Mario Cazzola
email: mario.cazzola@uniroma2.it

I Floriani

C P Page

The therapeutic efficacy of erdosteine in the treatment of chronic obstructive bronchitis: a meta-analysis of individual patient data

Keywords: Meta-analysis, chronic bronchitis, COPD, acute exacerbation, cough, expectoration

Erdosteine, a drug approved for the treatment of acute and chronic pulmonary diseases, has been shown to be an effective treatment for chronic bronchitis or COPD (CB/COPD) in several studies, although marked differences in the perception of its usefulness still remain.

Aim: to test the available evidence for the efficacy of erdosteine in adults with stable or exacerbated CB/COPD. Methods: meta-analysis of individual patient data from both published and unpublished randomised controlled trials (RCTs) comparing erdosteine with placebo/mucolytics, given for up to 10 days in association with standard therapy (RCTs used for regulatory drug approval). Individual patient data were provided by the manufacturer of erdosteine, Edmond Pharma (Milano, Italy).

Endpoints were symptom scores (cough frequency and intensity, sputum viscosity and purulence, difficulty to expectorate, catarrh rhonchi at auscultation, dyspnoea), a cumulative global efficacy index (cGEI), and an overall physician efficacy assessment (OA).

Results: individual data from 1046 patients from 15 RCTs (12 on exacerbated and 3 on stable CB/COPD) were obtained. Erdosteine induced a significant reduction of cGEI vs. comparators (-1.02; 95% CI: from -1.60 to -0.44; p = 0.0006), both placebo and mucolytics. On individual symptoms, it positively impacted on cough frequency (-0.19; 95% CI: from -0.34 to -0.03) and intensity (-0.30; 95%CI: from -0.44 to -0.17), sputum viscosity (-0.28; 95%CI: from -0.49 to -0.07), difficulty to expectorate (-0.24; 95%CI: from -0.40 to -0.08), and catarrh ronchi at auscultation (-0.35; 95%CI: from -0.60 to -0.10). The effects on dyspnoea were only significant vs. placebo, whereas sputum purulence was not significantly modified. The OA also favoured erdosteine, doubling the chance of success compared with placebo and mucolytics: OR (odds ratio) 2.06; (95%CI: from 1.27 to 3.33). The treatment with erdosteine was well tolerated. Adverse events, mainly gastrointestinal, were reported by 10.2% of patients compared to 11.0% in the reference groups.

Introduction

Chronic bronchitis (CB) is very often associated with airflow obstruction and is especially frequent in smokers, is considered to contribute to the airway mucus hypersecretory component of Chronic Obstructive Pulmonary Disease (COPD) [START_REF] Iqbal | Worldwide guidelines for chronic obstructive pulmonary disease: a comparison of diagnosis and treatment recommendations[END_REF], and is associated with considerable morbidity and high health-care costs [START_REF] Miravitlles | Costs of chronic bronchitis and COPD. A one year follow-up study[END_REF]. Patients with chronic bronchitis and COPD suffer from recurrent exacerbations, with an increase in volume and/or purulence of sputum, cough and dyspnoea which contribute to progressive clinical deterioration and account for a significant proportion of the cost of caring for such patients [START_REF] Donaldson | The relationship between exacerbation frequency and lung function decline in chronic obstructive pulmonary disease[END_REF][START_REF] Seemungal | Effect of exacerbation on quality of life in patients with chronic obstructive pulmonary disease[END_REF][START_REF] Miravitlles | Pharmacoeconomic evaluation of acute exacerbations of chronic bronchitis and COPD[END_REF].

There is evidence for inflammatory and morphological changes in the airways associated with loss of ciliary function and mucus gland hyperplasia, and the importance of mucus in contributing to airflow limitation and disease progression are underscored by recent studies [START_REF] De Marco | Incidence of chronic obstructive pulmonary disease in a cohort of young adults according to the presence of chronic cough and phlegm[END_REF][START_REF] Brusasco | Airway inflammation in COPD. Friend or Foe ?[END_REF].

The use of mucolytics as adjunctive treatment of both stable and exacerbated CB/COPD has been proposed to improve disease outcome, although the value of the use of such drugs is still considered uncertain [START_REF] Rabe | Global strategy for the diagnosis, management and prevention of Chronic Obstructive Pulmonary Disease: GOLD executive summary[END_REF].

Erdosteine is a a drug approved for the treatment of acute and chronic pulmonary diseases for more than 10 years which has been shown to improve sputum rheology in patients with mucus hypersecretion through an active metabolite (Met-I) having free thiol groups [START_REF] Moretti | Pharmacology and clinical efficacy of erdosteine in chronic obstructive pulmonary disease[END_REF]. Although a few studies have been published showing that CB/COPD patients may benefit from erdosteine, marked differences in the perception of its usefulness still remain.

The aim of the present systematic review is therefore to test the available evidence that erdosteine treatment in patients with CB/COPD may be effective and accompanied by clinically relevant improvements.

Methods

This systematic review was performed in accordance with the Quality of Reporting of meta-analyses (QUORUM) guidelines [START_REF] Moher | Improving the quality of reports of meta-analyses of randomized controlled trials: the QUORUM statement[END_REF].

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Types of studies

Randomized controlled trials (RCTs) focusing on the comparison between erdosteine and placebo or mucolytics which reported data on efficacy and safety after 7-10 days of treatment, were used for this meta-analysis.

Types of patients

Adults patients having a medical history of chronic bronchitis (CB), generally defined as the presence of cough and sputum production for at least three months a year over two consecutive years were included in the studies used in this meta-analysis. The three largest studies also included evidence for airway obstruction, defined as an FEV 1 /FVC ratio at least 10% below the normal theoretical value [11,12,13].

Patients were enrolled either at occurrence of an acute exacerbation or during the stable phase of the disease. The diagnosis of acute exacerbations was based on the occurrence of increased mucopurulent sputum, cough and fever.

Three studies additionally included the isolation of antibiotic-sensitive bacterial strains in sputum [11,14,15]. In two studies the inclusion of patients with hypersecretory acute bronchitis was also allowed [15,16].

Type of intervention

Erdosteine (300 mg capsule) was administered two or three times daily on top of background therapy, generally antibiotics and bronchodilators (beta 2agonists and aminophyllines) in patients with acute exacerbations, and bronchodilators in those with stable disease.

Placebo or mucolytics (ambroxol, N-acetylcysteine, carbocysteine, sobrerol) were administered with the same dosing schedules as erdosteine (i.e. two or three times daily) on top of background treatments.

Type of outcome measures

The following outcomes were investigated: i) cumulative global efficacy index (cGEI), the sum of all assessed respiratory symptom scores, ii) respiratory individual symptom scores (cough frequency and intensity, sputum viscosity and purulence, difficulty to expectorate, catarrh rhonchi at auscultation, dyspnoea), iii) overall assessment of efficacy (OA) by the Investigator, and

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frequency of adverse events. In the original studies, similar scoring systems were used for patient self-assessment of symptoms, usually categorised on a 0-3 scale from 0= absent to 3= worst. In all the studies, safety was evaluated in terms of incidence of adverse events reported during treatment, with particular regard to gastrointestinal complaints.

