H and PDE4L, for which rolipram has high and low affinity respectively [18, 21, 23 25]. The findings that the relative amount of each conformer varies considerably between cells and tissues, and that inhibition of PDE4L is generally associated with anti-inflammatory activity (e.g. inhibition of cytokine generation and oxidant production [26 29]) whereas adverse effects (emesis, gastric acid secretion [30, 31]) reflect the inhibition of PDE4H, had important implications with regard to understanding the mechanism of side-effects and the design of second generation inhibitors with improved therapeutic ratios. It was hypothesised that poorly tolerated compounds, such as rolipram, selectively interact with the conformer of PDE4 (i.e. PDE4H) that is highly expressed in parietal cells and the central nervous system (CNS), and responsible for nausea and vomiting. An extension of this idea was that PDE4 inhibitors that have a lower affinity at PDE4H but the same or an improved affinity at PDE4L should have a higher therapeutic ratio. PDE Inhibitors nearing the market Cilomilast (figure 1) is in Phase II (see below) and Phase III clinical development for asthma (Western Europe, US and Japan) and COPD (US) respectively [32 35]. It represents the most advanced and extensively studied PDE4 inhibitor in clinical development for COPD with Byk-Gulden providing the major competition with roflumilast (figure 1), about which relatively little is known (table 1). Bayer has also indicated the development of the 2-benzoylbenzofuran, BAY (figure 1), for COPD and Phase II trials were completed in June However, while Phase III studies were scheduled to begin at the end of 2001 the development of BAY has been deferred pending a complete analysis of the COPD patient data [36]. Pumafentrine (Byk-33043), SCH (D-4396) and YM-976 (figure 1) are other PDE inhibitors that are known to have been clinically evaluated in asthma and/or COPD [36]. According to Altana s reports of its research and development (R&D) pipeline, pumafentrine, a hybrid PDE3/PDE4 inhibitor, entered Phase II clinical trials for asthma and COPD in March 2001 and is predicted to reach the market in 2006 [36] although little data are in the public domain. The selective PDE4 inhibitors SCH , which Fig. 1. Molecular structures of PDE inhibitors in clinical trials for asthma and/or COPD. 50

4 PHOSPHODIESTERASE 4 IN ASTHMA AND COPD was licensed to Schering Plough by Chiroscience, and YM-976 are reported to have entered Phase I clinical trials but the results have not been disclosed [36]. The competition in the asthma arena is now less fierce than it was at the beginning of While roflumilast is in Phase III trials, the development of most of its immediate competitors including cilomilast, BAY and Napp s xanthine inhibitor, V-11294A, has probably been discontinued (table 1). The remainder of this review will concentrate on cilomilast and roflumilast. Biochemistry and enzymology Cilomilast Cilomilast is a potent (Ki = 92 nm), competitive and selective inhibitor of PDE4 [37, 38], which preferentially interacts with the magnesiumbound form (holoenzyme) of the enzyme when compared to free enzyme (apoenzyme) [39]; it is essentially inactive against PDEs 1, 2, 3, 5 and 7 at concentrations up to 10 µm [38]. In addition, cilomilast is 10-fold more selective for PDE4D than any of the other isoenzymes in this family (table 3) and has almost equal affinity at recombinant forms of PDE4H (IC 50 = 120 nm) and PDE4L (IC 50 = 95 nm), which is maintained across all variants [38, 40, 41]. This profile of activity contrasts markedly with rolipram, which is ~100-fold selective for PDE4H [38, 42], and is reflected by improved therapeutic ratios in a number of test systems [43]. Roflumilast Roflumilast and roflumilast N-oxide, a major metabolite in humans (see below), are highly potent (IC 50 s = 0.8 and 2 nm respectively), competitive and selective inhibitors of PDE4 [44] having essentially no activity against PDEs 1, 2, 3 and 5 at concentrations up to 10 µm [44]. Circumstantial evidence indicates that roflumilast and other benzamide PDE4 inhibitors preferentially interact with PDE4L rather than PDE4H and do not discriminate between PDE4A, PDE4B and PDE4D [44]. Like cilomilast, roflumilast demonstrates improved therapeutic ratios in a number of test systems when compared to rolipram [44, 45]. Table 3. Selectivity of cilomilast and rolipram for PDE4 isoenzymes PDE Isoenzyme Cilomilast Rolipram (IC 50 -nm) (IC 50 -nm) PDE4A PDE4B PDE4C PDE4D Data taken from [117]. Clinical efficacy of cilomilast PHASE I STUDIES In short- (9 days, 6-weeks) and long-term (12 months) studies, cilomilast is well tolerated up to 15 mg b.i.d. [46 48]. No drug-related adverse effects have been reported in haematology, clinical chemistry, heart rate, blood pressure, ECG (assessed by 12-lead Holter monitoring) and urinalysis. However, higher doses (20 mg b.i.d.) are associated with unacceptable side effects when compared to placebo including nausea and vomiting [46]. PHASE II STUDIES Asthma Nieman and colleagues have reported the results of a randomized, placebo-controlled, double blind crossover trial with cilomilast in 27 patients with exercise-induced asthma. Subjects were randomized to receive cilomilast (10 mg b.i.d.) or placebo for 7 days followed by a 7 day washout and then the alternative treatment for 7 days. The primary efficacy variable was the maximum percentage decrease (MPD) in forced expiratory volume in one second (FEV 1 ) in response to exercise. In the placebo group the mean fall in FEV 1 after exercise was 32.9%, which was significantly greater than the deterioration in lung function seen when the same subjects received a single dose of cilomilast (23.6% reduction in FEV 1 ). The improvement in lung function was incremental such that after 7 days of therapy the MPD FEV 1 was further reduced to 21.8% [49]. Improvements