MD Jens M Hohlfeld
email: hohlfeld@item.fraunhofer.de

Kerstin Schoenfeld

Mehyar Lavae- Mokhtari

Frank Schaumann

Meike Mueller

Dirk Bredenbroeker

Norbert Krug

Robert Hermann

Mehyar Lavae-Mokhtari

Roflumilast attenuates pulmonary inflammation upon segmental endotoxin challenge in healthy subjects: A randomized placebo-controlled trial

Keywords: randomized controlled trial, bronchoscopy, bronchoalveolar lavage, phosphodiesterase inhibitors, experimental lung inflammation

Rationale: Roflumilast, an investigational, targeted phosphodiesterase 4 inhibitor, reduces the in vitro and in vivo inflammatory activity of cells such as neutrophils, eosinophils, macrophages, and monocytes.

Objectives:

The aim of this study was to explore the anti-inflammatory properties of roflumilast in a human model of segmental bronchial endotoxin challenge.

Methods: In a randomized, placebo-controlled, double-blind, single-center parallelgroup study, 37 healthy subjects of either sex were treated for 28 days with either oral roflumilast 500 µg once daily or placebo. At Day 29, a baseline bronchoalveolar lavage was performed, followed by segmental endotoxin challenge (4 ng/kg) and saline control challenge.

After 24 h, bronchoalveolar lavage fluid was sampled from the challenged segments and cells were counted and differentiated.

Results: After endotoxin challenge, influx of total cells (difference from baseline) in bronchoalveolar lavage of roflumilast treated subjects was 36% lower than with placebo (p=0.02). Correspondingly, the influx of neutrophils and eosinophils of roflumilast treated subjects was 39% (p=0.02) and 74% (p=0.01) lower than with placebo, respectively. In contrast, endotoxin-induced influx of monocytes was not different between roflumilast and placebo treated subjects. No significant differences existed between the groups pertaining to endotoxin-induced influx of macrophages and lymphocytes. Roflumilast was well tolerated.

No unexpected or serious treatment-emergent signs and symptoms were observed.

Conclusions: Roflumilast attenuated the endotoxin-induced influx of neutrophils and eosinophils into the airways. This study demonstrates the anti-inflammatory properties of roflumilast on bronchoalveolar granulocytes in endotoxin-induced airway inflammation in healthy subjects.

A c c e p t e d m a n u s c r i p t A c c e p t e d m a n u s c r i p t INTRODUCTION

Chronic obstructive pulmonary disease (COPD) is a leading cause of chronic morbidity and death world-wide with a predicted further increase of prevalence and mortality in the coming decades [START_REF] Pauwels | Burden and clinical features of chronic obstructive pulmonary disease (COPD)[END_REF]. The disease is characterized by airflow limitation that is not fully reversible, progressive, and associated with an abnormal inflammatory airway response to inhaled noxa, mainly cigarette smoke [START_REF] Pauwels | Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease. NHLBI/WHO Global Initiative for Chronic Obstructive Lung Disease (GOLD) Workshop summary[END_REF]. The cellular inflammatory response in the airways is predominated by neutrophils [START_REF] Stanescu | Airways obstruction, chronic expectoration, and rapid decline of FEV1 in smokers are associated with increased levels of sputum neutrophils[END_REF], monocytes, and macrophages as well as CD8+ Tlymphocytes. These inflammatory cells produce a variety of cytokines and chemokines, which stimulate the inflammatory processes and attract further inflammatory cells to the site of inflammation. Apart from the aforementioned cells, eosinophils may also play a role in course of acute exacerbations of COPD [START_REF] Fujimoto | Airway inflammation during stable and acutely exacerbated chronic obstructive pulmonary disease[END_REF].

Endotoxin, a cell wall component of gram-negative bacteria, is ubiquitously present as contaminant on airborne particles including cigarette smoke [START_REF] Hasday | Bacterial endotoxin is an active component of cigarette smoke[END_REF]. Upon inhalation, endotoxin challenges the innate immune system thereby inducing a robust inflammatory response in human airways, which is predominated by neutrophils [START_REF] Thorn | The inflammatory response in humans after inhalation of bacterial endotoxin: a review[END_REF]. After segmental bronchial instillation of endotoxin, elevations of neutrophils, monocytes, macrophages, and lymphocytes as well as pro-inflammatory mediators such as interleukin (IL)-8 and tumor necrosis factor alpha (TNF-) occur in the bronchoalveolar lavage (BAL) fluid [START_REF] O'grady | Local inflammatory responses following bronchial endotoxin instillation in humans[END_REF]. Endotoxin could thus initiate and sustain the inflammation in smoking COPD patients and might be involved in the pathophysiology of gram-negative bacterial exacerbations of the disease.

Given the similarities between the endotoxin-induced inflammatory response in the lung and the airway inflammation seen in COPD patients, endotoxin challenge could be a valuable pharmacodynamic model for the characterization of the anti-inflammatory properties of novel investigational drugs developed for the treatment of COPD. Hitherto, the model of endotoxin-induced airway inflammation in humans has been used to determine the antiinflammatory drug effect of salmeterol, prednisolone and the PDE4 inhibitor cilomilast [START_REF] Maris | Antiinflammatory effects of salmeterol after inhalation of lipopolysaccharide by healthy volunteers[END_REF][START_REF] Michel | Evaluation of oral corticosteroids and phosphodiesterase-4 inhibitor on the acute inflammation induced by inhaled lipopolysaccharide in human[END_REF].

A c c e p t e d m a n u s c r i p t

While prednisolone and cilomilast failed to reduce airway inflammation in induced sputum, salmeterol reduced cell recruitment in bronchoalveolar lavage following endotoxin inhalation.

However, a proof for salmeterol or ß-agonists as antiinflammatory drugs in patients with COPD is missing.

Roflumilast is a targeted oral, once-daily phosphodiesterase 4 (PDE4) inhibitor under investigation for the treatment of COPD [START_REF] Reid | Roflumilast Altana Pharma[END_REF]. Inhibition of PDE4 increases intracellular cyclic adenosine monophosphate (cAMP) concentrations, which in turn leads to activation of protein kinase A. Subsequent phosphorylation and inactivation of target transcription factors ultimately results in attenuation of cellular inflammatory activity [START_REF] Lipworth | Phosphodiesterase-4 inhibitors for asthma and chronic obstructive pulmonary disease[END_REF][START_REF] Spina | Phosphodiesterase-4 inhibitors in the treatment of inflammatory lung disease[END_REF]. Accordingly, roflumilast inhibits the release of inflammatory cytokines and chemokines in human cells in vitro and in animal models of COPD and asthma in vivo [START_REF] Bundschuh | In vivo efficacy in airway disease models of roflumilast, a novel orally active PDE4 inhibitor[END_REF][START_REF] Hatzelmann | Anti-inflammatory and immunomodulatory potential of the novel PDE4 inhibitor roflumilast in vitro[END_REF][START_REF] Martorana | Roflumilast fully prevents emphysema in mice chronically exposed to cigarette smoke[END_REF][START_REF] Wollin | Inhibition of airway hyperresponsiveness and pulmonary inflammation by roflumilast and other PDE4 inhibitors[END_REF]. In these animal models, roflumilast also reduces the pulmonary accumulation of neutrophils, eosinophils, monocytes, and macrophages after endotoxin or allergen challenge. In recent clinical studies, roflumilast was shown to reduce eosinophils in induced sputum of patients with asthma [START_REF] Hargreave | The effect of roflumilast on sputum eosinophils in patients with asthma [abstract[END_REF] and to reduce the number of neutrophils and eosinophils in induced sputum of patients with COPD, which was paralleled by an improvement of post-bronchodilator FEV 1 [START_REF] Grootendorst | Treatment with PDE4 inhibitor roflumilast reduces sputum neutrophil and eosinophil numbers in patients with COPD [abstract[END_REF]. Furthermore, large clinical studies in COPD indicate that roflumilast 500 µg once daily results in significant improvements in lung function and may potentially reduce the rate of the exacerbation in patients with COPD [START_REF] Rabe | Roflumilast--an oral anti-inflammatory treatment for chronic obstructive pulmonary disease: a randomised controlled trial[END_REF].

The aim of this randomized, placebo-controlled, double-blind, single-center study in healthy subjects was to investigate the effects of roflumilast on the recruitment of inflammatory cells into the airways and associated inflammatory markers following segmental bronchial endotoxin challenge. (553 words)

A c c e p t e d m a n u s c r i p t

Study Design

This study was conducted according to a randomized, double-blind, placebocontrolled, parallel-group, single-center, 4-week design (Figure 1). Forty-three subjects were randomized to receive oral roflumilast 500 µg once daily or placebo for 28 ± 3 days. Study medication was taken at home and subjects were seen weekly to check treatment compliance by drug accountability and blood sampling for retrospective assessment of roflumilast and its primary active metabolite roflumilast N-oxide. On the last day of treatment, subjects underwent bronchoscopy with BAL and segmental instillation of endotoxin and saline.

A c c e p t e d m a n u s c r i p t

fluid from the challenged segments. The 24-h interval between endotoxin challenge and postchallenge BAL was chosen because the change of inflammatory cell counts from baseline was the main study objective and cellular changes in BAL fluid after endotoxin challenge were reported to be maximal at this time point [START_REF] O'grady | Local inflammatory responses following bronchial endotoxin instillation in humans[END_REF]. BAL fluid from both days was processed and analyzed for inflammatory cells and mediators as described below.