Study Search

Literature was search systematically for relevant clinical trials with no language restrictions (Pub Med, Google Scholar and Scirus with search terms "chronic bronchitis", "COPD" and "acute exacerbations" combined with "erdosteine").

Furthermore, the manufacturer of erdosteine (Edmond Pharma s.r.l., Italy)

was contacted and asked for any additional non-indexed publications and relevant unpublished studies. Individual patient data from the published and

Data extraction

For each of the selected trials, the following information was retrieved: first author, publication year, details of study design, studied treatments (type of drug, schedule, duration), patient characteristics (total number, age and sex distribution, number randomised and number included in the analysis), study endpoints, occurrence and type of adverse events.

The quality of the selected trials was assessed according to a five-point validated scale [START_REF] Jadad | Assessing the quality of reports of randomized clinical trials: is blinding necessary?[END_REF] measuring a range of factors that impact the quality of a trial: randomization methods, blinding and description of withdrawals and drop-outs. Two independent reviewers assessed the quality of the trials to be included. Differences in the evaluation were resolved by consensus, referring back to the original article/report.

Statistical analysis

Trials were grouped according to the type of erdosteine comparator (active or placebo), study quality (Jadad scale score 1-2 vs. 3-4-5) and whether the study was published or not.

The summary measure for the respiratory individual symptom scores, as well as for the c-GEI, was the difference between changes from baseline and the end of treatment mean values calculated in the two treatment arms.

For comparison of OA of the efficacy of erdosteine versus active or placebo group, events of interest were considered under the categories good, excellent or positive.

Global estimates of the effect of treatments over time on the selected outcomes and the corresponding 95% confidence intervals (CIs) were calculated using the inverse variance method for continuous variables and the Mantel-Haenszel method for dichotomous variables.

For the pooling of the estimates, either the fixed-effect or the randomeffect model were considered, depending on the presence of statistical

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heterogeneity. Statistical heterogeneity was defined as an I 2 statistic value >50% [START_REF] Higgins | Measuring inconsistency in meta-analyses[END_REF]. In order to assess the heterogeneity of the included trials, the Cochrane Q statistic [START_REF] Deeks | Statistical methods for examining heterogeneity and combining results from several studies in metaanalysis[END_REF] was calculated. For p-values <0.10, the assumption of homogeneity was deemed not valid.

Occurrence of adverse events was analysed descriptively.

All statistical analyses were made using SAS statistical software version 9.1 (SAS Institute Inc., Cary, NC, USA) and the software 'REVMAN 4.2'

provided by the Cochrane Collaboration.

Results

Study selection

Thirty-one potentially relevant studies conducted in patients with CB/COPD were retrieved. The study selection process is presented in Fig. 1. Of these, 16 were excluded for the following reasons: 3 because they only evaluated mucus rheology [START_REF] Marchioni | Effects of erdosteine on sputum biochemical and rheologic properties: Pharmacokinetics in chronic obstructive lung disease[END_REF][START_REF] Olivieri | Activity of erdosteine on mucociliary transport in patients affected by chronic bronchitis[END_REF][START_REF] Grandi | A dose effect study of oral erdosteine on rheologic properties of human mucus. Randomised double-blind study with comparison among 3 groups treated with 600 and 900 mg of erdosteine or with placebo[END_REF], 2 because they were dose-range finding studies [START_REF] Siquet | A dose effect study of oral erdosteine on rheological properties of human mucus[END_REF][START_REF] Nagaoka | Early phase II trial of KW-9144 on sputum excretion difficulty[END_REF], 5 because of inadequate design [START_REF] Inclimona | Therapeutic clinical trial with FARMO 83/R: Open design[END_REF][START_REF] Cattini | Efficacy and safety of erdosteine in chronic bronchitis in phase of clinical reactivation: open study[END_REF][START_REF] Deodato | /die per aerosol): open study in the treatment of chronic bronchitis in phase of clinical reactivation[END_REF][START_REF] De Castro Pereira | Efficacy and tolerability of erdosteine in chronic obstructive pulmonary disease[END_REF][START_REF] Rozborilova | Effect of erdosteine in patients suffering from chronic obstructive pulmonary disease[END_REF], 3 because of lack of symptom assessment up to 10 days [START_REF] Fioretti | Prevention of exacerbations in chronic bronchitic patients with erdosteine[END_REF][START_REF] Ghiringhelli | Cross-over clinical study of efficacy and tolerability of erdosteine in the treatment of chronic obstructive bronchial disease in stable hypersecretive phase controlled double-blind study vs placebo[END_REF][START_REF] Moretti | The effect of long-term treatment with erdosteine on chronic obstructive pulmonary disease: The Equalife study[END_REF], 1 because of a different formulation of erdosteine [START_REF] Rossi | ): double-blind study vs. N-acetylcysteine in the treatment of chronic bronchopneumopathy in phase of clinical reactivation[END_REF] and 2 because of insufficient efficacy information [START_REF] Kim | Clinical efficacy of erdosteine in patients with acute or chronic bronchitis: A randomized, double-blind, comparative study vs. ambroxol[END_REF][START_REF] Mohanty | Evaluation of efficacy and safety of erdosteine in patients affected by exacerbation of chronic bronchitis and receiving ciprofloxacin as basic treatment[END_REF]. Therefore, 15 RCTs were included in the final analysis.

Study characteristics

The main characteristics of the 15 selected RCTs which enrolled 1046 adult patients are summarized in Table 1.

Six studies involving 587 patients were randomized, double-blind trials comparing erdosteine vs. placebo, four studies were performed in patients with acute exacerbations of CB/COPD and two in patients with stable chronic obstructive bronchitis. Erdosteine 300 mg or placebo were administered two

Study quality

We characterized the studies according to a set of factors that reflect their methodological rigor. Overall, most studies were considered of good quality in that they were prospective, randomized, double-blind trials. Only 2 [14,16] were not randomized and 3 [16,18,[START_REF] Tellings | Erdosteine: Original secretolytic drug with mucociliar activity. A controlled clinical study versus ambroxol[END_REF] were single-blind studies. When applicable, the reasons for early withdrawal were adequately described.

Table 2 summarizes the results of the study quality assessment.

Patient characteristics

The majority of subjects (70.7%) had a diagnosis of acute exacerbation of CB/COPD with a few cases (3.3%) where features of asthma were also present. Patients with stable COPD disease accounted for 26.3% of the study population, while in a negligible proportion of subjects (2.9%) no unequivocal diagnosis of CB/COPD was reported. Rather a hypersecretory pulmonary disease (either acute bronchitis or pneumonia or restrictive lung disease). In patients with exacerbated disease concomitant antibiotics were administered, and 37% of patients received bronchodilator therapy. Use of corticosteroids was very limited. Smoking history was incompletely recorded, and lung function at inclusion was measured in all but 4 studies (72% of patients).

Information related to the patients included in the eligible RCTs is presented in Table 3.