in MPD peak expiratory flow rate (PEFR), time to recovery after exercise and percent protection against exercise-induced bronchoconstriction were also noted [49]. The results of a multicentre, placebo-controlled, double blind randomised parallel group study with cilomilast (5, 10 and 15 mg b.i.d. for 6 weeks) involving 303 patients (18 to 70 years) taking inhaled corticosteroids concurrently have been reported [47, 50]. All patients had an FEV 1 of approximately 66% (range 50 to 80%) of predicted, expressed a 12% or greater responsiveness to salbutamol and had asthma that was inadequately controlled with inhaled corticosteroids. Two hundred and sixty six patients completed the study. At the highest tolerated dose (15 mg b.i.d.) cilomilast (n = 79) as well as placebo (n = 72) increased FEV 1 from week 1 onwards and this effect was greater with the active treatment group (figure 2). However, the improvement in lung function failed to reach statistical significance at any time except at week 2 when the mean difference in trough FEV 1 was 210 ml greater than in the placebo group [47]. Improvements, relative to placebo, in forced expiratory flow at 25% to 75% of forced vital capacity (FEF ) and domiciliary PEFR were also detected but, again, statistical significance was not achieved [47]. However, in the physicians global assessment, 59% of patients taking cilomilast (15 mg b.i.d.) were rated as markedly improved 51

5 M.A. GIEMBYCZ Fig. 2. Effect of oral cilomilast on the mean change from baseline in clinic forced expiratory volume in 1 second (FEV 1 ) in patients with asthma. Subjects entered a one-week placebo run-in before being randomised (double blind) to receive cilomilast (15 mg b.i.d.; n = 79) or placebo (n = 72) for 6-weeks. At the end of each week of treatment trough (pre-dose) FEV 1 was measured. See text for further details. Data taken from [47]. * Statistically significant improvement in lung function relative to placebo. compared to 39% of patients given placebo, which was highly significant. Similarly, in the patients global assessment, 69% in the active treatment group (15 mg b.i.d.) indicated that they were markedly improved compared to 41% of patients that received placebo [47, 50]. An international (Germany, UK, France, South Africa), multicentred (21) Phase IIb double blind, parallel group 12 month efficacy, safety and tolerability study of cilomilast (10 and 15 mg b.i.d.) has also been evaluated in 211 asthmatic patients (male and female) aged between 19 and 70 years, which was an extension of three double blind randomized Phase II studies of 4 to 6 weeks duration [48, 51]. One hundred and fifty eight patients received cilomilast and the remainder were given placebo. Inclusion criteria included a history of episodic wheezing for at least 6 months, an FEV 1 45% and 90% of predicted for height, sex and weight, and responsiveness to salbutamol ( 12%) at time of screening. The safety and tolerability of this study are discussed below. With respect to efficacy, clinically relevant and statistically significant improvements above placebo were seen in forced vital capacity (FVC) and PEFR, which were sustained from week 1 to the end of month 12. A consistent improvement in FEV 1 was also noted but this did not reach statistical significance. Diary asthma symptom scores also indicated a reduction in cough, wheeze and breathlessness/chest tightness in the active treatment group when compared to placebo [51]. In November 2000 SmithKline-Beecham announced that further development of cilomilast for asthma was to be delayed for at least 12 months following disappointing results of a pivotal European trial. Although a positive outcome was reported from a similar multicentre study in the US, which prompted a further European investigation [52], the development of cilomilast for asthma is thought to have been abandoned. COPD In a European multicentre dose ranging study, 424 patients with moderate COPD (mean postbronchodilator FEV 1 = 46.8% of predicted; FEV 1 /FVC ratio = 0.55; mean sensitivity to salbutamol = 5.4% increase in FEV 1 ; mean smoking history = 39.7 pack years), were randomised to receive cilomilast (5 mg, n = 109; 10 mg, n = 102; 15 mg, n = 107 b.i.d.) or placebo (n = 106) for 6 weeks [53 55]. At the highest dose, cilomilast produced a progressive and highly significant increase in trough FEV 1 from week 1 to the end of the study period. At the end of week 6 cilomilast had increased trough FEV 1 by 160 ml, which represented an 11% improvement in lung function, when compared to subjects that received placebo [53 55]. Similar improvements at week 6, relative to placebo, were observed for the 15 mg b.i.d. dose in FVC, PEFR (mean differences compared to placebo of 190 ml and 34.1 L/min respectively), exertional dyspnea, rescue bronchodilator use and resting and post-exercise arterial oxygen saturation [53 55]. Lower doses of cilomilast (5 and 10 mg b.i.d.) produced negligible improvements in lung function, which was confirmed in a follow-up multicentered study conducted in the US involving 224 patients [56]. Quality of life assessments using the St. George s Respiratory Questionaire (SGRQ) and the Medical Outcomes Study 36-item short form health survey (SF-36) [57] were also recorded before and 6 weeks after therapy with cilomilast (5, 10 and 15 mg b.i.d.) or placebo [53, 58]. In agreement with the lung function data described above, consistent improvements approaching that defined as clinically relevant (-4 points) in the total and composite scores (symptoms, activity, impacts) of the SGRQ were recorded for those subjects that received 15 mg cilomilast when compared to the placebo group (table 4). Similar improvements were recorded for the physical composite score of the SF-36 in those subjects that were given 10 and 15 mg cilomilast [53, 58]. PHASE III STUDIES COPD The improvement in lung function and health status of COPD patients given cilomilast in Phase II clinical trials has prompted further evaluation of safety (see below) and efficacy in a Phase III program of studies of 6 months duration [59 61]. Six hundred and forty seven patients with mild to moderate COPD, defined as having an FEV 1 of between 30 and 70% of predicted and a reversibility with β 2 -adrenoceptor agonists of less than 15%, were recruited and given cilomilast (15 mg b.i.d., n = 431) or placebo (n = 216). Exacerbations [59], lung function [60] and health status [61] were monitored. Relative to placebo, cilomilast significantly reduced, by 39% and 45% respectively, the risk of a self-managed exacerbation and of an exacerbation requiring treatment by a physician or hospitalisation. Equally, there was a statistically 52