Bronchoscopic Procedure

Bronchoscopies were performed according to the Fraunhofer Institute study site standard operating procedure, which is based on guidelines for investigative bronchoscopies [START_REF]Workshop summary and guidelines: investigative use of bronchoscopy, lavage, and bronchial biopsies in asthma and other airway diseases[END_REF]. Details of the principal bronchoscopic procedure with BAL and segmental bronchial instillation have been described previously [START_REF] Erpenbeck | Natural porcine surfactant augments airway inflammation after allergen challenge in patients with asthma[END_REF][START_REF] Schaumann | Metal-rich ambient particles (particulate matter 2.5) cause airway inflammation in healthy subjects[END_REF]. Briefly, BAL was performed with 6 x 20 ml warmed saline (0.9%) whereof the first recovered aliquot was discarded. On the last day of treatment with roflumilast, baseline BAL was done in the anterior segment of the left lower lobe. Clinical Center Reference Endotoxin (National Institute of Health -Warren Grant Magnuson Clinical Center, Bethesda, MD, USA) at a dose of 4 ng/kg body weight was diluted in 10 ml of saline (0.9%) and instilled to the medial segment of the middle lobe using a sterile catheter introduced through the working channel of the bronchoscope. As a control challenge, 10 ml of saline (0.9%) were applied to the medial segment of the lingula using a new catheter for instillation. BAL was obtained 24 h later from both challenged segments.

Assessment of Anti-inflammatory Pharmacodynamic Effects

Total cells, neutrophils, eosinophils, monocytes, macrophages, and lymphocytes in BAL were counted and differentiated using light microscopy [START_REF] Erpenbeck | Natural porcine surfactant augments airway inflammation after allergen challenge in patients with asthma[END_REF]. Because monocytes could not be differentiated from macrophages by light microscopy, monocytes were counted by flow cytometry using granularity and expression of CD14 for identification as described

A c c e p t e d m a n u s c r i p t

before [START_REF] Schaumann | Metal-rich ambient particles (particulate matter 2.5) cause airway inflammation in healthy subjects[END_REF]. Cell-free BAL supernatants were stored frozen in aliquots at -70°C until further analysis.

BAL supernatants were analyzed for TNF-, IL-6, IL-8, matrixmetalloproteinase (MMP)-9 and monocyte chemoattractant protein (MCP)-1 using commercially available ELISA (enzyme-linked immunosorbent assays) kits (all obtained from R&D Systems GmbH, Wiesbaden, Germany except, MMP-9 from Amersham Biosciences, Freiburg, Germany).

Nitrate and nitrite were determined by using the Sievers Nitric Oxide Analyzer (NOA 280i ® , General Electric Analytical Instruments, Urmston, UK), which is a high-sensitivity detector for measuring nitric oxide based on a gasphase chemiluminescent reaction [START_REF] Erpenbeck | Local nitric oxide levels reflect the degree of allergic airway inflammation after segmental allergen challenge in asthmatics[END_REF].

Assessment of Safety

Physical examinations and electrocardiogramms (ECG) were conducted at the screening visit and at a final visit. Clinical laboratory parameters were also assessed at screening and end-of-study examination and they were additionally monitored weekly during the treatment phase. Vital signs (blood pressure, pulse rate) were measured at screening, before randomization, before bronchoscopy, and at a final visit. At each visit, subjects were asked whether or not they had experienced any adverse event (AE). Any use of concomitant medication was recorded.

Lung function was measured according to standard procedures [START_REF]Standardization of Spirometry, 1994 Update[END_REF] by spirometry (Masterscope, Viasys Healthcare, Höchberg, Germany) at screening, prior to bronchoscopy, and at study termination. Following bronchoscopy with segmental endotoxin instillation, monitoring of lung function was additionally performed every 2 h with a hand-held spirometer (Asthma Monitor AM1, Viasys Healthcare, Höchberg, Germany) until bedtime.

A c c e p t e d m a n u s c r i p t Sample Size and Statistics

The main interest of our study was to explore cellular changes and to assess potential effects of roflumilast on the influx of neutrophils, eosinophils, monocytes, macrophages and lymphocytes after endotoxin challenge.

Assuming a normal distribution of the aforementioned cell variables, a sample size of 34 subjects (total of both treatment groups) is considered sufficient to ensure a power of at least 80% in correctly detecting a difference of 20% between the two treatments under the following assumptions: =0.025 one-sided and a coefficient of variation of the respective target variable(s) of 20%.

The statistical comparisons between the two treatments were based on the mean differences of the target variables between baseline BAL and BAL obtained 24 h after endotoxin challenge. Statistical analysis was performed with an ANCOVA model.

A c c e p t e d m a n u s c r i p t

Roflumilast attenuates pulmonary inflammation Hohlfeld et al.

9

RESULTS

Study Population

Forty-eight subjects were enrolled into the study (Figure 2). Five subjects were not randomized at visit V1 because they were found ineligible according to predefined inclusion and exclusion criteria. Of the 43 subjects in the treatment phase, four subjects discontinued the study prematurely due to respiratory tract infection and one subject due to moderate headache. In addition, one subject discontinued the study due to elevated liver enzymes. This abnormality was already observed during baseline (at visit V0). The subject had been randomized by mistake and was excluded on the second day of treatment. The remaining 37 subjects underwent bronchoscopy with segmental endotoxin challenge on the last day of the treatment phase. After endotoxin challenge, in one subject of the roflumilast group BAL fluid was mistakenly obtained from a non-challenged segment. Therefore, the data was excluded from efficacy analysis. Subjects in both treatment groups were well matched with regard to demographic data and baseline characteristics (Table 1).

Analysis of Anti-inflammatory Pharmacodynamic Effects

Endotoxin but not saline induced a remarkable and significant influx of inflammatory cells into the airways. Neutrophils and eosinophils showed the most pronounced increases from baseline upon bronchial endotoxin challenge (about 885-and 440-fold, respectively, for placebo-treated subjects; Table 2). In contrast, lymphocytes and macrophages/monocytes were only modestly increased upon endotoxin challenge (Table 2). When the mean endotoxin-induced changes in the total number of inflammatory cells in BAL are considered, about 16-to 17-fold increases relative to baseline were noted.

Compared with placebo, roflumilast treatment showed a statistically significant reduction of the endotoxin-induced change from baseline of total cells in BAL by about 36% (p=0.02, Figure 3a). Analysis of the individual cell types indicated that the observed

A c c e p t e d m a n u s c r i p t

roflumilast effects on total cell count were mainly driven by a significant attenuation of neutrophil (-39%) and eosinophil (-74%) influx (change from baseline) when compared with placebo (p=0.02 and p=0.01, respectively, Figure 3b and 3c, respectively). Further, a numerical trend towards attenuation of monocyte influx by roflumilast could be noted, however, the difference between both treatment groups was not statistically significant (p=0.21, Figure 3d). Also for lymphocytes, no significant treatment difference between the roflumilast and placebo could be noted upon endotoxin challenge (5.44 ± 2.96 x 10 3 cells/ml versus 7.27 ± 2.78 x 10 3 cells/ml in BAL fluid, respectively; p=0.34).

Soluble inflammatory markers in BAL fluid at baseline and 24 h after saline or endotoxin challenge are presented in Table 3. Except for nitrate and nitrite, all inflammatory markers in BAL fluid were significantly increased after endotoxin challenge, but only slightly elevated after saline instillation in both treatment groups. There were no statistically significant differences between the treatments for any of the soluble inflammatory markers in BAL fluid.

Safety Analysis

Five subjects discontinued the study prematurely due to AEs: four subjects due to mild respiratory tract infections (three subjects in the roflumilast group and one subject in the placebo group) and one subject due to moderate headache (roflumilast group). All AEs resolved completely.

Adverse events were reported by [START_REF] Schaumann | Metal-rich ambient particles (particulate matter 2.5) cause airway inflammation in healthy subjects[END_REF]

A c c e p t e d m a n u s c r i p t

was not observed. These adverse events, which were observed with a higher frequency in the roflumilast treatment group, are part of the known safety and tolerability profile of roflumilast. All AEs were mild with the exception of three moderate AEs (one episode of headache in a subject of the roflumilast group and two episodes of fever following bronchoscopy with BAL in one subject of the placebo group). No AE was of severe intensity and there was no serious AE. All AEs in this study resolved completely.

Physical examination, blood pressure, heart rate, and laboratory measurements did not reveal any treatment-emergent alterations by the study medication, except for a trend towards a slight decrease of mean body weight in the roflumilast group with a difference of -1.4 ± 1.7 kg versus baseline when compared with the placebo group (-0.1 ± 1.8 kg). The subjects' lung function as measured at screening and at study termination was not altered.

DISCUSSION

Chronic airway inflammation is regarded as a key factor in the pathogenesis of COPD.

Currently available treatments for COPD allow to prevent and control symptoms, reduce exacerbations, increase exercise tolerance, and improve health status [START_REF] Barnes | Antiinflammatory effects of salmeterol/fluticasone propionate in chronic obstructive lung disease[END_REF]. However, many COPD patients do only benefit in terms of modest symptomatic improvements from currently available therapies. Therefore, new treatment approaches that target more effectively the specific inflammation characteristics in COPD are still warranted.