Outcomes

In the 1046 patients included in the analysis, erdosteine induced a significant reduction of cGEI versus comparators (-1.02; 95% CI: from -1.60 to -0.44; p = 0.0006). A higher effect was observed in comparison to placebo (-1.41; 95% CI: from -2.49 to -0.33; p = 0.01), although the reduction was also significant vs. active comparators (-0.66; 95% CI: from -1.30 to -0.02; p = 0.04) (Fig. 2).

M A N U S C R I P T A C C E P T E D

Data on cough frequency and intensity were available in 13 and 11 RCTs, respectively (972 and 496 participants). Erdosteine positively impacted on cough frequency overall (-0.19; 95% CI: from -0.34 to -0.03; p = 0.02) and achieved significance vs. placebo (-0.23; 95%CI: from -0.47 to 0.00; p = 0.05) (Fig. 3). With regard to cough intensity, the effect of erdosteine was significant overall (-0.30; 95% CI: from -0.44 to -0.17; p < 0.00001), vs.

placebo (-0.42; 95% CI: from -0.75 to -0.08; p = 0.01) and mucolytics (- 95% CI: from -0.60 to -0.10; p = 0.006), while the effects on dyspnoea were only significant vs. placebo (-0.17; 95% CI: from -0.30 to -0.05).

0
Outcome data related to individual respiratory symptom scores are shown in Table 4.

Data regarding treatment success, defined as physician OA equal to good/excellent/positive, were reported in 13 RCTs. The efficacy of treatment was considered as good/excellent/positive in 297 of 472 patients treated with erdosteine and in 239 of 465 patients treated with comparators.

Erdosteine provided a double chance of treatment success compared with placebo and mucolytics (OR 2.06; 95% CI: from 1.27 to 3.33 overall) (Fig. 5).

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Statistical analyses did not suggest potential bias either for study quality and for whether a study was published or not (data not shown).

Safety profile

All treatments were well tolerated. Adverse events were reported by 54 patients (10.2%) in the erdosteine group and in 57 patients (11.0%) in the reference groups. The most frequently occurring adverse events were gastrointestinal complaints, in particular nausea, epigastric pain or heartburn, diarrhoea or loose stool. Only one patient treated with erdosteine reported taste loss, and allergic reactions occurred in 3 patients treated with erdosteine and in 3 with reference treatments. A summary of reported adverse events is presented in Table 5.

Discussion

The present systematic review on erdosteine efficacy has been conducted on When considering the two studies conducted in patients with stable CB/COPD [12,[START_REF] Voisin | Controlled, double-blind, pilot study comparing erdosteine at the daily dose of 600 mg to placebo in two groups of patients affected by stabilized chronic obstructive bronchitis[END_REF], the benefit provided by erdosteine on cough and sputum scores was less evident, presumably due to the short duration of treatment in these studies. It should be noted however, that in another study, not considered for the present review, a more prolonged treatment of 28 days significantly improved chronic symptoms of cough and sputum in stable CB patients [START_REF] Ghiringhelli | Cross-over clinical study of efficacy and tolerability of erdosteine in the treatment of chronic obstructive bronchial disease in stable hypersecretive phase controlled double-blind study vs placebo[END_REF]. In the comparative studies vs. placebo, a significant effect on dyspnoea was also shown, suggesting that a facilitated mucus clearance with erdosteine has the potential to translate into improved quality of life for patients with CB. On the other hand, erdosteine was no more active on sputum purulence scores compared to antibiotic monotherapy, a finding that is perhaps not surprising given the major efficacy of antibiotics on this feature of exacerbations, rather than mucolytics.

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13

These results underline the potential relevance of interventions focused on mucus clearance for the treatment of CB and COPD. While smoking cessation, bronchodilators, glucocorticosteroids and antibiotics (especially for acute exacerbation of CB) provide an effective armamentarium for the treatment of the symptomatic and airflow abnormalities of CB/COPD in all phases of the disease, the mucus component of airway obstruction has generally received less attention than other reversible compounds of the condition [START_REF] Rabe | Global strategy for the diagnosis, management and prevention of Chronic Obstructive Pulmonary Disease: GOLD executive summary[END_REF]. Nonetheless, if mucus can be effectively cleared, both symptoms and airflow may be relevantly improved, given that sputum is also a reversible component of the disease. In this respect, recent studies have raised renewed interest in the relevance of mucus in airway diseases and the role of mucolytic-expectorant treatments as an additional therapeutic strategy in the treatment of CB/COPD [START_REF] Brusasco | Airway inflammation in COPD. Friend or Foe ?[END_REF].

The present findings indicate that erdosteine plus antibiotics is more effective than antibiotic monotherapy in patients with CB/COPD, especially for the treatment of acute infective exacerbations, and support the addition of erdosteine to the usual treatment in order to provide further resolution of symptoms and allow a more rapid recovery.

Alternative mucolytic treatments with proven efficacy in patients with CB are available [START_REF] Stey | The effect of oral N-acetylcysteine in chronic bronchitis: a quantitative systematic review[END_REF][START_REF] Poole | Mucolytic agents for chronic bronchitis or chornic obstructive pulmonary disease (Review). Cochrane Database of Systematic Reviews[END_REF]. In the present meta-analysis, the comparison versus other active mucolytics given at comparable doses and regimens (either bid or tid), provided evidence of a tendency to erdosteine having a better efficacy.

This evidence was prevalently obtained in patients suffering from acute exacerbations of CB which represented the vast majority of the study population versus active comparators used in the present meta-analysis [12] Aubier M, L Berdah. Étude multicentrique, controlée, en double aveugle, de l'efficacité et de la tolérance de vectrine® 8erdostéine) versus placebo dans le traitement de la bronchite chronique hypersecrétante stabilisée.

Rev Mal Respir 1999, 16 :521-528. Erdosteine, the second generation mucolytic:international up-dating of clinical studies (adults and paediatrics).

7 unpublished

 7 studies in patients with CB submitted for European marketing approval in 2005 were considered.

  or three times daily on top of background therapy. Nine RCTs involving 459 patients compared erdosteine to other mucolytics (ambroxol, Nacetylcysteine, carbocysteine, sobrerol), six studies were double-blinded and three single-blinded. The majority of patients presented with acute CB/COPD exacerbations, and study treatments were administered in association with antibiotics and bronchodilator therapy (beta 2 -agonists and aminophylline).