6 PHOSPHODIESTERASE 4 IN ASTHMA AND COPD Table 4. Results of St. George s Respiratory Questionnaire in subjects with COPD 6 weeks and 6 months after therapy with cilomilast 6 Weeks (n = 107) 6 Months (n = 431) Cilomilast (15 mg b.i.d.) Mean Difference P-Value Mean Difference P-Value Total Score >0.001 Symptoms Score Activity Score Impacts Score Data taken from [58, 61, 80]. significant improvement in lung function (figure 3) such that at the end of the study period FEV 1, FVC and trough FEV 1 were improved by 80 ml, 110 ml and 40 ml respectively relative to the placebo group. Other lung function parameters including trough FEF and trough FEV 6 also were improved by cilomilast (differences from placebo = 40 ml/s and 90 ml respectively) suggesting that a clinically important impact of the drug may have occurred in the small airways [60, 62]. It is noteworthy, that in this clinical trial cilomilast did not improve lung function but protected against a deterioration in FEV 1 when compared to those patients that received placebo (figure 3). These data clearly contrast, and are difficult to reconcile, with the results of the Phase II trials for COPD (figure 4) described above [54, 55], where lung function was improved by cilomilast. Changes in health status and global health status using the SGRQ and SF-36 respectively were made at baseline and 6 months after therapy with cilomilast (15 mg b.i.d.) and placebo [61]. In agreement with the lung function data, consistent improvements defined as clinically relevant and statistically significant in the total (-4.1 points) and composite scores (symptoms, activity, impacts) of the SGRQ were recorded for those subjects that received 15 mg cilomilast when compared to patients that were given placebo (table 4). At the end Fig. 3. Effect of oral cilomilast on the mean change from baseline in clinic FEV 1 in patients with COPD. Subjects entered a 4-weeks placebo run-in before being randomised (double blind) to receive cilomilast (15 mg b.i.d.; n = 431) or placebo (n = 216) for 24 weeks. At defined times after treatment trough (pre-dose) FEV 1 was measured. See text for further details. Data taken from [60]. * Statistically significant difference in lung function relative to placebo. Fig. 4. Effect of oral cilomilast on the mean change from baseline in clinic FEV 1 in patients with moderate COPD. Subjects entered a 2- weeks placebo run-in before being randomised (double blind) to receive cilomilast (5 mg, n = 109; 10 mg, n = 102; 15 mg, n = 107; b.i.d.) or placebo (n = 106) for 6-weeks. At defined times after treatment trough (pre-dose) FEV 1 was measured. Results with 15 mg cilomilast (b.i.d) are shown. See text for further details. Data taken from [53 55]. * Statistically significant improvement in lung function relative to placebo. of the study period significant improvements were also recorded for the physical function and general health perception scores of the SF-36 in the cilomilast-treated group [61]. Despite these clear improvements in lung function recorded from Phase III trials, SmithKline- Beecham announced in October 2000 that additional confirmatory trials were to be performed and, assuming a successful outcome, are certain to delay the launch of cilomilast for COPD [63]. It has since been reported that cilomilast may have anti-inflammatory activity [64]. Fifty nine patients with mild to moderate COPD (mean age and FEV years and 51.6% of predicted respectively; 7.5% mean reversibility with salbutamol) were recruited for a double blind, placebocontrolled, parallel group study and randomised to receive cilomilast (15 mg b.i.d.) or placebo for 12 weeks. Bronchial biopsies were taken at baseline and at week 10 and stained immunohistochemically for neutrophil, macrophage and T-lymphocyte markers. Relative to baseline, there were significantly less cells staining for CD4, CD8 and CD68 in those subjects that were given cilomilast compared to the placebo group indicating that T-lymphocytes and macrophage numbers were reduced. 53

7 M.A. GIEMBYCZ The number of sub-epithelial neutrophils was also reduced by cilomilast (39%) but this did not reach statistical significance [64]. a) Clinical efficacy of roflumilast Information on the effects of roflumilast in humans is scant and restricted to company statements and presentations at international conferences. No peer-reviewed publications on the clinical activity of roflumilast in asthma or COPD have appeared although efficacy of roflumilast in the treatment of allergic rhinitis was reported at the end of 2001 [65]. Asthma Sixteen patients with exercise-induced asthma were recruited for a placebo-controlled, randomised double blind, two-period cross-over study where placebo or roflumilast (500 µg o.d.) was administered in random order for 28 days [66]. FEV 1 was measured before and repeatedly up to 12 min after the end of the exercise challenge. Blood was taken for determination of lipopolysaccharide (LPS)-induced tumour necrosis factor-α (TNFα) released ex vivo as a surrogate marker of inflammatory cell activation. At the end of the study the