American Journal of Respiratory and Critical Care Medicine

Cilomilast (Ariflo), a new oral phosphodiesterase-4 selective inhibitor, improves lung function in chronic obstructive pulmonary disease (COPD). We have evaluated its antiinflammatory effects in 59 patients with COPD randomized to receive cilomilast, 15 mg two times a day, or placebo for 12 weeks. Induced sputum differential cell counts were obtained at baseline and at five further visits. Interleukin-8 and neutrophil elastase were measured in sputum supernatant. Bronchial biopsies obtained at baseline and at Week 10 were immunostained and counted for neutrophils, CD8+ and CD4+ T-lymphocyte subsets, and CD68+ macrophages. Cells expressing the genes for interleukin-8 and tumor necrosis factor–α were identified by in situ hybridization and quantified. Compared with placebo, analysis of variance (ANOVA) of the change from baseline showed that cilomilast did not alter any sputum endpoint or FEV1. However, bronchial biopsies demonstrated that cilomilast treatment was associated with reductions in CD8+ (p = 0.001; ANOVA) and CD68+ cells (p < 0.05; ANOVA). In addition, by Poisson analysis, comparison of cell counts analyzed as a ratio of active to placebo demonstrated reductions of CD8+ (48% p < 0.01) and CD68+ (47% p = 0.001) cells. This is the first demonstration of reduction by any agent of airway tissue inflammatory cells characteristic of COPD. Phosphodiesterase-4 inhibitors represent a promising new class of substances for use in antiinflammatory treatment of this disease.

It is predicted that chronic obstructive pulmonary disease (COPD) that was in the sixth place in 2000 in the global ranking of causes of death will move to the third place by 2020 (1). COPD is an inflammatory condition (25) characterized clinically by poorly reversible airflow limitation and accelerated rate of decline in lung function (6). The inflammation underlying COPD differs from that of asthma in the predominance of CD8+ cells and macrophages in airway and alveolar tissues of smokers with COPD (2, 3), and CD8+ cell numbers in all lung compartments correlate inversely with FEV1% of predicted (3, 7, 8). Increased numbers of neutrophils have been reported in bronchoalveolar lavage fluid and sputum (9, 10) and correlate with accelerated decline in FEV1 over 15 years (11). The cytokines interleukin (IL)-8 and tumor necrosis factor–α (TNF-α) have been shown in one study to be increased in induced sputum (12), and acting on their respective receptors, they may play a role in the selective recruitment of the inflammatory cells (6, 13, 14).

Current treatment modalities for COPD are limited. These include symptom relief by bronchodilators (15). Also, the appreciation that COPD is an inflammatory condition has led to the frequent prescription of inhaled corticosteroids (ICS). However, ICS do not alter the long-term decline of FEV1 (16), and there are conflicting data as to the effects of ICS on markers of inflammation in the sputum of patients with COPD (17, 18). A trial of ICS in COPD has failed to show changes in tissue CD8+, CD68+ cells, or neutrophils, albeit there is a reduction of subepithelial mast cells (19). The recently published Global Initiative for Chronic Obstructive Lung Disease guidelines identify a pressing need to develop agents that suppress the inflammation associated with COPD and prevent disease progression (6).

Cilomilast is a selective second-generation phosphodiesterase-4 (PDE4) inhibitor. Phosphodiesterases inactivate cAMP, and PDE4 is the predominant isoenzyme in inflammatory cells. Thus, it was proposed that selective PDE4 inhibitors might be effective in treating inflammatory lung conditions such as COPD (20). Indeed, experimental evidence suggests that cilomilast has antiinflammatory activity of relevance to COPD (21). In addition, data obtained from a recent relatively large-scale clinical trial demonstrate significant improvement in lung function after 6 weeks of treatment with cilomilast (22).

This study was designed specifically to evaluate the effects of cilomilast on inflammation in COPD and was not statistically powered to show differences in clinical endpoints such as FEV1. We hypothesized that cilomilast would reduce the characteristic airway inflammation in patients with COPD treated for 12 weeks in a parallel-group randomized, placebo-controlled study. Induced sputum and endobronchial biopsies were obtained from patients before and after treatment. Neutrophil numbers and concentrations of IL-8 and neutrophil elastase were examined in induced sputum and neutrophils, CD8+ and CD4+ T lymphocytes, and CD68+ macrophages, and gene expression of the proinflammatory mediators IL-8 and TNF-α were evaluated in bronchial biopsies.