9

 9 Dosing schedules were two or three times daily for both erdosteine and active comparators. Most of the RCTs included a mixed population of both inpatients and outpatients.

  individual patient data obtained in 15 both published and unpublished studies, selected from a dossier used for recent European registration by the manufacturing company and consistent with the clinical indication object of this meta-analysis, with the evaluation of a total of 1046 patients. Although a company-driven bias in the retrieval of the studies cannot be definitively ruled out and the overall number of patients was rather small, there are several positive features in the present work that support the validity of the observed findings. The direct access to individual patients' data and the possibility to include unpublished studies in our view have contributed to reduce the publication bias that sometimes may represent a limit of systematic reviews based on published data. Additionally, the methodology of meta-analysis can overcome a lack of power of single individual trials, as it was the case especially for the unpublished studies. In this respect, the sensitivity analysis performed to evaluate possible heterogeneity due to publication and/or study quality did not show significant differences.The results of the present meta-analysis, conducted on studies focusing on the comparison between erdosteine and placebo or mucolytics, indicate that the addition of erdosteine to background treatment of patients withM A N U S C R I P T A C C E P T E D ARTICLE IN PRESS12 CB/COPD can be beneficial with significant effects observed in the overall population included, both patients with acute infective exacerbations and with stable disease. A significantly better outcome was demonstrated in patients treated with erdosteine for the cumulative index of global efficacy and for each of the individual symptoms, except for sputum purulence and dyspnoea, and reflected by the significantly higher percentage of treatment success, as indicated by the physician's final efficacy assessment.The advantage provided by erdosteine was especially evident versus placebo. Since in these studies the majority of patients (namely those with acute respiratory infections) were treated with antibiotics, it derives that erdosteine combined with an antibiotic treatment is therapeutically more useful than antibiotic monotherapy[11,14,15,[START_REF] Bisetti | Mucolytic activity of erdosteine: Double blind clinical trial vs. placebo[END_REF]. The index of global efficacy and the physician's OA, when available, significantly favoured erdosteine in all of the studies. The individual symptom scores related to cough and expectoration, including the more objective assessment of catarrh ronchi at auscultation in two studies[11,[START_REF] Voisin | Controlled, double-blind, pilot study comparing erdosteine at the daily dose of 600 mg to placebo in two groups of patients affected by stabilized chronic obstructive bronchitis[END_REF], indicated a faster amelioration of this symptom during the 7-10 days of treatment in the patients also receiving erdosteine.

[14

  13,17,[START_REF] Marchioni | Clinical experience with erdosteine in the treatment of exacerbation of chronic obstructive bronchopneumopathies[END_REF][START_REF] Scarpazza | Controlled clinical trial of erdosteine in chronic bronchitis under exacerbation[END_REF][START_REF] Fumagalli | Erdosteine: A new molecule with mucolytic activity. Clinical and instrumental evaluation in patients with acute exacerbated chronic bronchopneumopathies[END_REF][START_REF] Materazzi | Evaluation of efficacy and safety of erdosteine in chronic bronchitis in phase of clinical reactivation[END_REF]. Taking each symptom individually, a general trend in favour of erdosteine was observed, except for sputum purulence and dyspnoea, and this translated into a significant reduction of the cumulative global efficacy score and a higher percentage of treatment success in patients treated with erdosteine. The more pronounced effect of erdosteine may be suggestive of a more rapid onset of activity, this being a feature already reported to characterize erdosteine pharmacologically[17,[START_REF] Busin | Erdosteine: Evaluation of mucorheological and immunosecretory parameters in patients with bronchial phlogistic pathology[END_REF][START_REF] Kharitonov | Fast onset of antioxidant actions of erdosteine and its potential use for acute treatment of COPD exacerbations[END_REF]. Mucolytic therapy has often been overlooked because of the difficulty of demonstrating effectiveness by objective criteria, the presence of clinical data that were sometimes contradictory, and uncertainty about the type of patients likely to benefit from their use.In this respect, possible drawbacks of the studies considered for this metaanalysis should be addressed. In the studies used in the present metaanalysis, respiratory symptoms have been subjectively assessed on Likerttype 0-4 scales (none to severe/almost constant). Although not fully validated, these scales are comparable to scoring systems employed in studies performed in similar patient populations when evaluating the effects of other therapeutic interventions, such as bronchodilators and/or inhaled corticosteroids ICSs[START_REF] Rossi | Comparison of the efficacy, tolerability and safety of formoterol dry powder and oral slow-release theophylline in the treatment of COPD[END_REF][START_REF] Szafranski | Efficacy and safety of budesonide/ formoterol in the management of chronic obstructive pulmonary disease[END_REF][START_REF] Calverley | Combined salmeterol and fluticasone in the treatment of chronic obstructive pulmonary disease: a randomised controlled trial[END_REF]. Although the patient's self-reporting on symptoms is regarded as an important measure to monitor disease activity and outcomes of care, a subjective symptom assessment may imply an incorrect estimate of treatment effectiveness and be unrelated to more objective measurements of disease improvement. The lack of a validated method to assess symptoms severity may therefore weaken the findings of the present review. It should be noted, however, that relatively few instruments are available in this respect, The more recent Breathlessness, Cough and Sputum Scale (BCSS), proposed as a reliable and responsive method for symptom monitoring[START_REF] Kline Leidy | The Breathlessness, Cough, and Sputum Scale: The development of empirically based guidelines for interpretation[END_REF], was not available at the time when the studies used in this meta-analysis were conducted; nevertheless it should be advisable to evaluate once more the symptomatic benefits provided by erdosteine through validated scoring methods to obtain more robust evidence for efficacy. Another possible limitation may be related to the concomitant treatments administered to the patients, namely the scarce intake of ICSs. Background treatments were mainly bronchodilators (beta 2agonists and theophylline) with antibiotics when patients were suffering from acute exacerbations. Since the most recent surveys report use of ICSs in a vast proportion of COPD patients in Europe, the present findings need to be confirmed in patients also receiving corticosteroids in order to ensure their generalization to the today's patient population and to assess the added M A N U S C R I P T A C C E P T E D ARTICLE IN PRESS 15 value that erdosteine (or other mucolytic treatments) may confer on top of already administered anti-inflammatory treatments. However there is no reason to disregard the possibility of a positive interaction between antiinflammatory and expectorant interventions. Furthermore, it should be remembered that bronchodilators are the recommended therapy for CB/COPD until the severe stages, and that the use of antibiotics represent the mainstay treatment for acute infective exacerbations. There is therefore still a not negligible number of CB/COPD patients who could certainly benefit from the addition of erdosteine to their usual therapy, as shown by the present results. In conclusion, the present meta-analysis supports the effectiveness of erdosteine in patients with CB/COPD, especially during acute exacerbations, and provides further strength to the published ACCP guidelines recommending the use of erdosteine on a short-term basis to increase mucous clearance [53]. Furthermore appropriately sized studies with fully validated endpoints should be undertaken to reinforce the present results and better define the longer-term benefit of erdosteine in patients with CB/COPD. Marchioni CF, Polu JM, Taytard A, Hanard T, Noseda G, Mancini C. Evaluation of efficacy and safety of erdosteine in patients affected by chronic bronchitis during an infective exacerbation phase and receiving amoxicillin as basic treatment (ECOBES, European Chronic Obstructive Bronchitis Erdosteine Study). Int J Clin Pharmacol Ther 1995, 33:612-618.

[ 13 ]

 13 Arnaud A. Clinical study of efficacy and tolerability of erdosteine in the treatment of chronic obstructive bronchitis with superifection. Controlled double-blind study vs. N-acetylcysteine. Clinical Study Report EC/ER9022P, Data on File 1991. Partly published in: Mancini C.