The identification and development of novel anti-inflammatory treatment concepts in COPD until proof-of-concept is lengthy, labor-intensive, and costly. This is mainly due to the apparent lack of well-established human pharmacodynamic models that would allow the identification of promising drug candidates in short-term studies with a reasonably low number of subjects. The model of induced pulmonary inflammation by segmental instillation of endotoxin in healthy subjects as first described by O'Grady et al. [START_REF] O'grady | Local inflammatory responses following bronchial endotoxin instillation in humans[END_REF] could be a useful approach to effectively characterize the clinical potential of an apparent anti-inflammatory

A c c e p t e d m a n u s c r i p t

drug candidate already at an early development stage. However, to the best of our knowledge, segmental endotoxin challenge has not been used and validated as yet for the characterization of anti-inflammatory COPD treatment effects. Therefore, it was the purpose of our study to apply this model for the placebo-controlled assessment of treatment effects of roflumilast, a targeted PDE4 inhibitor. Further, we aimed at putting the results into perspective with previously observed roflumilast effects in pre-clinical COPD models as well as clinically obtained anti-inflammatory characteristics in COPD patients.

In our study, we found that roflumilast -compared with placebo -significantly reduced the endotoxin-induced influx of neutrophils, eosinophils, and total cells into the broncho-alveolar compartment. In contrast to the effect on granulocytes, endotoxin-induced recruitment of monocytes/macrophages and lymphocytes was only numerically lowered by roflumilast, but did not achieve statistical significance versus placebo. This predominance of roflumilast effects towards inhibition of granulocyte influx rather than lymphocyte and monocyte/macrophage increase has also been observed in animal models after cigarette smoke and endototoxin exposure [START_REF] Martorana | Roflumilast fully prevents emphysema in mice chronically exposed to cigarette smoke[END_REF][START_REF] Fitzgerald | Effect of roflumilast on pulmonary inflammation in guinea pigs exposed to cigarette smoke [abstract[END_REF][START_REF] Wollin | Inhibition by roflumilast versus cilomilast of pulmonary leukocyte accumulation and TNF release in a rat model of LPS-induced pulmonary neutrophilia [abstract[END_REF]. Further, the roflumilast effect pattern in BAL observed in response to the segmental endotoxin challenge is also consistent with the previously demonstrated ability of roflumilast to reduce the number of neutrophils and eosinophils in induced sputum of patients with COPD and asthma, respectively, whereby the reduction of sputum neutrophilia in COPD patients was paralleled by improvements in lung function [START_REF] Grootendorst | Treatment with PDE4 inhibitor roflumilast reduces sputum neutrophil and eosinophil numbers in patients with COPD [abstract[END_REF][START_REF] Rabe | Roflumilast--an oral anti-inflammatory treatment for chronic obstructive pulmonary disease: a randomised controlled trial[END_REF]. Taken together, the modulation of cellular inflammatory responses upon endotoxin challenge by roflumilast appears to be largely consistent with the previously shown pre-clinical and clinical anti-inflammatory characteristics of this targeted PDE4 inhibitor.

However, a randomized controlled clinical study in COPD patients with cilomilast, another investigational PDE4 inhibitor, showed that treatment for 12 weeks compared with placebo was associated with reductions in CD8+ T-lymphocytes and CD68+ macrophages in bronchial biopsies. In contrast to roflumilast, cilomilast did not alter the number or fraction of

A c c e p t e d m a n u s c r i p t

neutrophils in bronchial biopsies nor in induced sputum [START_REF] Gamble | Antiinflammatory effects of the phosphodiesterase-4 inhibitor cilomilast (Ariflo) in chronic obstructive pulmonary disease[END_REF]. For a more detailed understanding whether this apparent discrepancy between predominant inhibition of neutrophilic versus monocytic inflammation may signify specific compound characteristics (roflumilast versus cilomilast) within the class of PDE4 inhibitors or rather reflecting differential PDE4 inhibitor effects in different compartments (bronchial mucosa versus BAL), the conduct of a roflumilast bronchial biopsy study in COPD patients would be required. We have not taken bronchial biopsies in our study because we enrolled healthy subjects in whom airway inflammation is virtually absent at baseline and bronchial biopsies after segmental instillation of endotoxin is hampered due to the fact that biopsy sampling from the subsegmental level (distal from endotoxin instillation) is very limited and often of poor quality.

In contrast to the observed significant and consistent treatment effects of roflumilast on cellular outcome variables in our study, cytokine/chemokine markers in BAL were apparently unaffected by roflumilast. However, the study by O'Grady and colleagues [START_REF] O'grady | Local inflammatory responses following bronchial endotoxin instillation in humans[END_REF] indicated that the time courses of cellular and cytokine/chemokine inflammatory responses in BAL upon segmental endotoxin challenge are distinctly different, with maximum cytokine/chemokine increases occurring already at 6 h post challenge, while maximum cellular responses only occurring at 24 h post challenge. This observation implies that the investigation of drug effects on inflammatory cell and cytokine/chemokine responses by endotoxin challenge would require post-challenge bronchoscopies at different time points (i.e. at 6 h and 24 h). From this methodological perspective, the present study using the 24-h bronchoscopy interval was therefore designed to primarily and adequately address the influence of roflumilast on cellular inflammatory markers but not on cytokine/chemokine responses, the latter were assessed only for exploratory purposes. This view is confirmed by our finding that post-challenge cytokines/chemokines at 24 h were numerically close to baseline levels.

A c c e p t e d m a n u s c r i p t

Two recent studies used a controlled clinical setting in humans with inhaled endotoxin to study the anti-inflammatory effect of salmeterol, prednisolone and the PDE4 inhibitor cilomilast [START_REF] Maris | Antiinflammatory effects of salmeterol after inhalation of lipopolysaccharide by healthy volunteers[END_REF][START_REF] Michel | Evaluation of oral corticosteroids and phosphodiesterase-4 inhibitor on the acute inflammation induced by inhaled lipopolysaccharide in human[END_REF]. Currently, there are no data published on the use of segmental endotoxin challenge for the characterization of anti-inflammatory effects of either established drugs or new drug candidates for the treatment of chronic inflammatory airway diseases such as COPD. Therefore, it is at present not possible to directly compare the pattern of the observed roflumilast effect in the endotoxin challenge model with already established COPD treatments (e.g. corticosteroids). However, our group conducted a study with a novel monoclonal MCP-1 antibody (ABN912) in patients with mild COPD. Here, we used the identical methodological approach of segmental instillation of endotoxin (i.e. at the identical dose of 4 ng/kg) to investigate the anti-inflammatory characteristics of the compound in the airways.

Interestingly, ABN912 significantly reduced BAL monocytes upon endotoxin challenge when compared to placebo, but did not alter the number of BAL neutrophils, macrophages, and lymphocytes [START_REF] Krug | Anti-MCP-1-monoclonal antibody (ABN912) attenuates[END_REF]. This observation of distinctly different cellular effects of two antiinflammatory compounds with a different mode of action strongly suggests that the endotoxin challenge model indeed allows to distinguish between as well as to properly characterize differential anti-inflammatory drug effects.

In our study, roflumilast 500 µg once daily was generally well tolerated. Safety results did not reveal any unexpected adverse treatment-emergent effect in terms of frequency or intensity of AEs. The observed trend towards a modest decrease of body weight may need further attention in future clinical studies with PDE4 inhibitors.

In conclusion, roflumilast significantly reduced the endotoxin-induced influx of total cells, neutrophils, and eosinophils into the airways of healthy subjects. This finding is consistent with anti-inflammatory properties of roflumilast shown in previous pre-clinical COPD models and clinical studies in COPD patients. The demonstrated anti-inflammatory potential of roflumilast may provide therapeutic benefits for the treatment of COPD, but MMP-9 = matrixmetalloproteinase 9; MCP-1 = monocyte chemoattractant protein 1, SD = standard deviation. † p<0.01 compared to baseline. No significant differences between groups were noted.

A c c e p t e d m a n u s c r i p t

A c c e p t e d m a n u s c r i p t

8 †

 8 Definition of abbreviations: SD = standard deviation; * p<0.025 for mean difference between roflumilast and placebo of change from baseline to 24 h after endotoxin challenge. † p<0.01 compared to baseline.

A c c e p t e d m a n u s c r i p t

Figure 1 :Figure 2 :

 12 Figure 1:Study design

Figure 3 :

 3 Figure 3:Change of bronchoalveolar lavage cells after endotoxin challenge: change

Figure 3 AFigure 3

 33 Figure 3 A

TABLE 1 .

 1 Demographic data and lung function characteristics of the study population.

	Placebo	Roflumilast 500 µg
	(n=19)	(n=18)

Definition of abbreviations: BMI = body mass index; FEV 1 = forced expiratory volume in one second; FVC = forced vital capacity; SD = standard deviation.

* Data presented as mean ± SD. No significant differences between groups were found.

TABLE 2 .

 2 Cell counts in bronchoalveolar lavage fluid at baseline and following segmental bronchial challenge with saline or endotoxin in healthy subjects treated with placebo or roflumilast. Differential cell counts were analysed by light microscopy, while monocytes † were additionally counted by flow cytometry. Values are given as mean SD.