Subjects

Patients were aged 40 to 80 years with stable COPD, a smoking history of at least 10 pack-years, and fixed airflow obstruction. Only inhaled short-acting β2-agonists and anticholinergic treatments were used during the study. Subjects were volunteers who gave informed written consent, and approval was given by each local research ethics committee.

Study design

This was a 12-week, randomized, placebo-controlled, double-blind, parallel-group study. After screening, there was a 4-week single-blind placebo run-in period before randomization. Bronchoscopy was performed 2 weeks into the placebo run-in period and after 10 weeks of treatment. Any patient taking ICS had these withdrawn at least 4 weeks before screening, i.e., at least 6 weeks before the first bronchoscopy. Patients were randomized if study medication compliance was 80 to 120% during the run-in phase and FEV1 was stable. Eligible subjects were randomly assigned to receive double-blind cilomilast, 15 mg, or placebo twice daily. Evaluations were performed after 1, 2, 4, 8, 10, and 12 weeks of treatment and 7 to 10 days after completion. Figure 1

shows the study design, and Figure 2 shows the numbers of patients in the study at each stage. Pulmonary function was measured at trough levels of cilomilast.

Sputum Induction and Processing

This was performed according to a previously published method (23). Differential cell counts were performed at the central laboratory (DP at Glenfield Hospital, Leicester, UK). An ELISA technique was used to measure IL-8 (R&D Systems, Minneapolis, MN) and TNF-α (Amersham Pharmacia, Piscataway, NJ) concentrations in the sputum supernatant. Free neutrophil elastase was measured using a spectrofluorimetric assay as previously described(24). Although TNF-α was recoverable from standards, it was not recovered from the study sputum samples; thus, this assay was unsuccessful.

Bronchoscopy Procedure

Bronchoscopy was performed according to the American Thoracic Society guidelines, as previously described (3). Six endobronchial biopsies were obtained from the carinae of the right middle/lower lobar bronchus and subsegmental airways. Cup forceps (Olympus FB 20-C; Olympus, Tokyo, Japan) were replaced after every five bronchoscopies to maintain specimen quality.

Processing of Bronchial Biopsies

Biopsies were fixed immediately in fresh 4% paraformaldehyde at room temperature for 4 hours before transfer to phosphate-buffered saline at 4°C overnight. After dehydration and clearing, biopsies were embedded in paraffin wax. All further processing and quantification were performed in the central laboratory (Lung Pathology, Royal Brompton Hospital, London, UK). Five-micrometer–thick sections were stained with hematoxylin and eosin and the following monoclonal antibodies: neutrophil elastase (neutrophils), CD8 (cytotoxic/suppressor T lymphocytes), CD68 (monocytes/macrophages), OPD4 (CD4+ T-helper lymphocytes). In situ hybridization was performed to identify cells' messenger RNA+ for IL-8 and TNF-α. Positive cells were counted using light microscopy and tissue areas measured using computerized image analysis.

Statistical Analysis

The study was designed to randomize 60 patients in a ratio of 1:1. This provided at least 90% likelihood of detecting a treatment-related 20% change in sputum neutrophil count, with a SD of 15% and a significance level of 0.05. The primary efficacy variable was change in neutrophil percentage in induced sputum. Secondary efficacy variables were biopsy numbers of subepithelial CD8+ cells, CD68+ cells, epithelial and subepithelial neutrophils, and FEV1. All endpoints were analyzed using an analysis of variance model. Biopsy cell counts were also analyzed using a Poisson model that was most suited to the distribution of the biopsy data and allowed for comparison of relative cell counts in the active compared with placebo groups. Additional details are provided in the online supplement.