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  300 mg TID N-Acetylcysteine 200 mg TID MC = Multicenter; DB = double-blind; SB = single-blind; RCT = randomised controlled trial; CT = Controlled trial; E / C = Erdosteine / Comparators ^ In all cases, placebo capsules are administered with same frequency and duration M A N U S C R I P T A C C E P T E D

Table 1 Main characteristics of RCTs included in the meta-analysis

 1

	Ref.	Study	Patient N		Study Population		Schedule	Schedule
		design	Randomised	Dropouts	Median Age, yr./	Diagnosis at inclusion	Concomitant medication	Erdosteine	Comparator
			/Analysed	(E / C)	Proportion Male,
					%
	Erdosteine versus placebo
	Ricevuti 14	DB, CT	24 / 24	0 / 0	57.0 / 42	Acute infective exacerbation of chronic	Amoxicillin 500 mg tid	300 mg TID	Placebo^
						bronchitis (amoxicillin-sensitive bacteria
						isolated from sputum)
	Voisin 40	MC, DB, RCT	88 / 82	4 / 2	57.5 / 74	Stable chronic obstructive bronchitis	Not available	300 mg BID	Placebo^
						(FEV1/FVC at least 10% below normal
						theoretical value)
	Hotzinger 15	DB, RCT	40 / 40	0 / 0	49.0 / 75	Hypersecretory infective bronchitis (acute	Co-trimoxazole 160+800 mg	300 mg TID	Placebo^
						bronchitis or relapses of chronic bronchitis)	bid
	Bisetti 39	DB, RCT	28 / 27	0 / 1	62.0 / 68	Acute exacerbation of chronic bronchitis	Antibiotics	300 mg BID	Placebo^
							Xanthines, beta2-agonists
	Marchioni 11	MC, DB, RCT	237 / 226	6 / 5	66.0 / 76	Acute exacerbation of chronic obstructive	Amoxicillin 500 mg tid	300 mg BID	Placebo^
						bronchitis (amoxicillin-sensitive bacteria	Xanthines, beta2-agonists
						isolated from sputum; FEV1/FVC at least
						10% below normal theoretical value)
	Aubier 12	MC, DB, RCT	170 / 166	1 / 3	59.0 / 58	Stable chronic obstructive bronchitis	Xanthines, beta2-agonists	300 mg BID	Placebo^
						(FEV1/FVC at least 10% below normal
						theoretical value)
	Erdosteine versus mucolytics
	Marchioni 43	DB, RCT	30 / 30	0 / 0	64.5 / 93	Acute exacerbation of chronic bronchitis	Antibiotics	300 mg BID	Sobrerol
							Xanthines, beta2-agonists,		200 mg BID
							steroids
	Scarpazza 44	DB, RCT	30 / 27	1 / 2	64.0 / 43	Acute exacerbation of chronic bronchitis	Antibiotics	150 mg TID	Sobrerol
							Xanthines, beta2-agonists		100 mg TID
	Fumagalli 45	DB, RCT	30 / 30	0 / 0	65.0 / 53	Acute or chronic exacerbated bronchitis	Antibiotics	300 mg BID	Ambroxol
							Xanthines, beta2-agonists,		30 mg BID
							steroids
	Ginesu 16	SB, CT	30 / 30	0 / 0	62.5 / 100	Bronchopulmonary diseases characterised	Antibiotics	150 mg TID	Ambroxol
						by expectoration and cough	Xanthines, beta2-agonists

Table 3 Patient Characteristics

 3 Data are mean (SD) ^ Data from 1 study vs. placebo and 1 study vs. mucolytics: not available.

	Erdosteine	Comparator

Table 4 Outcome data on individual respiratory symptoms from the RCTs included in the meta-analysis

 4

	Ref.	Cough	Cough	Sputum	Sputum	Difficulty	Catarrh ronchi at	Dyspnoea
		frequency	intensity	viscosity	purulence	to expectorate	auscultation
	Erdosteine versus placebo
	Ricevuti 14	NA	-0.33 [-0.85, 0.19]	-0.58 [-1.03, -0.13]	-0.34 [-0.65, -0.03]	NA	NA	-0.42 [-1.03, 0.19]
	Voisin 40	0.17 [-0.15, 0.49]	NA	0.07 [-0.20, 0.34]	0.19 [-0.07, 0.45]	0.25 [-0.04, 0.54]	NA	-0.12 [-0.44, 0.20]
	Hotzinger 15	-0.55 [-1.06, -0.04]	-0.40 [-0.81, 0.01]	NA	0.00 [-0.35, 0.35]	-0.65 [-1.23, -0.07]	NA	NA
	Bisetti 39	-0.83 [-1.40, -0.26]	-0.89 [-1.33, -0.45]	NA	-0.33 [-0.88, 0.22]	-0.95 [-1.56, -0.34]	-0.83 [-1.14, -0.52]	NA
	Marchioni 11	-0.18 [-0.39, 0.03]	NA	-0.41 [-0.60, -0.22]	-0.43 [-0.61, -0.25]	-0.32 [-0.53, -0.11]	-0.41 [-0.62, -0.20]	-0.21 [-0.41, -0.01]
	Aubier 12	-0.19 [-0.38, 0.00]	-0.14 [-0.37, 0.09]	-0.24 [-0.46, -0.02]	NA	-0.17 [-0.39, 0.05]	NA	-0.14 [-0.32, 0.04]
	Subtotal	-0.23 [-0.47, 0.00]	-0.42 [-0.75, -0.08]	-0.27 [-0.51, -0.03]	-0.18 [-0.46, 0.10]	-0.29 [-0.60, 0.03]	-0.60 [-1.01, -0.19]	-0.17 [-0.30, -0.05]
	Erdosteine versus Mucolytics
	Marchioni 43	-0.53 [-0.99, -0.07]	-0.26 [-0.60, 0.08]	NA	-0.27 [-0.83, 0.29]	-0.27 [-0.68, 0.14]	-0.40 [-0.80, -0.00]	NA
	Scarpazza 44	-0.17 [-0.58, 0.24]	NA	NA	0.06 [-0.33, 0.45]	-0.18 [-0.60, 0.24]	0.22 [-0.09, 0.53]	NA
	Fumagalli 45	0.06 [-0.46, 0.58]	-0.07 [-0.60, 0.46]	NA	0.11 [-0.50, 0.72]	-0.44 [-1.28, 0.40]	NA	NA
	Ginesu 16	-0.20 [-0.59, 0.19]	-0.27 [-0.64, 0.10]	-0.54 [-0.98, -0.10]	NA	-0.40 [-0.83, 0.03]	-0.34 [-0.69, 0.01]	-0.06 [-0.45, 0.33]
	Tellings 38	-0.40 [-0.82, 0.02]	-0.20 [-0.56, 0.16]	NA	NA	NA	NA	NA
	Materazzi 46	NE	NE	NE	NA	NE	NE	NA
	Zanasi 17	NA	-0.44 [-0.83, -0.05]	NA	-0.20 [-0.55, 0.15]	-0.16 [-0.43, 0.11]	-0.24 [-0.56, 0.08]	NA
	Franco 18	-0.25 [-0.95, 0.45]	-0.25 [-0.90, 0.40]	-0.75 [-1.40, -0.10]	-0.25 [-0.89, 0.39]	-0.66 [-1.28, -0.04]	-0.50 [-1.13, 0.13]	NA
	Arnaud 13	0.22 [-0.03, 0.47]	NA	0.06 [-0.21, 0.33]	0.20 [-0.04, 0.44]	0.04 [-0.20, 0.28]	NA	0.23 [-0.01, 0.47]
	Subtotal	-0.15 [-0.38, 0.08]	-0.26 [-0.43, -0.10]	-0.36 [-0.87, 0.16]	0.02 [-0.15, 0.19]	-0.19 [-0.34, -0.03]	-0.21 [-0.48, 0.05]	0.13 [-0.14, 0.40]

Acknowledgements

The Authors would like to acknowledge Edmond Pharma for providing information on unpublished studies and allowing direct access to original patients' data.