		Subjects	Total cells	Macrophages	Neutrophils	Eosinophils	Lymphocytes	Monocytes †
				/ Monocytes
		(n)	(x 10 3 /ml)	(x 10 3 /ml)	(x 10 3 /ml)	(x 10 3 /ml)	(x 10 3 /ml)	(x 10 3 /ml)
	Placebo
	Baseline	19	100 50.0	97.1 49.1	1.42 2.00	0.02 0.08	5.33 6.20	2.06 1.50
	Saline	19	110 60.0	96.4 49.6	5.32 10.3	0.20 0.33	4.33 3.90	2.65 2.38
	Endotoxin	19	1720 960 †	447 153 †	1256 870 †	8.83 11.0 †	13.3 14.7 †	148 85.3 †
	Roflumilast 500 µg
	Baseline	18	80.0 40.0	79.5 36.6	0.64 0.85	0.07 0.14	3.02 2.72	1.33 1.14
	Saline	18	140 160	128 139	7.02 18.8	0.09 0.20	4.67 5.17	4.35 9.63
	Endotoxin	17	1280 480* †	435 239 †	836 314* † 3.43 7.10*	7.71 11.2	126 59.

TABLE 3 .

 3 Inflammatory markers in bronchoalveolar lavage fluid at baseline and following segmental bronchial challenge with saline or endotoxin in healthy subjects treated with placebo or roflumilast. Values are given as mean SD. Definition of abbreviations: TNF-= tumor necrosis factor alpha; IL-6 = interleukin 6; IL-8 = interleukin 8;

		Subjects	TNF-	IL-6	IL-8	MMP-9	MCP-1	Nitrate	Nitrite
		(n)	(pg/ml)	(pg/ml)	(pg/ml)	(ng/ml)	(pg/ml)	(µmol/l)	(µmol/l)
	Placebo
	Baseline	19	16.2 2.9	4.7 2.2	30.5 2.8	1.6 1.2	35.8 13.5 0.7 0.3 0.1 0.1
	Saline	19	16.2 2.6	7.1 8.0	31.5 2.7	2.0 0.9	36.6 16.3 0.5 0.2 0.2 0.2
	Endotoxin	19	55.2	191 119 † 67.1 21.6 † 7.2 2.5 † 77.9 45.0 † 0.6 0.3 0.2 0.2
			22.2 †
	Roflumilast 500 µg
	Baseline	18	16.1 1.6	3.7 1.9	31.2 0	1.0 0	33.4 9.50 0.6 0.3 0.3 0.5
	Saline	18	16.0 1.6	7.0 6.8	31.4 3.9	1.5 0.9	40 7 23.3 0.6 0.3 0.1 0.1
	Endotoxin	17	56.5	219 147 † 79.7 34.2 † 7.3 2.7 † 81.0 37.0 † 0.7 0.3 0.1 0.1
			20.8 †

LPS-induced monocyte recruitment into the lung in patients with COPD[abstract]. Proc Am Thorac Soc 2006;3:A849

Acknowledgements

The authors thank Arnd Manegold, ALTANA Pharma AG, Konstanz, Germany for conceptual contribution and support in the conduct of the study and Angela Schilling, ALTANA Pharma AG, Konstanz, Germany for helpful suggestions during the preparation of this article. The authors thank Anthony F. Suffredini, National Institute of Health, Bethesda, MD, USA for his support to obtain National Institute of Health Clinical Center Reference Endotoxin.

needs to be further characterized at the level of cytokine/chemokine involvement and in additional compartments relevant to airway inflammation such as bronchial mucosal. Finally, we propose that the segmental endotoxin challenge model allows for a rapid, safe, and wellstandardized characterization and differentiation of anti-inflammatory drug effects in human airways.

Conflict of Interest

KS, DB, and RH are employees of ALTANA Pharma AG, Konstanz, Germany. The study described in this article has been designed, conducted, analyzed, and reported within the scope of their employment. JMH, MLM, FS, MM, and NK are employees of Fraunhofer ITEM, Hannover, Germany, which has received a research grant for this clinical trial from ALTANA Pharma AG and for other clinical trials in COPD from Novartis, GSK and Boehringer Ingelheim. JMH has been reimbursed by ALTANA Pharma AG for attending a conference and presenting the results of this study.

A c c e p t e d m a n u s c r i p t

Roflumilast attenuates pulmonary inflammation Hohlfeld et al.


Guglielmo De Laurentiis

M.D Mauro Maniscalco
email: mauromaniscalco@hotmail.com

Flavia Cianciulli

Anna Stanziola

Serafino Marsico

Jon O Lundberg

Eddie Weitzberg

Matteo Sofia

Exhaled nitric oxide monitoring in COPD using a portable analyzer

Keywords: exhaled nitric oxide, airway inflammation, chronic obstructive pulmonary disease, hand-held analyzer, exacerbation

BACKGROUND

Exhaled nitric oxide (FeNO) is a non-invasive marker of airway inflammation in asthma. A very recent statement has suggested FeNO as potential outcome in chronic obstructive pulmonary disease (COPD). Recently, a new hand-held FeNO analyzer (NIOX MINO) has been developed.

PATIENTS AND METHODS

We have evaluated the NIOX MINO in COPD patients and monitored FeNO levels during 1-year assessment in the outpatient setting. Short-term variability in FeNO was compared using a NIOX MINO and a stationary chemiluminescence analyser (NOA, Sensormedics) in healthy volunteers and COPD patients on two consecutive months. Long-term FeNO variability was assessed on a cohort of 70 COPD outpatients measuring FeNO for one year. The intra-individual FeNO coefficient of variation (eNOCoV) was taken as index FeNO long-term variability.

RESULTS

In COPD there were no significant differences between NIOX MINO and NOA FeNO values recorded at baseline and one month later. 95% limits of agreement between NIOX MINO and NOA were -2.7 and 1.9 ppb with significant reliability (r=0.96, p<0.0001). Mean FeNO at baseline was 15.0±9.5 ppb. Over the one year period the overall mean FeNO was 15.5±10.1 ppb. The long term eNOCoV was 33.9±16.4 % (range 8.1-83.1 %), and it was significantly associated with exacerbation rate (r= 0.57, p<0.0001).

CONCLUSION

FeNO electrochemical handheld analyser is feasible in COPD showing good agreement with stationary chemiluminescence analyser. COPD patients exhibit a wide range of FeNO levels and a A c c e p t e d m a n u s c r i p t 3 high variability of FeNO over time, which was positively associated with the number of exacerbations.

Exhaled NO (FeNO) has been proposed as an useful non-invasive marker of lower airways inflammation in asthma [START_REF] Dupont | Prospective evaluation of the validity of exhaled nitric oxide for the diagnosis of asthma[END_REF] and specific guidelines have been developed for standardized measurements [START_REF]ATS/ERS recommendations for standardized procedures for the online and offline measurement of exhaled lower respiratory nitric oxide and nasal nitric oxide, 2005[END_REF]. The most common way of measuring exhaled NO is by chemiluminescence [START_REF] Archer | Measurement of nitric oxide in biological models[END_REF].

Recently, a novel handheld NO analyser based on electrochemical sensor has been developed and it has been tested in healthy subjects and patients with asthma showing an high reproducibility and a good correlation with other methods [START_REF] Alving | Performance of a new hand-held device for exhaled nitric oxide measurement in adults and children[END_REF][START_REF] Hemmingsson | Novel hand-held device for exhaled nitric oxide-analysis in research and clinical applications[END_REF][START_REF] Gill | Validation study of fractional exhaled nitric oxide measurements using a handheld monitoring device[END_REF][START_REF] Mcgill | Validation of a hand-held exhaled nitric oxide analyzer for use in children[END_REF][START_REF] Menzies | Portable exhaled nitric oxide measurement: Comparison with the "gold standard" technique[END_REF][START_REF] Khalili | Reliability of a new hand-held device for the measurement of exhaled nitric oxide[END_REF][START_REF] Maniscalco | Validation study of nasal nitric oxide measurements using a hand-held electrochemical analyser[END_REF].

Differently from asthma, studies of exhaled NO in patients with COPD are somewhat conflicting [START_REF] Torok | Bronchial hyper-responsiveness and exhaled nitric oxide in chronic obstructive pulmonary disease[END_REF][START_REF] Agusti | Serial measurements of exhaled nitric oxide during exacerbations of chronic obstructive pulmonary disease[END_REF]. Interestingly, FeNO levels have been reported to increase significantly in a group of COPD patients during cold weather and at the onset of exacerbation [START_REF] Bhowmik | Effects of exacerbations and seasonality on exhaled nitric oxide in COPD[END_REF], suggesting that the monitoring of FeNO variation in COPD might have clinical implications [START_REF] Montuschi | Exhaled carbon monoxide and nitric oxide in COPD[END_REF]. Furthermore, a very recent statement has suggested FeNO as potential outcome in COPD [START_REF] Cazzola | Outcomes for COPD pharmacological trials: from lung function to biomarkers[END_REF].

While in healthy people short and long term FeNO variations have been evaluated [START_REF] Ekroos | Exhaled nitric oxide and its long-term variation in healthy non-smoking subjects[END_REF][START_REF] Ekroos | Short-term variability of exhaled nitric oxide in young male patients with mild asthma and in healthy subjects[END_REF], the feasibility of FeNO monitoring in COPD is still unknown.