Patient Characteristics

Fifty-nine patients met the eligibility criteria for randomization. One patient was lost to follow-up 3 days after randomization, and another was withdrawn for noncompliance 32 days after randomization, leaving 57 patients with available data in the intention-to-treat population. Four patients were withdrawn after adverse events (see below), leaving 53 patients who completed the study. The groups were comparable with respect to baseline demographic characteristics, lung function, and smoking history (Table 1)

TABLE 1. Demographic characteristics (intention-to-treat population)


Characteristics

Placebo

Cilomilast
Sex
Male2325
Female54
Age, yr*61 (7)62 (11)
Smoking
Current1615
Ex1214
Pack-years*46 (18.2)46 (20.4)
Chronic bronchitis, MRC criteria2324
FEV1/FVC*0.53 (0.10)0.56 (0.09)
FEV1 prebronchodilator, L*1.62 (0.43)1.81 (0.41)
FEV1, % predicted*53.9 (12.4)58.2 (8.2)
FVC prebronchodilator, L*3.08 (0.62)3.30 (0.82)
Albuterol reversibility, %*
7.9 (5.9)
7.0 (6.2)

*Mean (SD), otherwise actual numbers are given.

Definition of abbreviation: MRC = Medical Research Council.

Albuterol reversibility as percentage of change from prebronchodilator FEV1.

. All patients were given albuterol for use as required, and 14 of 59 used ipratropium bromide at a constant dosage (eight in the placebo group, six in the cilomilast group). Before screening, six patients in each group had used ICS. According to the tablet count data, 93.1% of patients taking cilomilast achieved 80 to 120% compliance with the study medication compared with 96.7% of patients receiving placebo.

Sputum

Sputum neutrophil percentage did not change significantly from baseline in the cilomilast group or in the placebo group. Furthermore, the changes in neutrophil percentage were not different between the active and placebo groups (Table 2)

TABLE 2. Results of sputum and biopsy cell counts (intention-to-treat population)


Cell Type

Placebo

Cilomilast

p Value
Sputum
Total cell count, 106/ml
Baseline1.62 (0.60)2.96 (0.59)
Endpoint3.57 (1.9685)5.86 (1.92)
Change1.95 (1.750)2.90 (1.72)0.700
Neutrophils, %
Baseline76.0 (4.0)70.5 (3.8)
Endpoint74.9 (3.6)73.7 (3.5)
Change−1.1 (3.6)3.3 (3.5)0.372
Bronchial biopsy
Epithelial neutrophils/0.1 mm2
Baseline5.3 (0–26.7)3.1 (0.7–174)
Week 102.8 (0–28.5)2.6 (0–30.3)
Mean change−0.8 (2.2)0.9 (2.4)0.597
Subepithelial neutrophils/mm2
Baseline37.5 (0–188)47.3 (0.7–174)
Week 1030.9 (0–287.4)35.8 (0–171.8)
Mean change18.3 (15.8)3.2 (17.1)0.496
Subepithelial CD8+/mm2
Baseline202.8 (8.4–1289.1)240.9 (9.6–1351.1)
Week 10302.4 (4.4–851.6)134.9 (20.0–860.6)
Mean change27.7 (45.2)−185.0 (49.7)0.001*
Subepithelial CD68+/mm2
Baseline55.7 (4.0–190.4)62.3 (6.8–235.9)
Week 1073.5 (7.1–370.7)49.1 (7.8–166.7)
Mean change25.3 (15.0)−21.1 (13.2)0.039*
Subepithelial CD4+/mm2
Baseline126.0 (26–651)61.2 (20–618)
Week 1090.1 (12.3–428.0)52.5 (7.3–575.5)
Mean change2.2 (33.3)−29.4 (36.0)0.496
Subepithelial IL-8+/mm2
Baseline24.3 (0–284.6)16.7 (0–117.5)
Week 108.6 (0–186.9)17.5 (0–69.1)
Mean change−16.0 (10.7)0.7 (11.6)0.270
Subepithelial TNF-α+/mm2
Baseline215.7 (20–1,440)232.0 (17–944)
Week 10237.5 (31–891)190.8 (17–1,478)
Mean change
42.1 (72.3)
−2.4 (78.1)
0.659

*Denotes a statistically significant difference by analysis of variance for the change from baseline between cilomilast and placebo groups.

Definition of abbreviations: IL = interleukin; TNF-α = tumor necrosis factor–α.

Mean (SEM) sputum values before and after treatment.

Median (range) biopsy values before and after treatment.

Mean change (SEM) for biopsy (at Week 10) and sputum (at endpoint) values.