Karin J Velthove Pharmd

PharmD Hubert G M Leufkens
email: h.g.m.leufkens@uu.nl

PhD Patrick C Souverein

MD PhD René C Schweizer

PhD Madelon Bracke

PhD Wouter W Van Solinge

Effects of glucocorticoids on the neutrophil count: a cohort study among hospitalized patients

Keywords: cohort studies, glucocorticoid, immunology, neutrophil DTA, difficult-to-treat asthma, GC, glucocorticoid, UPOD, Utrecht Patient

Background: Systemic glucocorticoids are often used in clinical practice for a large variety of indications. Clinical observations have shown that patients using glucocorticoids often have higher neutrophil counts. Debate remains whether this observed neutrophilia is associated with glucocorticoid use or that other factors, like disease and severity of disease, should be considered. The objective of this study was to investigate the effect of systemic glucocorticoids on the absolute neutrophil count in hospitalized patients.

Methods: A cohort study was conducted using data from the Utrecht Patient Oriented Database which comprises clinical data of patients of the University Medical Center Utrecht.

We identified all adult patients, hospitalized in 2005 with at least two blood samples for hematological testing during admission and compared in hospital glucocorticoid use with nonuse.

Results: A total of 809 glucocorticoid users and 2,658 non-users were included in the study with comparable neutrophil counts at admission (8.2.10 9 /l for glucocorticoid users and 8.0.10 9 /l for non-users). Overall analysis showed a slight association between glucocorticoid use and an increase in neutrophil count (RR 1.3; 95% CI 1.1-1.5). However, within diagnostic subgroups there was no increase in neutrophil count in glucocorticoid users. Furthermore, among all no dose response relationship, no effect of time between the two samples, and no effect of anti-inflammatory/sodium retaining potency was found.

Conclusion:

Observed neutrophilia in users of systemic glucocorticoids is probably associated with underlying disease, rather than glucocorticoid use itself.

Introduction

Glucocorticoids (GCs) have a widespread and complex mechanism of action. They have many effects, depending on disease and cell type [START_REF] Czock | Pharmacokinetics and pharmacodynamics of systemically administered glucocorticoids[END_REF][START_REF] Umland | Review of the molecular and cellular mechanisms of action of glucocorticoids for use in asthma[END_REF]. Glucocorticoids reduce the number of many inflammatory cells, in particular increase the rate of apoptosis of eosinophils [START_REF] Czock | Pharmacokinetics and pharmacodynamics of systemically administered glucocorticoids[END_REF][START_REF] Umland | Review of the molecular and cellular mechanisms of action of glucocorticoids for use in asthma[END_REF][START_REF] Jayaram | Determining asthma treatment by monitoring sputum cell counts: effect on exacerbations[END_REF][START_REF] Carpagnano | Exhaled markers in the monitoring of airways inflammation and its response to steroid's treatment in mild persistent asthma[END_REF][START_REF]From the Global Strategy for Asthma Management and Prevention. Global Initiative for Asthma (GINA)[END_REF].

Neutrophils were found to be less responsive to these drugs [START_REF] Barnes | How do corticosteroids work in asthma?[END_REF][START_REF] Sampson | The role of eosinophils and neutrophils in inflammation[END_REF][START_REF] Jatakanon | Neutrophilic inflammation in severe persistent asthma[END_REF]. In vitro studies have shown that glucocorticoids inhibit neutrophil apoptosis [START_REF] Czock | Pharmacokinetics and pharmacodynamics of systemically administered glucocorticoids[END_REF][START_REF] Umland | Review of the molecular and cellular mechanisms of action of glucocorticoids for use in asthma[END_REF][START_REF] Zhang | Beclomethasone, budesonide and fluticasone propionate inhibit human neutrophil apoptosis[END_REF][START_REF] Liles | Glucocorticoids inhibit apoptosis of human neutrophils[END_REF][START_REF] Belvisi | Regulation of inflammatory cell function by corticosteroids[END_REF]. However, in vivo studies mainly have focused on healthy volunteers and short-term effects of glucocorticoids on the neutrophil count and showed normalization of the neutrophil count within 24 hours [START_REF] Steele | Functional capacity of marginated and bone marrow reserve granulocytes[END_REF][START_REF] Chakraborty | Pharmacoimmunodynamic interactions of interleukin-10 and prednisone in healthy volunteers[END_REF]. Therefore, the clinical effect of glucocorticoids on the neutrophil count is controversial.

Clinical observations have shown that patients using GCs often have higher neutrophil counts, particularly in respiratory disease. It is well known that granulocytes play an important role in asthma and COPD [START_REF] Caramori | Pharmacology of airway inflammation in asthma and COPD[END_REF][START_REF] Franciosi | Markers of disease severity in chronic obstructive pulmonary disease[END_REF] but although neutrophilia has been associated with asthma severity, debate remains whether neutrophilia is a characteristic of asthma severity or results from glucocorticoid treatment [START_REF]From the Global Strategy for Asthma Management and Prevention. Global Initiative for Asthma (GINA)[END_REF][START_REF] Jatakanon | Neutrophilic inflammation in severe persistent asthma[END_REF][START_REF] Douwes | Non-eosinophilic asthma: importance and possible mechanisms[END_REF][START_REF] Nguyen | Increase in airway neutrophils after oral but not inhaled corticosteroid therapy in mild asthma[END_REF][START_REF] Louis | The relationship between airways inflammation and asthma severity[END_REF][START_REF] Wenzel | Asthma: defining of the persistent adult phenotypes[END_REF][START_REF] Chanez | Severe asthma in adults: What are the important questions?[END_REF][START_REF] Wenzel | Proceedings of the ATS workshop on refractory asthma. Current understanding, recommendations, and unanswered questions[END_REF][START_REF] Chung | Difficult/therapyresistant asthma. The need for an integrated approach to define clinical phenotypes, evaluate risk factors, understand pathophysiology and find novel therapies. ERS Task Force on Difficult/Therapy-resistant asthma[END_REF][START_REF] Shannon | Differences in airway cytokine profile in severe asthma compared to moderate asthma[END_REF][START_REF] Jang | Factors influencing the responsiveness to inhaled glucocorticoids of patients with moderate-to-severe asthma[END_REF]. More severely ill asthma patients receive often higher doses of glucocorticoids and glucocorticoids are highly used among hospitalized asthma patients. Therefore, in this observational study, we compared neutrophil counts in a variety of diseases in patients using GCs with non-users, taking into account several approaches to adjust for disease bias on these counts.