mean fall in FEV 1 in response to exercise was significantly reduced (by 41%) when compared to placebo. Similarly, the median TNFα concentration was reduced by 21% during roflumilast treatment whereas it remained constant under placebo [66], indicating that an anti-inflammatory plasma concentration of the drug had been achieved. A randomised, two-period cross-over phase II study in 12 patients with mild asthma who responded to allergen with early and late phase responses has also been conducted where the effect of a single dose of roflumilast (1000 µg) and placebo (given 1 h before challenge) was examined. The subjects had a positive skin prick test to allergen, a FEV 1 >70% predicted and a histamine provocative concentration(pc)20 <16 mg/ml. The only medication allowed was β 2 -adrenoceptor agonist. In this trial the maximum late asthmatic response was reduced by 62% and there was also a trend for inhibition of the early response [67]. COPD The efficacy and side-effect profile of roflumilast has been reported in a randomised, placebocontrolled double blind trial involving 657 patients with COPD. Of the subjects that were recruited, 516 entered the study (mean FEV 1 of ~54% of predicted) and after a 2 weeks run-in were randomised to receive roflumilast (250 µg n = 175; 500 µg o.d., n = 169) or placebo (n = 172) for 26 weeks. Four weeks after treatment discernible increases in lung function (FEV 1, FVC and morning PEFR) were apparent in the active treatment groups (figure 5) with the incidence of adverse events not exceeding the number reported in placebo-treated patients (table 5). At the end of the study a 100 ml increase in FEV 1 was reported with b) c) Fig. 5. Effect of oral roflumilast on lung function in patients with COPD. Subjects entered a 2-weeks placebo run-in before being randomised to receive roflumilast (250 µg, n = 175; 500 µg o.d., n = 169) or placebo (n = 172) for 26 weeks. At defined times after treatment FEV 1 (a), forced vital capacity (FVC) (b) and morning peak expiratory flow rate (PEFR) (c) were measured and are shown as the median difference from baseline. See text for further details. Data taken from [68]. both dosing regimens. Additionally, both doses reduced the need for rescue medication although the 500 µg dose was more effective in reducing the number of exacerbations, by 48%, when compared to the lower dose (8%) [68]. Pharmacokinetics of cilomilast and roflumilast The absorption, distribution, metabolism and excretion (acronym ADME) of cilomilast have been investigated in a number of pharmacokinetic studies involving healthy Caucasian adult volunteers who were non-smokers [69 71]. 54

8 PHOSPHODIESTERASE 4 IN ASTHMA AND COPD Table 5. Adverse effects of oral roflumilast in patients with COPD in a six months phase II clinical trial Roflumilast Placebo 250 µg o.d. 500 µg o.d. (n = 172) (n = 175) (n = 169) Headache 2% 2% 4% Abdominal pain 1% 2% 3% Nausea 2% 1% 3% Diarrhea 0% 3% 3% Back pain 0% 2% 3% Insomnia 1% 1% 3% Dizziness 1% 1% 2% Vomiting 1% 0% 1% Data show percentage of patients reporting adverse effetct. Data taken from: [68]. In the fasted state the absorption of cilomilast was rapid and complete following oral administration with the peak plasma concentration (Cmax) being reached after approximately 1 to 2 h irrespective of dose [70, 71] (table 6). Cilomilast, given orally or by intravenous infusion, was almost completely bioavailable [mean absolute bioavailability in solution = 96% (range %)], had a plasma elimination half-life (t1/2) of 6 to 8 h that was doseindependent and was subject to negligible first-pass hepatic metabolism [69 71]. Plasma clearance (CL; 1.5 to 2 L/h) and mean volume of distribution at steady state (Vss; ~10 to 16 L) were low indicating that cilomilast was not distributed widely in tissues [71]. A dose-escalating study, in 4 healthy non-smoking volunteers aged 29 to 47 years, established that intravenous infusion of cilomilast (1, 2 and 4 mg over 1 h) provided dose-proportional systemic exposure indicative of linear pharmacokinetics [71]. Similar results were obtained in 13 healthy male subjects (aged years) that were randomised to receive 3 progressively rising oral doses of cilomilast (0.2, 0.5, 1, 2, 4, 6 and 7 mg) in the fasted state, with a period of at least 7 days between doses [70]. Administration of single and multiple doses of cilomilast (up to 30 mg/dose) after a fatrich meal significantly reduced Cmax and prolonged the time taken to reach Cmax (tmax), although overall systemic exposure (defined as area under the plasma concentration-time curve from time zero to infinity; AUC0- ) and the elimination phase t1/2 were unchanged [70, 71]. The pharmacokinetics of roflumilast following a single oral dose (500 µg) and after intravenous infusion (150 µg over 15 min) have been investigated in 24 healthy male subjects (age range 22 to 44 years) in 2 open, randomised 2-period cross-over studies [72, 73]. In the fasted state the absorption of roflumilast is rapid and complete following oral administration with Cmax (~7.5 µg/l) being reached after approximately 1 h. Roflumilast given orally or by intravenous infusion is readily bioavailable [mean absolute bioavailability in solution = 79% (range 69-92%)] and has an elimination phase t1/2 of between 10 and 16 h [72, 73]. In subjects fed a fat-rich meal the Cmax was significantly reduced (from 6.52 to 3.86 µg/l) and tmax was doubled (from 0.96 to 1.96 h) when compared to individuals that received roflumilast in the fasted state [73]. However, there was no effect on the t1/2 or AUC indicating that the availability of roflumilast was similar in fed and fasted subjects [73]. Effect of age Chronic bronchitis and emphysema afflict elderly patients for the most part and it is important to determine if the pharmacokinetics of any new therapy for COPD are affected by age. To this end Zussman and colleagues have conducted