. Similarly, there were no significant differences in the changes of sputum macrophage percentage, total cell counts, IL-8, or neutrophil elastase concentrations (see Table E1 in the online supplement).

Bronchial Biopsies

Figures 3 and 4

show examples of biopsy tissue sections immunostained for CD8+ cells and neutrophils, respectively. The mean area per biopsy section was 0.69 mm2. The mean length of reticular basement membrane per section was 3.63 mm. The data obtained for all cell counts in the cilomilast or placebo groups at baseline and at Week 10 are shown in Table 2. Compared with placebo, analysis of variance of the change from baseline demonstrated significant reductions in counts of bronchial biopsy CD8+ (p = 0.001) and CD68+ (p = 0.04) cells by cilomilast. Subepithelial cell counts for CD8+ and CD68+ cells for each patient at baseline and at Week 10 by treatment group are shown in Figures 5 and 6 . Post hoc Poisson regression analysis confirmed the reductions in the cilomilast group compared with the placebo group in bronchial biopsy counts for CD8+ (48%, p = 0.004) and CD68+ (55%, p < 0.001) cells. In addition, by the post hoc analysis there were reductions of subepithelial CD4+ cells (42%, p = 0.025) and neutrophils (37%, p = 0.049) (Figure 7) . There were no significant treatment-related effects seen in the numbers of epithelial neutrophils, IL-8 messenger RNA+, or TNF-α messenger RNA+ cells.

Lung Function

At baseline, the mean FEV1 in the cilomilast group was 210 ml higher than in the placebo group (no significant difference). At 8 weeks, there was a 340-ml difference in FEV1 in favor of the cilomilast group (p = 0.02), with a 40-ml rise in FEV1 in the cilomilast group and a 90-ml fall in the placebo group. By the end of the study, the mean FEV1 in the placebo group had fallen, from its baseline at the start of the study, by 60 ml (SEM, 0.04), whereas it had risen by a mean of 10 ml (SEM, 0.03) in the cilomilast group. This difference did not achieve statistical significance (p = 0.16). In those subjects who showed a cilomilast-associated reduction in CD8+ cell count, there was a positive albeit weak correlation with the improvement in FEV1 (r = 0.36), which did not reach statistical significance (p = 0.18). There was no treatment difference in Borg breathlessness score.

Adverse Events

There were no side effects of cilomilast on hematology, biochemistry, vital signs, and ECG findings. Diarrhea occurred in six (20.7%) patients in the cilomilast group and in four (13.3%) patients in the placebo group (no significant difference). The diarrhea was mild to moderate in severity and did not result in treatment withdrawal. Three patients in the cilomilast group and two in the placebo group complained of nausea. Four patients in the cilomilast group were withdrawn from the study due to myocardial infarction, COPD exacerbation, dyspepsia, abdominal pain, and/or nausea (p < 0.05). One patient in the placebo group developed pancreatitis.

In COPD, the extent of inflammation has been linked to severity, and CD8+ T-cell numbers have been shown to correlate inversely with lung function (3, 25). In this study, we have demonstrated treatment-related reductions of inflammatory cells in bronchial biopsy tissue in COPD. Although glycophosphopeptical has been recently reported to be associated with alterations in natural immunity in COPD (26), no agent has been shown to reduce tissue CD8+ and CD68+ cell numbers in this disease. Cilomilast has previously been shown to improve lung function over 6 weeks at trough levels of the drug (22). Phosphodiesterase inhibitors promote the accumulation of intracellular cAMP, a second messenger that suppresses activity of immune cells and induces airway smooth muscle relaxation. PDE4 is the predominant phosphodiesterase isoenzyme in immune and inflammatory cells and cilomilast, an agent shown to have antiinflammatory effects in vitro (21), and is selective for the inhibition of this isozyme.

One key function of the CD8+ cell is the lysis of virally infected or altered host cells. However, it has been shown experimentally that excessive or inappropriate CD8+ activation can cause extensive tissue damage (27). In patients with similar smoking histories, the severity of COPD and of emphysematous lung destruction has been linked to the amplification of inflammation due to the persistence of (latent) viral infection (28). Cilomilast may attenuate such CD8+ cell–mediated damage by reducing the numbers of these cells.