Material and methods

Study design and setting

A cohort study was conducted using data from the Utrecht Patient Oriented Database (UPOD). UPOD is an infrastructure of relational databases comprising administrative data on patient characteristics, laboratory test results, medication orders, discharge diagnoses and

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4 medical procedures for all patients treated at the University Medical Center Utrecht, a 1,042 bed tertiary teaching hospital in the center of the Netherlands. Each year, approximately 165 000 patients are treated during more than 28 000 hospitalizations, 15 000 day care treatments, and 334 000 outpatient visits. UPOD data acquisition and data management is in accordance with current Dutch privacy and ethical regulations. A more complete description of UPOD has been published elsewhere [START_REF] Berg | Linking laboratory and medication data: new opportunities for pharmacoepidemiological research[END_REF].

We identified all adult patients (≥ 18 years) who were hospitalized in the University Medical Center Utrecht in 2005 and had at least two hematological blood tests during hospitalization.

Exposed patients used systemic glucocorticoids during admission, where glucocorticoid use started before or after withdrawal of the first blood test; unexposed patients did not use systemic glucocorticoids during admission. The study design is summarized in Figure 1. For all participants the discharge diagnose was defined according to the ICD-9-CM code [START_REF]Anonymous. International Classification of Diaseses, Ninth Revision, Clinical Modification (ICD-9-CM)[END_REF].

Neutrophil testing

For each glucocorticoid user, the first blood test during admission and the last blood measurement during in-hospital glucocorticoid use were selected for analysis, where these samples should cover at least a one day period. Up to four unexposed patients were sampled to each glucocorticoid user according to calendar time (with a maximum of 15 days before or after the test date of the user), neutrophil count at time of admission (max 2.10 9 neutrophils/l around the neutrophil count of the user) and days between the two blood samples (max two days around the number of days for the glucocorticoid user). We used data on eosinophil counts in the same patients measured on the same moments in time as a reference procedure as the effect of glucocorticoids on the absolute eosinophil count is widely known [START_REF] Czock | Pharmacokinetics and pharmacodynamics of systemically administered glucocorticoids[END_REF][START_REF] Umland | Review of the molecular and cellular mechanisms of action of glucocorticoids for use in asthma[END_REF][START_REF] Jayaram | Determining asthma treatment by monitoring sputum cell counts: effect on exacerbations[END_REF][START_REF] Carpagnano | Exhaled markers in the monitoring of airways inflammation and its response to steroid's treatment in mild persistent asthma[END_REF][START_REF]From the Global Strategy for Asthma Management and Prevention. Global Initiative for Asthma (GINA)[END_REF]. Data on glucocorticoid use was retrieved from the medication file from UPOD and daily dose

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exposure was expressed as systemic prednisone equivalents, using defined daily dosages [START_REF] Anonymous | ATC Classification index with DDDs[END_REF]. Moreover, glucocorticoids were categorized according to their anti-inflammatory/sodium retaining potency for subgroup analyses [START_REF] Hardman | Goodman & Gilman's the pharmacological basis of therapeutics[END_REF].

Data analysis

Student t-tests, Mann-Whitney tests, and chi-square tests were used, as appropriate.

Glucocorticoid dose and change in neutrophil and eosinophil counts were associated through a non-linear relationship. Therefore, the glucocorticoid dose was categorized into tertiles. The change in neutrophil and eosinophil count was also categorized into tertiles to obtain three equally sized groups. These three groups were defined as an increase, decrease or no change in neutrophil or eosinophil count. Potential confounders included in the analysis were age, gender, diagnosis, duration of hospitalization, length of period between two blood samples, CRP levels, and death during hospitalization. Subsequently, unconditional multivariate Cox proportional hazards analysis was used to estimate the strength of the association between glucocorticoid exposure and either an increase versus no change, or a decrease compared to no change in the absolute neutrophil or eosinophil count, expressed as relative risk (RR) with 95% confidence intervals (CI). All variables that changed the regression coefficient of glucocorticoid use with less than ten percent were excluded from the model. All analyses were conducted using SPSS for Windows, version 14.0 (SPSS Inc., Chicago, Illinois, USA).

Results

From the UPOD database, we identified 809 glucocorticoid users and 2,658 non-users. The age and gender distributions of the glucocorticoid users were comparable to patients not using

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systemic glucocorticoids, as well as the duration of hospitalization and the number of deaths during hospitalization. CRP levels were comparable for GC users and non-users at time of admission, but CRP levels were lower for GC users in the second blood sample compared to non-users (Table 1). Glucocorticoid users had a lower eosinophil count than non-users, and non-users had an increase in eosinophil count during hospitalization (Table 1, Figure 2). The absolute neutrophil count at admission was comparable for users and non-users. During hospitalization, glucocorticoid users had an overall minor increase of 0.3.10 9 neutrophils/l, where non-users had an overall slight decrease of 0.3.10 9 /l (Table 1). However, differences in neutrophil count between the two blood samples for each patient were equally distributed among glucocorticoid users and non-users, with means close to zero (Figure 2). As shown in Table 1, glucocorticoid use was not randomly distributed among the diagnosis groups.

As shown in Table 2, 28.1% of users and 32.5% of non-users had a decrease of more than 2.10 9 neutrophils/l, 40.7% of users and 42.4% of non-users remained unchanged and 31.3% of users and 25.1% of non-users had an increase of more than 2.10 9 neutrophils/l. Overall, use of systemic glucocorticoids was associated with a slight increase in the neutrophil count (crude RR 1.2; 95% CI 1.0-1.4). After adjustment for diagnosis, the adjusted RR yielded a value of 1.3 (95% CI 1.1-1.5). However, subgroup analysis for the major diagnostic groups showed that there is no association between glucocorticoid use and an increase in neutrophil count among diagnostic subgroups. The RR for neoplasms was 1.2 (95% CI 0.8-1.6), for circulatory disease 1.2 (95% CI 0.8-1.6), and for respiratory diseases 1.1 (95% CI 0.6-2.1).

Glucocorticoid use was not associated with a decrease in the absolute neutrophil count in overall analysis or in subgroup analyses (Table 2). With respect to the absolute eosinophil count 220 (27.3%) of users and 534 (20.1%) of non-users had a decrease of more than 0.05.10 9 /l, 366 (45.4%) of users and 893 (33.6%) of non-users remained unchanged and 220

M A N U S C R I P T A C C E P T E D ARTICLE IN PRESS 7 (27.3%
) of glucocorticoid users and 1,227 (46.2%) of non-users had an increase of more than 0.05.10 9 /l. Use of systemic glucocorticoids was inversely associated with an increase in the eosinophil count (adjusted RR 0.6; 95% CI 0.6-0.7). There was no clear association of glucocorticoid use and a decrease in eosinophil count (adjusted RR 1.2, 95% CI 1.0-1.4).