In this study we aimed at validating the feasibility of a hand-held FeNO analyzer in COPD patients and at examining the short and long-term variability of exhaled NO in COPD outpatients.

A c c e p t e d

m a n u s c r i p t 6

MATERIALS AND METHODS

Patients

The study was conducted between November 2005 and November 2006 on a cohort of COPD patients visiting our Department as outpatients. They have received COPD diagnosis in the past according to Global Obstructive Lung Disease (GOLD) guidelines [START_REF] Rabe | Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease: GOLD executive summary[END_REF]. Patients with a history of atopy or bronchial asthma, severe co-morbidities (heart and/or liver and/or kidney diseases, recent (< 2 weeks) upper airway infection or ongoing acute exacerbations at first visit were excluded.

All patients gave the informed consent and the study protocol was approved by the Ethics Committee of the Monaldi Hospital.

FeNO measurement validation

We compared the electrochemical FeNO device (NIOX MINO, Aerocrine, Sweden) to the chemiluminescence analyser (NOA, Sensormedics, Italy). Twenty subjects, including 10 COPD patients and 10 age-matched healthy controls were examined (HC). All COPD patients did not show clinical or spirometric features of present or past (at least 4 weeks before) pulmonary exacerbation event. HC subjects were all not smokers, with not history of atopy or bronchial asthma or recent upper airway infection.

All participants were invited to perform the FeNO measurement with both analysers, exhaling for 10 seconds with a constant expiration flow (50 ml/sec) against a mean of 15 cmH 2 O mouth pressure. Standard FeNO was measured before any forced expiratory maneuvers, according to current guidelines [START_REF]ATS/ERS recommendations for standardized procedures for the online and offline measurement of exhaled lower respiratory nitric oxide and nasal nitric oxide, 2005[END_REF]. Maneuvers not resulting in a FeNO plateau or those with irregular pressure tracings were excluded. The recorded ambient NO concentration was always less than 5 ppb.

The FeNO measurements with the handheld analyser were obtained following the manufacturer's instructions. Subjects performed a 10-second slow steady exhalation, which was assisted by visual and audio biofeedback systems located on the device. The mean of the measurements in each device, or the first approved measurement in the NIOX MINO was used for agreement studies [START_REF] Alving | Performance of a new hand-held device for exhaled nitric oxide measurement in adults and children[END_REF]. Moreover, to assess comparatively short term variability of FeNO measurement with two devices in COPD patients and in controls, the FeNO measurements with NIOX MINO and with NOA analyzers were obtained on two different occasions (at the first visit and after one month). On both occasions NIOX MINO measurements were acquired using six and ten seconds schedules of expiratory time.

Long term FeNO variability

FeNO levels, spirometry, pulsoxyemoglobin saturation and clinical status including previous exacerbation were assessed over 1 year in 70 COPD patients (table 1).

Clinical evaluation was based on a score of breathlessness, sputum, cough and night awakenings (BSCN) rated on a scale from 0 to 4 [START_REF] Calverley | Relationship between respiratory symptoms and medical treatment in exacerbations of COPD[END_REF] being calculated as the mean value of the last three days.

We also collected relevant clinical data including drug utilization. The numbers of exacerbation during the previous month before the visit were also collected. Accordingly, event-based exacerbation was defined, using data recorded monthly, as the use of oral corticosteroids and/or antibiotics and/or hospitalization for a worsening in the patient's respiratory symptoms at the discretion of their usual physician. Furthermore, recent exacerbation was defined if the eventexacerbation occurred within the week before or the week after the visit.

The FeNO monthly intra-subject variability, as individual coefficient of variability (CoV) percent, was retrospectly assessed by calculating the CoV (mean/SD) x (100).

At each visit spirometry (MasterScreen PFT, Jaeger, Sensormedics, Italy) and oxyhemoglobin saturation (Nonin, Palmsat 2500) were measured.

Validation of the handheld NO analyzer

Eighty FeNO measurements were taken with both analysers in 10 HC and in 10 COPD patients.

The FeNO range was 5 -25 ppb with the NIOX MINO device and 4.7 -20.9 ppb with the NOA device. The mean values for the NIOX MINO and NOA were 14.8 ± 5.7 ppb and 14.2 ± 5.9 ppb (total readings = 80) respectively with a significant reliability (r=0.96, p<0.0001) when comparing the individual mean values in two devices (Figure 1a).

The Bland-Altman plot showed good agreement between NIOX MINO and NOA when comparing the mean of two exhaled NO measurements (mean value = -0.4 ppb, 95 % limits of agreement -2.7 and 1.9 ppb, (figure 1b). Paired samples t-test confirmed the high reliability of two devices (p=0.002) with a mean difference of 0.6 ppb with a significant correlation (r= 0.97, p<0.0001).The mean of the intra-subjects FeNO difference was 0.21 (range -4.1 to + 2.1) suggesting that NIOX MINO FeNO measurements were generally slightly higher than NOA FeNO readings. A good degree of agreement (95 % degree limits -2.8 and 3.3 ppb) was showed when comparing the mean of two approved exhaled NO measurements in the NOA and the first approved measurement in the NIOX MINO in the clinical setting.

There were no significant differences in COPD patients between NIOX MINO and NOA FeNO values recorded at baseline (15.3 ± 6.4 ppb and 15.0 ± 6.3 ppb respectively, p=0.9) and one month later (16.9 ± 5.9 ppb and 15.9 ± 6.9 ppb respectively, p=0.7).

A good correlation degree was showed between FeNO NIOX MINO 10 seconds and 6 expiratory time schedules when considering among overall group (14.8 ± 5.7 ppb and 14.4 ± 5.9 ppb respectively, p<0.0001, r = 0.92) and both groups separately (13.5 ± 5.0 ppb and 13.2 ± 5.9 ppb in HC, p<0.0001 r=0.91, and 16.0 ± 6.1 ppb and 15.5 ± 6.4 ppb in COPD, p<0.0001 r=0.92).

However, the intra-individual CoV between the two monthly sessions was 6.5±5.1% in HC and 26.3±30.2% in COPD patients (p=0.0082).

A c c e p t e d m a n u s c r i p t 10 4.2 Long term FeNO variability in COPD

Eleven patients (10M/1F) did not attend at least three visits (4 patients) or did not manage to perform a correct expiratory manouvre (2 patients with no adherence with device, 5 patients with FEV1= 0.6 L and they were not included in the data analysis).

The clinical characteristics of remaining COPD patients included in the data analysis are reported in the table 1. The FeNO validation COPD group did not significantly differ in terms of smoking habit, drug usage and FeNO levels in comparison to the long-term cohort.

Baseline (first visit) mean

Over the one year period of the study mean FeNO measured from a total of 426 visits was 15.5 ± 10.1 ppb.

In COPD patients mean CoV FeNO was 34.4 ± 17.9 % (95 % CI 22.3-39.5) and it was significantly higher than in HC (6.2 ± 5.1 %, 95 % CI 3.8-8.6) (p<0.0001).

Both FeNO concentration and FeNO CoV were unrelated to BSCN, oxygen saturation and FEV 1 .

There was a significant correlation between individual exacerbation rates and FeNO CoV (r=0.57, p<0.0001) (figure 2). Moreover, the COPD patients with eNO CoV -fold increase in exacerbation rate as compared to the COPD with eNO CoV less than 40% (table 2), with the highest FeNO values close to the exacerbation (figure 3).

There was not significant difference between smoking habit between the two groups (p=0.61, data shown in table 2).

There were not significant differences between two COPD groups in inhaled corticosteroids and long acting beta2 agonist consumption. In order to validate the reproducibility and the reliability of the electrochemical handheld device in COPD patients, we have evaluated the NIOX MINO measurements against the gold standard chemiluminescence method, finding a good agreement between the two methods.

Previous studies have shown NIOX MINO reproducibility and accuracy in healthy people at different ages and in asthma [START_REF] Alving | Performance of a new hand-held device for exhaled nitric oxide measurement in adults and children[END_REF][START_REF] Hemmingsson | Novel hand-held device for exhaled nitric oxide-analysis in research and clinical applications[END_REF][START_REF] Mcgill | Validation of a hand-held exhaled nitric oxide analyzer for use in children[END_REF][START_REF] Menzies | Portable exhaled nitric oxide measurement: Comparison with the "gold standard" technique[END_REF][START_REF] Khalili | Reliability of a new hand-held device for the measurement of exhaled nitric oxide[END_REF], but no data on COPD patients are available.

In our study the upper and lower specifications on the Altman-Bland plots comparing data from the two analyzers equate to almost 2 ppb on the arithmetic scale. Given that the differences between values for HC and COPD patients were not statistically significant at the conventional flow rate of 50 mL/s, this degree of accuracy appeared to be acceptable. Moreover, the median of the intrasubjects FeNO difference suggested that FeNO measurements by NIOX MINO were generally slightly higher than NOA FeNO readings, similar to data obtained from previous studies [START_REF] Alving | Performance of a new hand-held device for exhaled nitric oxide measurement in adults and children[END_REF][START_REF] Menzies | Portable exhaled nitric oxide measurement: Comparison with the "gold standard" technique[END_REF].

The use of handheld analyzer at standard 50ml/sec flow rate allowed for easy monitoring of wide ranges of FeNO in outpatients COPD, which could be of interest when comparing the measurements in healthy people and patients with asthma.