a singledose, non-randomized, open, parallel group study in young (18-45 years, n = 16) and elderly (65 to 84 years, n = 15) male and female volunteers where oral cilomilast (10 mg) was given after a light carbohydrate-rich breakfast [70]. In elderly subjects systemic exposure (AUC0- ) and Cmax were 21% and 12% higher respectively when Table 6. Summary of derived pharmacokinetic data following intravenous and oral administration of cilomilast to human male volunteers Pharmacokinetic Intravenous Administration Oral Administration Parameter 1 (4 mg; n = 16) (15 mg; n = 15) AUC0- (ng h -1. ml -1 ) 2236 (551) (2103) Cmax (ng ml -1 ) 385 (88) 1113 (229) tmax (h) 1.50 ( ) t1/2 (h) 7.95 (1.94) 8.14 (1.63) CL (l h -1 ) 1.94 (0.49) ND 2 Vss (l) (3.3) ND 2 1. Data represent mean (± SD) except for tmax [median (range)]; 2. Not determined for oral doses; 3. n = 15. Pharmacokinetic parameters determined included maximum observed plasma concentration (Cmax), the time taken to reach Cmax (tmax), the elimination phase half life (t1/2), the area under the plasma concentration-time curve from time zero to infinity (AUC0- ), clearance (CL) and mean volume of distribution at steady state (Vss). Data from [71]. 55

9 M.A. GIEMBYCZ compared to the same parameters calculated in the younger population group, although there was no significant change in tmax. A small (<1 h) prolongation of the elimination phase t1/2 was also found, which is indicative solely of reduced clearance given that the bioavailability of cilomilast is 100%. As cilomilast was well tolerated the authors concluded that dose-adjustments would not be necessary in elderly patients [70]. Morning vs. evening dosing Symptoms of asthma and COPD can vary depending on the time of day and are often worse at night. Consequently, as circadian differences in gastric emptying, gastrointestinal motility and altered posture during sleep can influence the therapeutic response of some drugs the pharmacokinetics of cilomilast have been assessed after morning and evening dosing [71, 74]. Twenty four healthy, non-smoking male volunteers (18 to 50 years) were recruited for an open-label, 2 period crossover study and received 2 single oral doses of cilomilast (15 mg) one in the morning (08.30 h) and the other in the evening (20.30h), 10 minutes after eating a fat-rich meal. No significant difference in Cmax, tmax or AUC0- was detected in the fed state. A small reduction (0.81 h) in the t1/2 of cilomilast was noted following evening dosing but this was considered to be of no clinical significance [71, 74]. Drug-drug interactions COPD has a high mortality and morbidity and, given the lack of effective therapies, patients with this disease require substantial pharmacological intervention during their lives. The possibility that cilomilast and/or roflumilast, like the non-selective PDE inhibitor, theophylline (see below), could interact with drugs commonly used in COPD has, therefore, been evaluated. With respect to cilomilast, initial studies suggest a low potential for drug-drug interactions as none of the pathways that metabolise this compound involve, to any great extent, cytochrome P450 enzymes (CYP1A2, CYP2D6, CYP3A4) most susceptible to competitive inhibition by other drugs [69]. Indeed, the only P450 enzyme implicated (CYP2C8), which catalyses hydroxylation, has few other substrates or inhibitors [69]. Moreover, cilomilast did not inhibit any important hepatic cytochrome P450 enzymes in vitro [69]. These data are supported by the finding that, at steady state, cilomilast (15 mg b.i.d.) had no clinically meaningful effect on the pharmacokinetics of digoxin (375 µg o.d.), theophylline (240 mg o.d. or individualised dose to achieve plasma concentrations of between 10 and 15 µg/ml) or prednisolone (10 mg o.d.) [75 79]. Conversely, neither theophylline nor Maalox Plus, an antacid commonly used in the elderly that contains salts of calcium, magnesium and/or aluminium that can alter the absorption or bioavailability of some drugs, had any significant influence on the pharmacokinetics of cilomilast (15 mg b.i.d.) [71, 80]. Similar open, randomised 3-period changeover studies involving 12 healthy male subjects, have shown that roflumilast (500 µg t.i.d. for 7 days) given orally has no significant effect on the pharmacokinetics of salbutamol or prednisolone (200 µg and 800 µg t.i.d. for 7 days respectively) [81, 82]. Thus, taken together these data demonstrate that neither cilomilast nor roflumilast interact with commonly prescribed medications for COPD indicating that these PDE4 inhibitors can be safely co-administered with these drugs. Effect of cigarette smoking Polycyclic aromatic hydrocarbons present in cigarette smoke are known to induce drug metabolising enzymes including CYP1A1 and CYP1A2 [83 86]. Moreover, theophylline, at concentrations (~100 µm) achieved therapeutically, is principally metabolized by CYP1A2 [87 94] such that dose adjustments are often necessary to compensate for the increased clearance in cigarette smokers [95 99]. Two preliminary studies have been conducted to establish if the pharmacokinetics of cilomilast are altered in male cigarette smokers who are otherwise healthy [76, 100]. Twenty five subjects were recruited for 2 groups comprising 12 smokers (defined as smoking 20 cigarettes per day) and 13 non-smokers. Each subject received a single oral dose of cilomilast (15 mg) and the pharmacokinetics were monitored over 24 h. There was no clinically relevant difference between the AUC0-, elimination phase t1/2 and CL between the two treatments [76, 100]. A small reduction in the Cmax was found in subjects who smoked compared to non-smokers but the authors of the study argue that this is unlikely to be of any clinical importance in the absence of any change in overall exposure [100]. The pharmacokinetics of roflumilast have also been compared in smokers (defined as smoking >18 cigarettes/day for at least 2 years) and nonsmokers [101]. In a parallel group study of 12 matched pairs, the pharmacokinetics (AUC0-, Cmax, t1/2) of a single oral dose of roflumilast (500 µg) were not significantly different between smokers and non-smokers [101]. Thus, cigarette smoking does not produce any significant drug interaction with cilomilast or roflumilast indicating that, unlike patients taking theophylline, no dose-adjustment with either of these PDE4 inhibitors will be necessary in smokers with COPD. Metabolism of cilomilast and roflumilast The metabolism of cilomilast has been investigated in four healthy human male volunteers aged 41 to 59 years [69]. Subjects were dosed in the fasted state both orally (10 mg of [ 14 C]cilomilast MBq in solution) and by intravenous infusion (4 mg [ 14 C]cilomilast MBq; in isotonic saline) over 1 h on 2 occasions 28 days apart. Three subjects completed the study and 1 subject received the oral dose only. Urine and feces were collected for 168 h after dose administration [69]. After intravenous and oral dosing of [ 14 C]cilomi- 56

10 PHOSPHODIESTERASE 4 IN ASTHMA AND COPD last the mean recovery of radioactivity was essentially complete (97% and 99% respectively). For both routes of administration the majority of radioactivity (91-92%) was recovered in the urine, with the remainder (6-8%) being found in the feces [69]. At least 11 metabolites of cilomilast have been detected by radio-high performance liquid chromatography/mass spectrometry in pooled urine samples, and 7 metabolites have been found in feces. Over 85% of each dose of cilomilast has been structurally identified and no significant qualitative or quantitative differences in radiolabeled metabolite profiles are apparent after oral or intravenous dosing [69]. The metabolism of cilomilast is extensive with unchanged drug accounting for less than 1% of the administered dose in urine and feces. Decyclopentylation (M6), acyl glucuronidation (M19 and M34) and 3-hydroxylation (M9) of the cyclopentyl ring were the major routes (figure 6) [69]. A large proportion of M6 also underwent subsequent glucuronidation (figure 6). While most metabolites were also detected in plasma they were essentially inactive as inhibitors of PDE4 and did not accumulate. Indeed, 24 h after dosing, these metabolites accounted for no more than 10% of the drug-related material with unchanged cilomilast being the major component [69]. Little is published on the metabolism of roflumilast although, as with many pyridylbenzamide PDE4 inhibitors, roflumilast underwent metabolic oxidation of the pyridyl nitrogen in humans and some animal species to form the N-oxide [44]. This metabolite retained the activity of the parent compound (see above) and it is suggested, based on as yet unpublished pharmacokinetic data, to contribute to the overall activity of roflumilast in vivo [36, 44]. Safety and tolerability of cilomilast Acute administration The safety and tolerability of single and repeated oral doses of cilomilast have been assessed in a randomised, double-blind, placebo-controlled, Fig. 6. Proposed pathway for the metabolism of cilomilast in human male volunteers. Subjects were dosed in the fasted state both orally (10 mg of [ 14 C]cilomilast MBq - in solution) and by intravenous infusion (4 mg [ 14 C]cilomilast MBq - in isotonic saline) over 1 h on 2 occasions 28 days apart. Blood samples were taken pre-dose and at intervals up to 72 h after dosing. Urine and feces were sampled over a period of 168 h after drug administration and metabolites were detected by radio-hplc/ms. Data taken from [69]. Bold arrows: major routes of biotransformation. Fine arrows: minor routes of biotransformation. Unfilled arrows: like route of biotransformation by gut microflora. 57

11 M.A. GIEMBYCZ parallel group study in six cohorts of normal healthy male volunteers over a period of 9 days [46]. Subjects were given a single dose of cilomilast in the fasted state followed, 24 h later, by a 6.5 days regimen of cilomilast at 2, 4, 7, 10, 15 and 20 mg b.i.d. Doses of cilomilast of up to 10 mg b.i.d. were associated with an adverse event profile that was similar to that found with placebo whereas higher doses (15 and 20 mg b.i.d.) produced gastrointestinal side-effects (nausea and vomiting) on the first dose of the single or repeat dose phase. Interestingly, subjects apparently became rapidly tolerant to these adverse events since nausea and vomiting were not observed on repeat dosing [46]. In a separate study the tolerability of a steep doseescalation regimen was determined in 17 healthy male subjects (aged years) that were randomised to receive cilomilast (10 mg b.i.d. on days 1 to 3; 20 mg b.i.d. on days 4 to 6; 30 mg b.i.d. on days 6 to 9) or placebo after food [70]. Administration of cilomilast after food significantly delayed tmax and Cmax, and hence improved tolerability in the absence of any change in overall systemic exposure and the elimination phase t1/2. Adverse events included nausea and headache that were generally mild, transient and self resolving. No clinically relevant changes in cardiographical parameters were noted [70]. Similarly, 6-weeks double blind treatment of 303 asthmatic patients (range: 18 to 70 years, mean: 42.3 years; FEV 1 range: 50 to 80% predicted; mean: 66% predicted; 12% reversibility with salbutamol) that were inadequately controlled with inhaled corticosteroids (mean use: 652 mg/day) with cilomilast (5, 10 or 15 mg b.i.d.) or placebo demonstrated good tolerability with a similar proportion (37.5 to 43.1%) of patients in each group reporting adverse events [47]. The side-effects that occurred in three or more patients in any treatment group are shown in table 5, with headache, nausea and dyspepsia being the most prevalent [47]. Chronic Administration An international, double-blind, placebo-controlled, multicentred Phase IIb, parallel