Increased numbers of tissue macrophages are an early change reported in young smokers and in COPD (3, 29, 30). It is now realized that alveolar macrophages have the capacity to produce proteinases capable of the tissue destruction seen in emphysema (31). In experimental infection, CD8+ T-cell recognition of epithelial cell surface–expressed antigen has been shown to induce alveolar epithelial cell release of macrophage chemoattractants resulting in tissue infiltration and tissue damage by macrophages (32). We have shown in this study that cilomilast also reduces the numbers of tissue macrophages, which might attenuate such damage.

Neutrophilia is often reported in induced sputum of patients with COPD (33, 34). In contrast, tissue neutrophilia has been observed in some but not all studies (3, 30). Two studies of patients with COPD have reported a reduction in sputum neutrophils after ICS treatment (18, 35), but these results were at variance with two further reports (17, 36). A reduction in sputum neutrophils has been demonstrated after treatment with theophylline, a nonselective phosphodiesterase inhibitor (37). Although 3 months of treatment with ICS reduces biopsy mast cell numbers in COPD, there are no significant effects on the numbers of CD8+ T cells, CD68+ macrophages, or neutrophils (19). PDE4 is the predominant cAMP-metabolizing enzyme in human neutrophils (38). Although we found no effect on sputum neutrophils, our post hoc analyses demonstrated a reduction of tissue neutrophils and CD4+ cells and confirmed the reductions in CD8+ and CD68+ cells seen with analysis of variance. Inflammatory cell numbers in sputum and bronchial biopsies are poorly correlated (39) but provide complementary information. Moreover, the differences in treatment effect found between sputum and biopsy in our study would indicate that analyses of airway tissue should be included in any future interventional studies of COPD.

Our study was designed to investigate the mechanism of action of cilomilast in terms of its antiinflammatory activity. As a biopsy study, it was necessarily small. Yet, the number of patients in our study was larger than many previous bronchial biopsy studies. This study was not statistically powered to show changes in pulmonary function—this might explain the weak associations and lack of statistical significance between the fall in CD8+ cells and improvements in FEV1. The placebo group showed a large (mean 60 ml) fall in FEV1 during the 12 weeks of the study. All the patients met the European Respiratory Society criteria for the diagnosis of COPD (43). The change in FEV1 was not related to smoking status, reported exacerbations, or earlier steroid use. Most of the patients were recruited and followed up over winter months, and it is possible that some subjects had unreported (subclinical) exacerbations resulting in a fall in FEV1. This may also explain why we failed to show a treatment-associated effect on FEV1. Another study of 424 patients with COPD who were treated with cilomilast has shown a significant maximum difference in trough FEV1 of 160 ml compared with placebo after 6 weeks of therapy (22). Four patients in the cilomilast group and none in the placebo group were withdrawn from this study due to serious adverse events. However, the larger study of cilomilast in COPD by Compton and coworkers (22) found that the withdrawal rate for serious adverse events in the cilomilast 15 mg two times a day group (107 patients) was not significantly greater than that in the placebo group.

In summary, this is the first study to evaluate the antiinflammatory effect of a selective PDE4 inhibitor in this increasingly common disease. We have, for the first time, shown a significant reduction by oral cilomilast treatment of tissue CD8+ T lymphocytes and CD68+ monocytes/macrophages in COPD. There is growing support for the hypothesis that these cells are the key effector cells responsible for the airway and lung damage of COPD and that this distinct pattern of inflammation requires alternative antiinflammatory treatment to that used in asthma. Finally, in contrast to many existing COPD treatments, oral cilomilast is available systemically. Such delivery may have added value in influencing the systemic aspects of COPD (4042) and also may be more effective in targeting the inflammatory process in small airways and lung parenchyma, the predominant anatomic sites responsible for the airflow obstruction.

The authors thank Drs. Daniella Haefner, P. Reinhard Grahmann, Franco Mirabella, Xavier Munoz, Sergi Marti, and M. Jesus Cruz, and Phil Lake for their valuable contributions to the study and Mr. Andrew Rogers for assistance with the illustrations.

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Correspondence and requests for reprints should be addressed to Peter K. Jeffery, Lung Pathology Unit, Imperial College, Royal Brompton Hospital, Sydney Street, London SW3 6NP, UK. E-mail:

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