The comparison between analyzers gave similar results after one month and when the handheld analyzer was used employing different expiratory flow times from six to ten seconds. We used these different expiratory times for two reasons: as the handheld analyzer does not allow the visual inspection of expiratory curve, abnormalities in the plateau phase leading to an increase in FeNO should be unmasked decreasing expiratory time. Furthermore, six second expiratory time might be a valid option for COPD patients with very low FEV 1 . For these reasons our data indicate that COPD patients (with FEV 1 greater than 0.85 L/sec) could handle the device using a 6 seconds time expiratory schedule as patients with almost normal lung function use the 10 seconds schedule.

Accordingly, previous studies concerning the comparability of different types of chemiluminescence NO analyzers have shown that significant differences could exist but they were mainly related to differences in gas calibration/procedure. Our data suggest that FeNO method of Levels of FeNO in COPD patients, at date, are not univocally determined and their relationship with disease exacerbation is not fully clarified.

Several studies have documented that FeNO levels during acute exacerbations were significantly higher than during a stable phase of COPD as Browmik et al. in a large cohort study have demonstrate to obtain paired stable and exacerbation FeNO measurements from 38 patients [START_REF] Bhowmik | Effects of exacerbations and seasonality on exhaled nitric oxide in COPD[END_REF].

Accordingly, Ekroos and coworkers have evaluated a change in FeNO by 30-35% or more within a 1-3 weeks interval as being abnormal and proposed this value as cut-off for a significant fluctuation in relation to FeNO in healthy subjects [START_REF] Ekroos | Exhaled nitric oxide and its long-term variation in healthy non-smoking subjects[END_REF]. In our study the cut-off in exhaled nitric oxide variation > 40% was chosen as the upper limit of 95 % of CI of the mean of the overall COPD group (39.5%). In this way, a cut-off in FeNO variation > 40-50% when using a handheld A limit of our study was that we did not analyze cause-effect relationship between FeNO variation and rate of exacerbation in COPD as the aim of our study was to examine the long term FeNO variability in stable COPD. Therefore we have not data to investigate whether the temporal relationship between exacerbation and FeNO elevations could reflect an early rise in FeNO prior to disease exacerbation.

In conclusion in COPD exhaled NO measurement with an electrochemical handheld analyser shows good agreement with the standard stationary chemiluminescence analyser. In outpatients COPD FeNO shows a greater variation than reported in healthy people, with a significant association to the rate of exacerbation. Although the underlying mechanisms of these FeNO fluctuations in apparently

A c c e p t e d m a n u s c r i p t 7 Using

 7 both the stationary and the electrochemical analysers all participants repeated the manoeuvre until two acceptable tests were performed.

A c c e p t e d m a n u s c r i p t 8 2. 4 Statistic.

 84 Data are presented as arithmetic mean ± standard deviation (SD). For comparison between analysers, Bland-Altman and scatter plots were constructed.Student's paired t-test was used to compare the mean number of measurements in the two devices.Long term FeNO data and CoV are presented as mean with 95 % CI. Comparison between groups was evaluated using parametric tests (paired and unpaired t-test and Pearson correlation test)

  FeNO in COPD was 15.0 ± 9.5 ppb, and it was not significantly different from HC (13.5 ± 5.0 ppb, p=0.60). The subgroup of COPD current smokers (n=12), showed significantly lower FeNO than COPD ex smoker (12.6 ±7.3 ppb vs 18.1 ± 11.0 ppb, p<0.0001).

A c c e p t e d m a n u s c r i p t 13 analysis

 13 does not significantly influence the results of measurements. The intra-individual reliability of NIOX MINO measurements obtained after one month when comparing the measurements performed both with stationary and handheld analyzer in healthy controls and in COPD showed a good agreement. During the validation study in COPD group there were four patients who presented a high FeNO levels variability between two months, with no apparently differences in clinical or spirometric data with other COPD subjects. On these bases we have designed a long-term study to evaluate the variability of FeNO in COPD patient, monitoring the impact of multiple factors on the disease natural history.Differences in FeNO between COPD and healthy controls have been reported with conflicting results postulating a role for airway inflammation, as NO levels are usually reported only slightly increased, except during exacerbation.Our data indicate that COPD patients have a greater intra-individual FeNO variation over time during relatively stable clinical conditions despite mean FeNO values comparable to those obtained from healthy subjects. The variation in FeNO was greater in COPD patients compared to controls during the long-term follow-up over one year. Moreover, this intra-individual FeNO variability was positively associated with the number of exacerbations during the study period.

A c c e p t e d m a n u s c r i p t 14 analyser

 14 seems to be reasonable as an index of previous mild to moderate exacerbation. Actually, this FeNO CoV was not influenced by the baseline FeNO, number of visits, GOLD stage, while a significant correlation with the rate of exacerbations was found. It could be reasonable that all the variability in FeNO may reflect disease exacerbation. However, we cannot exclude other possible causes of larger FeNO variation in stable COPD such as airway persistent inflammation outside of the clinical overt exacerbation. Indeed cell sputum composition, abnormal mechanics, oxidative stress and alveolar inflammation are all factors potentially involved in changes over the time of airway NO output in apparently stable COPD[START_REF] Ricciardolo | Multiple roles of nitric oxide in the airways[END_REF]. Accordingly, in another study on FeNO long term variability in lung transplant recipients Antus et al. [21] have reported that FeNO variation grater than 30% was a specific marker of acute infection during a two years period of follow-up. On the other hand, our data on long term eNO variability in COPD differ from what reported in asthma, where inhaled corticosteroid treatment was the main factor responsible for decrease of exhaled NO long term variation [22]. Noteworthy the differences in FeNO variation were not influenced by smoking habit. This may depend on the limited number of smokers included in the study; however, according to previous reports [23, 24] in our study COPD current smokers had significant lower FeNO levels measured by hand-held analyzer than COPD ex smokers.

A c c e p t e d m a n u s c r i p t A c c e p t e d m a n u s c r i p t 20

 20 Starnes JD, 3rd, Shearer M, Kennedy R, Way A, Arif A, Prabhakar S: Exhaled nitric oxide in asthma: variability, relation to asthma severity, and peripheral blood lymphocyte cytokine expression. J Asthma 2006, 43(2):95-99.23.Corradi M, Majori M, Cacciani GC, Consigli GF, de'Munari E, Pesci A: Increased exhaled nitric oxide in patients with stable chronic obstructive pulmonary disease. Thorax 1999, 54(7):572-575. 24. Sterk PJ, De Gouw HW, Ricciardolo FL, Rabe KF: Exhaled nitric oxide in COPD: glancing through a smoke screen. Thorax 1999, 54(7):565-567.

Figure 1 .a)

 1 Figure 1.

Figure 2 .

 2 Figure 2.

Figure 3 .A c c e p t e d m a n u s c r i p t

 3 Figure 3.

Figure A c c e p t e d m a n u s c r i p tFigureA c c e p t e d m a n u s c r i p tFigure

  Figure

Table 1 .

 1 Overall COPD patients characteristics at baseline. Data are represented as mean ± SD

	Male/Female	52/7
	Age (years)	66.3 ± 8.6
	Current Smokers number (pk/year)	12 (51.4 ± 19.3)
	FEV 1 (L)	1.4 ± 0.5
	GOLD stage (score)	2.6 ± 0.9
	BMI (kg /m 2 )	27.1 ± 5.5
	FeNO (ppb)	15.0 ± 9.6
	Visit rate (number)	7.2
	BSCN (score)	5.5 ± 1.7
	SaO2 (%)	92.1 ± 3.8

Table 2 .

 2 Anthropometrics, pulmonary function parameters (FEV 1 , GOLD stage), exhaled nitric oxide levels (FeNO) at the first visit and analysis results of monthly intra-subject variability, as individual variability coefficient (CoV) percent, of clinical and functional parameters in a cohort of 59 COPD outpatients during one year of study.

	eNO CoV	eNO CoV < 40 %
		p
	(n =17)	(n = 42)

stable COPD patients requires further studies, FeNO measurement with a handheld analyzer is feasible for FeNO monitoring in COPD.

ACKOWLEDGEMENTS

We would like to thank Aerocrine AB for providing the NIOX MINO analyzer and sensors.


Eva Millqvist
email: eva.millqvist@medfak.gu.se

Ewa Ternesten-Hasséus

Mats Bende

Inhaled ethanol potentiates the cough response to capsaicin in patients with airway sensory hyperreactivity

Keywords: capsaicin, ethanol, chemical sensitivity, cough, human, sensory hyperreactivity

A suggested explanation for airway symptoms induced by chemicals and scents is sensory hyperreactivity (SHR) of airway mucosal nerves. Patients with SHR have increased cough sensitivity to inhaled capsaicin, mediated by transient receptor potential (TRP) ion channels.

In animal experiments, some TRP receptors are potentiated by ethanol, which is why in this study, the aim was to evaluate whether a pre-inhalation of ethanol could influence the capsaicin cough response in patients with SHR. Fifteen patients with SHR and 15 healthy controls were provoked on three occasions with two concentrations of inhaled capsaicin. Before each capsaicin provocation, a preinhalation of saline or one of two concentrations of ethanol was given in a doubleblind, randomized fashion. The participants reacted in a dose-dependent way with cough on the capsaicin inhalations. Among the patients, but not in the control group, pre-inhalation of ethanol increased the cough response dose-dependently. The results suggest that the pathophysiology of SHR is related to airway mucosal TRP receptors in the sensory nerves. In scented products the combination of ethanol as a solvent and perfume may augment an airway reaction in sensitive individuals.