group 12 month efficacy, safety and tolerability study of cilomilast (10 and 15 mg b.i.d.) has also been evaluated in 211 asthmatic patients (male and female), which was an extension of 3 double blind randomized Phase II studies of 4 to 6 weeks duration [48]. Inclusion criteria and the results of the efficacy limb of the study are described above [51]. In this trial, which permitted the use of inhaled β 2 - adrenoceptor agonists and corticosteroid as required, cilomilast was well tolerated with generally fewer adverse events reported in the active treatment group [48]. Nevertheless, long term exposure of asthmatic subjects to cilomilast was associated with a small increase in the incidence of nausea and headache but not the addition of any new adverse effects over those seen in the 4 to 6 week studies (table 7) [48]. Five patients that took cilomilast and 2 patients that received placebo experienced serious adverse events although none of these were considered to be related to the study medication. In the cilomilast group three subjects experienced asthma exacerbations, together with single cases of nasal polyposis and cardiac arrest [48]. Uterine hemorrhage and a suspected overdose accounted for the serious adverse effects in the placebo group [48]. Table 7. Adverse effects of oral cilomilast in asthmatic patients taking inhaled corticosteroids in a 12 months safety and tolerability study. Cilomilast Parameter Placebo 5 mg b.i.d. 10 mg b.i.d. 15 mg b.i.d. (n = 72) (n = 80) (n = 72) (n = 79) Patients with Adverse effects 27 (37.5) 31 (38.8) 31 (43.1) 33 (41.8) Headache 4 (5.6) 4 (5.0) 7 (9.7) 11 (3.9) Asthma Exacerbations 5 (6.3) 5 (6.3) 6 (8.3) 7 (8.9) Nausea 6 (8.3) 2 (2.5) 3 (4.2) 8 (10.1) Bronchitis 2 (2.8) 5 (6.3) 4 (5.6) Diarrhoea 3 (4.2) 2 (2.5) 1 (1.4) 3 (3.8) Rhinitis 1 (1.4) 4 (5.0) 2 (2.8) 1 (1.3) Dyspepsia 1 (1.4) 1 (1.3) 1 (1.4) 4 (4.1) Upper Respiratory Tract Infection 1 (1.4) 2 (2.5) 1 (1.4) 3 (3.8) Abdominal Pain 3 (4.2) 1 (1.4) 2 (2.5) Urinary Tract Infection 1 (1.4) 3 (3.8) 1 (1.4) 1 (1.3) Supraventricular Extrasystole 3 (3.8) 1 (13) Insomnia 1 (1.3) 3 (3.8) Data show number and pecentage (in parenthesis) of patients reporting adverse effects. Data taken from [47]. 58

12 PHOSPHODIESTERASE 4 IN ASTHMA AND COPD A multinational Phase III program has been conducted to assess the safety of cilomilast over 6 months in a large population (2049) of patients with mild to moderate COPD [80]. Inclusion criteria included an FEV 1 of between 30 and 70% of predicted and less than 15% reversibility with β 2 - adrenoceptor agonists. Subjects received either cilomilast (15 mg b.i.d., n = 1374) or placebo (n = 684). The most common adverse event was acute exacerbation of COPD, although this was less prevalent in cilomilast-treated patients (30.7%) when compared to subjects that received placebo (38.9%). Other side effects in the cilomilast-treated group, which included diarrhea, nausea, abdominal pain, upper respiratory tract infection, headache, dyspepsia and vomiting, were generally self-limited and of mild or moderate intensity [80]. In addition, Holter and 12-lead ECG, vital signs and clinical laboratory data obtained at each visit did not indicate any adverse effects related to cilomilast. Pharmacodynamic drug-drug interactions Several studies have been conducted to determine if cilomilast evokes adverse effects when coadministered with drugs commonly prescribed for asthma and COPD. As regards potential cardiovascular actions, cilomilast when co-administered with theophylline failed to produce clinically significant tachycardia, changes in cardiac rhythm or supine or erect blood pressure, 12-lead ECG time intervals or morphology, or hand tremor [102]. Further investigations demonstrated that cilomilast did not potentiate the pharmacodynamic effects of nebulized salbutamol (tremor, heart rate) digoxin (ECG wave form and rate), predisolone (diurnal variation of endogenous cortisol) or warfarin (normalised pro-thrombin time) [76 78, 102]. Safety and tolerability of roflumilast The cardiovascular activity and tolerability of repeated oral doses of roflumilast have been assessed in a randomised, double-blind, placebocontrolled, cross-over study involving 12 healthy male volunteers [103]. Each subject underwent 2 periods of treatment of 5 days duration during which time roflumilast (500 µg o.d.) or placebo was administered. The washout time before crossover was 3 to 5 weeks. On day 4 of the study no significant differences in cardiac output and heart rate-corrected total electromechanical systole were found between placebo- and roflumilast-treated subjects either pre-dose or 2 h post dose [103]. In addition, excess AUC of heart rate on day 5 was not influenced by roflumilast at 2 and 4 h post dosing [103]. All other cardiography parameters, exercise and resting ECG, blood pressure and heart rate were unaltered by roflumilast [103]. The most common adverse events were headache (4/12 subjects) and gastrointestinal irritation (2/12 subjects). Mild to moderate tiredness and muscle ache of the back were reported by one subject in each roflumilast arm of the study [103]. Concluding remarks The decision by the pharmaceutical industry to develop selective PDE4 inhibitors for the treatment of airways inflammatory diseases is based on a conceptually robust hypothesis that is now supported by a wealth of pre-clinical and clinical data [18, 21, 41, 43]. Therefore, it is highly likely that, if approved and shown to be potentially diseasemodifying, PDE4 inhibitors will become physicians drugs of choice for patients in whom lung function is compromised by emphysema and/or bronchitis. A critical question that still defies explanation is why second generation PDE4 inhibitors demonstrate efficacy in COPD but are generally inactive, or less active, at the same doses in asthma. If drugs such as cilomilast and roflumilast are