Upper and lower airway symptoms induced by chemicals and scents are a common problem in society, and sometimes the experienced symptoms are excessive, leading individuals to seek health care [START_REF] Caress | A review of a two-phase population study of multiple chemical sensitivities[END_REF][START_REF] Kipen | Measuring chemical sensitivity prevalence: a questionnaire for population studies[END_REF]. A suggested explanation for this condition is a hyperreactivity of the sensory nerves of the entire airways. The condition is therefore called "sensory hyperreactivity (SHR)", and it affects more than 6% of the adult population in Sweden [START_REF] Johansson | Relationship between self-reported odor intolerance and sensitivity to inhaled capsaicin: proposed definition of airway sensory hyperreactivity and estimation of its prevalence[END_REF]. Common symptoms are nasal blockage, rhinorrhea, eye irritation, heavy breathing, cough, hoarseness, and phlegm, as well as general symptoms, such as headache and nausea [START_REF] Johansson | Capsaicin inhalation test for identification of sensory hyperreactivity[END_REF][START_REF] Ternesten-Hasseus | Quality of life and capsaicin sensitivity in patients with airway symptoms induced by chemicals and scents: a longitudinal study[END_REF]. After capsaicin inhalation, changed levels of nerve growth factor in nasal secretions have been detected, indicating a neurochemical imbalance in the airways, which is related to SHR [START_REF] Millqvist | Changes in levels of nerve growth factor in nasal secretions after capsaicin inhalation in patients with airway symptoms from scents and chemicals[END_REF]. Cough sensitivity to capsaicin is a measure of airway sensory reactivity [START_REF] Karlsson | Afferent neural pathways in cough and reflex bronchoconstriction[END_REF][START_REF] Midgren | Capsaicin-induced cough in humans[END_REF]. Patients with SHR react more strongly than healthy individuals to provocation with inhaled capsaicin, and the cutoff values for a positive reaction have been determined [START_REF] Johansson | Capsaicin inhalation test for identification of sensory hyperreactivity[END_REF]. The capsaicin inhalation test in the diagnosis of SHR has good short-term and long-term reproducibility [START_REF] Ternesten-Hasseus | Quality of life and capsaicin sensitivity in patients with airway symptoms induced by chemicals and scents: a longitudinal study[END_REF][START_REF] Millqvist | Sensory hyperreactivity -a possible mechanism underlying cough and asthma-like symptoms[END_REF].

Capsaicin, the main, pungent ingredient in chili, stimulates the unmyelinated C-fibers of the sensory nervous system, giving a burning sensation by activating the ion channel of transient receptor potential vanilloid subunit 1 (TRPV1) [START_REF] Caterina | The capsaicin receptor: a heat-activated ion channel in the pain pathway[END_REF]. This receptor is also activated by other stimuli, including noxious heat, lipids, and protons [START_REF] Cortright | Biochemical pharmacology of the vanilloid receptor TRPV1. An update[END_REF][START_REF] Pingle | Capsaicin receptor: TRPV1 a promiscuous TRP channel[END_REF].

An increased expression of TRPV1 is found in several conditions characterized by general hypersensitivity, such as thermal hyperagesia, vulvodynia, rectal hypersensitivity, esophagitis, and chronic cough [START_REF] Pogatzki-Zahn | Heat hyperalgesia after incision requires TRPV1 and is distinct from pure inflammatory pain[END_REF][START_REF] Tympanidis | Increased vanilloid receptor VR1 innervation in vulvodynia[END_REF][START_REF] Chan | Sensory fibres expressing capsaicin receptor TRPV1 in patients with rectal hypersensitivity and faecal urgency[END_REF][START_REF] Mitchell | Expression and characterization of the intracellular vanilloid receptor (TRPV1) in bronchi from patients with chronic cough[END_REF][START_REF] Matthews | Increased capsaicin receptor TRPV1 nerve fibres in the inflamed human oesophagus[END_REF][START_REF] Groneberg | Increased expression of transient receptor potential vanilloid-1 in airway nerves of chronic cough[END_REF]. Recently, it has been found that ethanol potentiates the response of TRPV1 to capsaicin in different organs in rats

Subjects

Fifteen non-smoking patients, 14 women and one man, 23-64 years old (mean age 53), with a history of at least 4 (mean 12) years of upper and lower airway symptoms induced by chemicals and scents, took part in the study. All had increased sensitivity to inhaled capsaicin and reached the cutoff for the diagnosis of SHR [START_REF] Johansson | Capsaicin inhalation test for identification of sensory hyperreactivity[END_REF]. None of the patients demonstrated spirometric reversibility or variability in pulmonary function.

Asthma was excluded by a negative methacholine test, in accordance with international guidelines [START_REF] Sterk | Airway responsiveness. Standardized challenge testing with pharmacological, physical and sensitizing stimuli in adults. Report Working Party Standardization of Lung Function Tests, European Community for Steel and Coal. Official Statement of the European Respiratory Society[END_REF]. All patients tested negative to the skin prick test with a standard panel of ten allergen sources common to Sweden.

The control group consisted of 15 non-smoking individuals, 14 women and one man, 21-62 years of age (mean age 42). None had a history of airway symptoms in response to exercise, scents, chemicals, or allergies, and none was taking any medication for the airways.

Informed consent was obtained from all subjects at the start of the investigation. The study was approved by the Regional Ethical Review Board of Gothenburg, Sweden.

Study design

Each participant visited the clinic on three occasions. A capsaicin inhalation test was performed at each visit. Before each test a pre-inhalation was given in a double-blind and randomized fashion, of either 1 mL saline, or 1 mL of 5% or 25% ethanol. The test was initiated with a pre-inhalation of either 1 mL saline, or 1 mL ethanol (5% or 25%) for 6 minutes, followed by 4 minutes of rest, as previously described [START_REF] Johansson | Relationship between self-reported odor intolerance and sensitivity to inhaled capsaicin: proposed definition of airway sensory hyperreactivity and estimation of its prevalence[END_REF][START_REF] Ternesten-Hasseus | Quality of life and capsaicin sensitivity in patients with airway symptoms induced by chemicals and scents: a longitudinal study[END_REF]. Thereafter, the subject was provoked in the same manner with two concentrations of capsaicin, namely, first 0.4 and then 2.0 µmol/L, in a 1 mL solution.

Each dose of 1 mL was inhaled through a mouthpiece without a nose clip by tidal volume breathing to completion, or for a maximum of 6 min, followed by 4 min of rest. The total number of evoked coughs was counted for 10 minutes from the onset of each inhalation. Forced expiratory volume in 1 second (FEV 1 ) was measured using a spirometer (Vitalograph, Buckingham, UK) before and after each provocation for evaluation of any bronchial obstruction.

Statistics

The Mann-Whitney U-test was used for non-paired data, and the Wilcoxon's signedrank test for paired data. Data are presented as means with 95% confidence intervals (CIs). Results were considered significant at p<0.05. All data were analyzed using StatView 5.0.1.0 (SAS Institute, Inc., Cary, NC, USA).

A c c e p t e d m a n u s c r i p t

Results

At each provocation, the patients and controls coughed dose-dependently as an effect of the capsaicin inhalations. The number of coughs after 0.4 and 2.0 µmol/L capsaicin differed significantly (Figure 1), with a probability value of <0.001 for patients and <0.05 for controls. The basic mean FEV 1 value was 101% of predicted value (95% CI 93-109) among the patients and 100% of predicted value (95% CI 94-105) among the controls and did not change significantly after any of the provocations (data not shown).

The effect of ethanol on the capsaicin inhalation test

Pre-inhalation of ethanol increased dose-dependently the effect of capsaicin (2.0 µmol/L) in the patients (Figure 2). After the use of saline as pre-inhalation, the mean number of cough on 2.0 µmol/L capsaicin was 38 (95% CI, 26 to 50), after 5% ethanol 47 (95% CI, 31 to 63) and after 25% ethanol 62 (95% CI, 40 to 83). The increase induced by 5% and 25% ethanol was statistically significant, p<0.05 and p<0.01, respectively. No such effect of ethanol was seen in the control group, where after pre-inhalation of saline, the mean number of cough on 2.0 µmol/L capsaicin was 6 (95% CI, 2 to 10), after 5% ethanol 6 (95% CI, 3 to 9) and after 25% ethanol 7 (95% CI, 3 to 11) (Figure 2).

A c c e p t e d m a n u s c r i p t 4. Discussion

In this study, patients with SHR showed to be more cough sensitive to inhaled capsaicin when the provocation was preceded by an inhalation of ethanol. A preinhalation of 5% or 25% ethanol enhanced the cough reaction to capsaicin. A control group without airway symptoms did not have the corresponding reactions and coughed significantly less than the patients in accordance with earlier studies [START_REF] Johansson | Relationship between self-reported odor intolerance and sensitivity to inhaled capsaicin: proposed definition of airway sensory hyperreactivity and estimation of its prevalence[END_REF][START_REF] Ternesten-Hasseus | Quality of life and capsaicin sensitivity in patients with airway symptoms induced by chemicals and scents: a longitudinal study[END_REF][START_REF] Ternesten-Hasseus | Inhalation method determines outcome of capsaicin inhalation in patients with chronic cough due to sensory hyperreactivity[END_REF].

Since there were no changes of FEV 1 after any provocation the cough reactions can not be regarded as due to bronchoconstriction and asthma.

During the last years there has been an increasing interest for the family of transient receptor (TRP) ion channels. They are in important by means for multiple organ systems to interact with their environment [START_REF] Kim | Sensing the air around us: the voltage-gated-like ion channel family[END_REF][START_REF] Montell | The TRP superfamily of cation channels[END_REF]. They are able to sense temperature, stretch, osmolarity and pain, among others and may be involved in different diseases by an increased level of channel expression [START_REF] Montell | The TRP superfamily of cation channels[END_REF][START_REF] Nilius | Transient receptor potential cation channels in disease[END_REF]. The TRPV1, belonging to one of the six subfamilies of TRP ion channels, is not only activated by capsaicin but also by noxious stimuli and heat, and potentiated by extracellular acidic pH, and interacts with vanilloid-like compounds [START_REF] Caterina | Sense and specificity: a molecular identity for nociceptors[END_REF][START_REF] Caterina | The vanilloid receptor: a molecular gateway to the pain pathway[END_REF][START_REF] Tominaga | Capsaicin receptor in the pain pathway[END_REF]. Patients with chronic cough have shown a five-folded increase of TRPV1-staining nerve profiles and, furthermore, a significant correlation between capsaicin response and the number of TRPV1positive nerves [START_REF] Groneberg | Increased expression of transient receptor potential vanilloid-1 in airway nerves of chronic cough[END_REF]. Hopefully, specific TRPV1 antagonists could be useful in treating disorders such as pain, chronic cough, and irritable bowl syndrome [START_REF] Szallasi | The vanilloid receptor TRPV1: 10 years from channel cloning to antagonist proof-of-concept[END_REF]. Also the ion channel TRPA1 (Transient receptor potential channel ankyrin subunit 1) is activated by a variety of noxious stimuli, including cold temperatures, pungent natural compounds, and environmental irritants [START_REF] Macpherson | Noxious compounds activate TRPA1 ion channels through covalent modification of cysteines[END_REF]. Many "sensory compounds" presumed to be specific have a promiscuous relationship with different TRP channels [START_REF] Macpherson | More than cool: promiscuous relationships of menthol and other sensory compounds[END_REF].

A c c e p t e d m a n u s c r i p t 10

The present outcome also agrees with earlier findings in animals. In biopsies from rat spinal cord, oesophagus and skin, ethanol potentiated the response of TRPV1 to capsaicin and protons and lowered the threshold for heat activation of TRPV1 [START_REF] Trevisani | Ethanol causes inflammation in the airways by a neurogenic and TRPV1-dependent mechanism[END_REF][START_REF] Trevisani | Ethanol elicits and potentiates nociceptor responses via the vanilloid receptor-1[END_REF].

In guinea pig airways, in vitro, ethanol stimulated peptidergic primary sensory neurons and caused TRPV1-dependant inflammation which was interpreted as a possible proof of the role of the TRPV1 in asthma [START_REF] Trevisani | Ethanol causes inflammation in the airways by a neurogenic and TRPV1-dependent mechanism[END_REF]. Although it has been proven in many studies that inhaled capsaicin causes powerful bronchoconstriction in guinea pigs [START_REF] Forsberg | Cough and bronchoconstriction mediated by capsaicin-sensitive sensory neurons in the guinea-pig[END_REF][START_REF] Kroll | Capsaicin-induced bronchoconstriction and neuropeptide release in guinea pig perfused lungs[END_REF] this connection is not that evident in humans [START_REF] Doherty | Capsaicin responsiveness and cough in asthma and chronic obstructive pulmonary disease[END_REF][START_REF] Fujimura | Airway cough sensitivity to inhaled capsaicin and bronchial responsiveness to methacholine in asthmatic and bronchitic subjects[END_REF][START_REF] Millqvist | Relationship of airway symptoms from chemicals to capsaicin cough sensitivity in atopic subjects[END_REF][START_REF] Ternesten-Hasseus | Sensitivity to methacholine and capsaicin in patients with unclear respiratory symptoms[END_REF]. However, there seems to be a similar pattern in different organ systems and in different species that ethanol enhances stimuli with a potential to be irritating but to our knowledge this has The tidal breathing method of capsaicin challenge, used in the present study, was developed for SHR and has been modified with time and has shown good reproducibility [START_REF] Ternesten-Hasseus | Quality of life and capsaicin sensitivity in patients with airway symptoms induced by chemicals and scents: a longitudinal study[END_REF][START_REF] Millqvist | Sensory hyperreactivity -a possible mechanism underlying cough and asthma-like symptoms[END_REF][START_REF] Ternesten-Hasseus | Inhalation method determines outcome of capsaicin inhalation in patients with chronic cough due to sensory hyperreactivity[END_REF][START_REF] Millqvist | Cough provocation with capsaicin is an objective way to test sensory hyperreactivity in patients with asthma-like symptoms[END_REF]. The testing of cough sensitivity with capsaicin inhalation is otherwise often performed with a "single-breath" technique using increasing concentrations of capsaicin until the individual coughs, for example, two or five times (C2 or C5), which has shown a good reproducibility [START_REF] Prudon | Cough and glottic-stop reflex sensitivity in health and disease[END_REF][START_REF] Morice | ERS guidelines on the assessment of cough[END_REF]. Others, who used both techniques, found that the tidal breathing method with a fixed time and concentration

A c c e p t e d m a n u s c r i p t

showed good reproducibility and dose-response relationship in the short term, as well as in the long term [START_REF] Midgren | Capsaicin-induced cough in humans[END_REF][START_REF] Hansson | Effects of inhaled lignocaine and adrenaline on capsaicin-induced cough in humans[END_REF]. The single-breath technique was more dependent on a standardized inhalation technique compared to the fixed time and concentration technique [START_REF] Hansson | The human cough reflex in health and disease[END_REF]. One potential problem with using the "single-breath" technique is that patients with SHR have an easily evoked cough reflex, and even inhalation of saline can induce coughing [START_REF] Millqvist | Sensory hyperreactivity -a possible mechanism underlying cough and asthma-like symptoms[END_REF][START_REF] Millqvist | Cough provocation with capsaicin is an objective way to test sensory hyperreactivity in patients with asthma-like symptoms[END_REF]. The occurrence of a tachyphylaxis problem after performing the test with the single-breath technique speaks in favour of the tidal breathing technique used in this study, which has also been discussed by other authors [START_REF] Cho | Elevated substance P levels in nasal lavage fluids from patients with chronic nonproductive cough and increased cough sensitivity to inhaled capsaicin[END_REF].

In conclusion, our present results suggest that the pathophysiology behind SHR is related to TRP receptors of the sensory nerves in the airway mucosa and that patients with SHR differ from healthy controls in that respect.

FEV 1 = 1 A

 11 Abbreviations:

A c c e p t e d m a n u s c r i p t 5 andA c c e p t e d m a n u s c r i p t 6 2.

 56 guinea pigs[START_REF] Trevisani | Ethanol causes inflammation in the airways by a neurogenic and TRPV1-dependent mechanism[END_REF][START_REF] Trevisani | Ethanol elicits and potentiates nociceptor responses via the vanilloid receptor-1[END_REF][START_REF] Nicoletti | Ethanol causes neurogenic vasodilation by TRPV1 activation and CGRP release in the trigeminovascular system of the guinea pig[END_REF][START_REF] Vetter | Mechanisms involved in potentiation of transient receptor potential vanilloid 1 responses by ethanol[END_REF]. If a corresponding effect could be achieved in humans, this would strongly indicate the role of the transient receptor potential (TRP) ion channels in the pathogenesis of SHR. In the future, the TRP ion channels may be an important key to understanding airway symptoms induced by chemicals and scents, our knowledge of which is so far limited. The aim of this study was therefore to evaluate whether pre-inhalation of ethanol could influence the capsaicin response in patients with SHR. Materials and Methods

A c c e p t e d m a n u s c r i p t 7 2. 3 .

 73 Capsaicin inhalation test A compressor (Pariboy 36; Paulritzau Pari-werk KG, Starnberg-am-See, Germany) and a nebulizer (Pari Inhalerboy, No. 36.75; Paulritzau Pari-werk KG, Starnberg-am-See, Germany) were used for inhalation of aqueous dilutions of the odorless capsaicin solution.

  not earlier been shown in human. In scented products the combination of ethanol as a solvent and perfume may augment an airway reaction in sensitive individuals. The results of the present study indicate an ethanol-mediated modulation of the TRPV1 or TRPA1 function. Still it is unclear whether this effect is specific like that in animal studies or nonspecific, as the fact that the patients coughed even after saline inhalations indicate.

A c c e p t e d m a n u s c r i p t A c c e p t e d m a n u s c r i p t 13 LegendsFigure 1

 131 Figure 1

Figure 2 Mean

 2 Figure 2

Acknowledgements: This study was supported by grants from the Vårdal Foundation, the Regional Health Care Authority of West Sweden, the Swedish Asthma and Allergy Association Foundation and the Swedish Heart and Lung Foundation. We are grateful to Inger Winberg for excellent help with the recordings.