diseasemodifying, as implied in a recent study [64], then one explanation for this paradox is that the inflammation in asthma is less sensitive to PDE4 inhibitors than the inflammatory response in COPD. Indeed, this idea could account for the apparent efficacy of roflumilast in asthma since dose-limiting side effects appear to be less troublesome with this compound than with other PDE4 inhibitors including cilomilast. In contrast, if the primary mechanism of action of PDE4 inhibitors is bronchodilatation then the discrepancy is more difficult to explain and may relate to the nature of the mediator(s) that regulates airways smooth muscle tone. However, the later explanation is not supported by a recent study, which showed that the improvement in lung function seen after long-term dosing of COPD patients with cilomilast was not due to the maintenance of acute bronchodilatation [104]. The basis of this conclusion was that cilomilast did not produce a ready increase in FEV 1 (indicative of bronchodilatation) in subjects who otherwise responded rapidly to salbutamol and ipratropium bromide. Despite the advent of second generation PDE4 inhibitors, dose-limiting side effects still hamper their clinical development, although they are seemingly less pronounced with cilomilast and roflumilast. With respect to the former compound it has been speculated that the combination of charge at physiological ph, dose-related linear pharmacokinetics and an improved PDE4H:PDE4L ratio over compounds such as rolipram may explain the improved therapeutic ratio [41]. Nevertheless, many questions still remain that could have relevance to the design of PDE4 inhibitors in the future. One of these relates to the selective targeting of PDE4 subtypes. Cilomilast is 10-fold selective for PDE4D over PDE4A, B and C, and this property was speculated to contribute to its improved therapeutic ratio over first generation compounds [41]. However, the results from in vitro and in vivo studies are inconsistent with this hypothesis and localization studies of PDE4 isoforms in the CNS provide circumstantial evidence that PDE4D may, in fact, be involved in the emetic response [105] (see below). Studies by WANG et al. have shown that PDE4B predominates in pro-inflammatory cells including 59

13 M.A. GIEMBYCZ human neutrophils and monocytes suggesting that this subtype may be the target for anti-inflammatory PDE4 inhibitors [106]. This idea is supported by the finding that the inhibitory concentration(ic)50 values of drugs that selectively inhibit PDE4A/PDE4B correlate strongly with their ability to suppress LPS-induced TNFα release from human monocytes and antigen-induced T-cell proliferation, whereas no such correlation was found when PDE4D-selective compounds were tested [107]. Moreover, an elegant series of experiments conducted by Hansen and colleagues demonstrated that allergen-induced eosinophilia and IgE production, as well as splenocyte proliferation and interleukin-4 release ex vivo, were completely unaffected in sensitised mice that lacked the PDE4D gene [108]. Arguably these data provide the best evidence that PDE4 inhibitors do not evoke their antiinflammatory activity by interacting with PDE4D and, by exclusion, implicate one or more of the other isoforms as the primary target of these drugs. However, this concept assumes that PDE4A and/or PDE4B do not mediate the adverse effects of PDE4 inhibitors, which would limit the amount of drug tolerated. An alternative interpretation is that multiple PDE4 subtypes need to be inhibited for efficacy to be realised. In this respect compounds like cilomilast and roflumilast may be optimal with regard to subtype selectivity, and changes to other aspects of the pharmacophore to increase the affinity for PDE4L relative to PDE4H, might be a more profitable approach to enhance the therapeutic ratio. Evidence to support the idea that inhibition of PDE4D may mediate emesis has recently emerged. In mice and humans, PDE4D is found in the area postrema and the nucleus tractus solitarious (NTS) [109, 110], brain structures that are implicated in the emetic response [111, 112]. Indeed, lesions of the area postrema attenuate vomiting evoked by most emetic drugs [111]. Similar results were recently reported in the squirrel monkey where PDE4D was found in most neurones in the medulla and detected in multiple structures including the NTS, area postrema and locus ceruleus [105]. Significantly, PDE4C was not detected in the medulla and PDE4A had a more restricted distribution being abundantly expressed in glia. The additional findings that a centrally-, but not peripherally-, acting neurokinin-1-receptor antagonist (CP 99,994) abrogated PDE4 inhibitor-induced emesis [113, 114] and that some PDE4D-reactive neurones in the medulla are innervated by substance P-enriched nerve terminals [105] provides circumstantial evidence to link emesis with inhibition of PDE4D. Further support for this idea is the finding that PDE4D, but not PDE4A or PDE4C, are expressed in the nodose ganglia [105], which houses the cell bodies of the major afferent (sensory) input from the stomach to the medulla. As part of the area postrema and nodose ganglia are believed to reside outside of the blood brain barrier [111], the expression of PDE4D by these structures could account for the peripheral component of PDE4 inhibitor-induced emetic response [114]. The recent description of subtype-selective PDE4 inhibitors [107, 115, 116] should resolve the importance of PDE4D in emesis in the near future. An exciting prospect for future pharmaceuticals relates to the recent merger of SmithKline- Beecham and GlaxoWellcome. The new company is in a unique position to evaluate the clinical potential of combining cilomilast with either the long-acting β 2 -adrenoeceptor agonist, salmeterol, or with the steroid, fluticasone. 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