The number of days between the two blood samples for each patient varied between one and 77 days between user-non-users pairs. As shown in Figure 3, there was no effect of the time between the blood samples for each patient and the change in neutrophil count, nor were there any differences for glucocorticoid users and non-users. Regarding short-term effects, there was a wide variation in the change in neutrophil counts for glucocorticoid users as well as for glucocorticoid non-users with a one day period between the two blood samples. A sensitivity analysis revealed that limiting the maximum period between the two blood samples to seven days did not influence the results (data not shown).

Furthermore, different diagnoses require treatment with different doses of glucocorticoids, which could cause confounding in studying the relationship between glucocorticoids and the neutrophil count. Among glucocorticoid users, no dose response relationship could be found, where adjustment occurred for diagnosis (Table 3). Of the glucocorticoid users 474 (58.6%) used glucocorticoids at time of the first blood sample, 335 (41.4%) of the users started glucocorticoid treatment after withdrawal of the first blood sample. Stratification on this parameter did not influence the overall results with an RR 1.9 (95% CI 1.4-2.5) for an increase versus no change compared with 1.3 (95% CI 1.1-1.5) when including all patients.

For a decrease versus no change, the RRs were 0.7 (95% CI 0.5-1.0) for only users that started glucocorticoid use after the first blood sample compared with 0.9 (95% CI 0.8-1.1) when all patients were included in the model. Subgroup analyses for the major diagnostic groups

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among patients that started glucocorticoid use after the first blood sample showed no association between glucocorticoid use and an increase or decrease in neutrophil count (data not shown). Stratifying glucocorticoids according to their anti-inflammatory/sodium retaining potency did not yield different results (data not shown).

Discussion

In this study, we showed that observed neutrophilia in users of systemic glucocorticoids is probably associated with underlying disease, rather than with the use of glucocorticoids itself.

Overall analysis yielded a 30% increase in the risk of an increase in the neutrophil count for GC users (adjusted RR 1.3, 95% CI 1.1-1.5). However this is the summed effect of several factors, next to the possible effect of systemic glucocorticoids, e.g. diagnosis, disease severity, dose and type of glucocorticoid, and the studied time window. These factors are considered one by one.

Regarding diagnosis, subgroup analysis in the major diagnostic groups showed that there was no association between glucocorticoid use and a change in the absolute neutrophil count among diagnostic subgroups.

Concerning disease severity, glucocorticoid users have a higher absolute neutrophil count in the second blood sample, compared to non-users, and this count is above the upper limit of our laboratories normal reference range of 1.6 -8.3.10 9 neutrophil/l, possibly indicating that glucocorticoid users were more severely ill than non-users (Table 1). However, the mean change in absolute neutrophil count for GC users, as well as for non-users was close to zero (Figure 2) and demographic parameters and disease severity markers, like duration of hospitalization and death during hospitalization, were comparable between GC users and non-

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9 users, CRP levels were lower in the second blood sample for GC users compared to non-users (Table 1).

Another argument showing that neutrophilia is not fully due to glucocorticoid use is that effect of dosing and anti-inflammatory potency of the glucocorticoid was not found in our study. Also, a sensitivity analysis showed that varying the cut-off value of a change in neutrophil count from 1 to 10.10 9 /l did not have major effects on the associations found (data not shown). The inverse association found between glucocorticoid use and an increase in eosinophil count is reassuring as it served as an internal reference procedure of our database.

The time window of studying the effect of glucocorticoids on the neutrophil count is important. Early, non-genomic effects occur within minutes to seconds after administration, genomic mechanisms of action take 30 minutes to 18 hours [START_REF] Czock | Pharmacokinetics and pharmacodynamics of systemically administered glucocorticoids[END_REF][START_REF] Buttgereit | Rapid glucocorticoid effects in immune cells[END_REF][START_REF] Goulding | The molecular complexity of glucocorticoid actions in inflammation -a four-ring circus[END_REF]. In vivo studies mainly focused on healthy volunteers and short-term effects of glucocorticoids on the neutrophil count within 24 hours [START_REF] Steele | Functional capacity of marginated and bone marrow reserve granulocytes[END_REF][START_REF] Chakraborty | Pharmacoimmunodynamic interactions of interleukin-10 and prednisone in healthy volunteers[END_REF]. Because of the clinical relevance of a prolonged change in the neutrophil count in this study there is at least a one day period between the two blood samples of each patient. The second blood sample for users was selected during glucocorticoid use.

Our findings are in accordance with other studies that found that neutrophilia is not fully due to glucocorticoid treatment. Green et al. concluded that, in some asthma subjects at least, neutrophilia is not due to the glucocorticoid treatment [START_REF] Green | Analysis of induced sputum in adults with asthma: identification of subgroup with isolated sputum neutrophilia and poor response to inhaled corticosteroids[END_REF]. Also Louis et al. found in their study that severe asthmatics, treated with systemic glucocorticoids, had a lower absolute neutrophil count in sputum compared to severe asthmatics who did not use systemic glucocorticoids [START_REF] Louis | The relationship between airways inflammation and asthma severity[END_REF]. Further research is needed to study inflammation in order to create more insight into the mechanistic role of neutrophils in asthma severity.

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One might argue that the results of this study should be verified in a randomized controlled trial. However, such a trial will probably not be approved by any medical ethical committee, because severe asthma patients cannot be deprived of glucocorticoids since these drugs are essential in the treatment of asthma and there is no alternative treatment at this time.

There are several studies on the cellular mechanism of glucocorticoids in general [START_REF] Umland | Review of the molecular and cellular mechanisms of action of glucocorticoids for use in asthma[END_REF][START_REF] Barnes | How do corticosteroids work in asthma?[END_REF][START_REF] Sin | Corticosteroids and adrenoceptor agonists: the compliments for combination therapy in chronic airways diseases[END_REF][START_REF] Adcock | Corticosteroid-insensitive asthma: molecular mechanisms[END_REF][START_REF] Barnes | Corticosteroids: the drugs to beat[END_REF][START_REF] Stahn | Molecular mechanisms of glucocorticoid action and selective glucocorticoid receptor agonists[END_REF]. In spite of the discussions about the glucocorticoid effect on neutrophils, this mechanism is not well-understood and should be studied in more detail. There are several mechanisms by which glucocorticoids could theoretically influence the absolute neutrophil count. By increasing the neutrophil production in the bone marrow, by demargination from the blood vessel wall, by increasing the life span, by limiting neutrophil emigration from the blood, or by a combination of these factors [START_REF] Czock | Pharmacokinetics and pharmacodynamics of systemically administered glucocorticoids[END_REF][START_REF] Zhang | Beclomethasone, budesonide and fluticasone propionate inhibit human neutrophil apoptosis[END_REF][START_REF] Liles | Glucocorticoids inhibit apoptosis of human neutrophils[END_REF][START_REF] Steele | Functional capacity of marginated and bone marrow reserve granulocytes[END_REF][START_REF] Hardman | Goodman & Gilman's the pharmacological basis of therapeutics[END_REF]. The bone marrow could be activated to produce more neutrophils [START_REF] Steele | Functional capacity of marginated and bone marrow reserve granulocytes[END_REF][START_REF] Hardman | Goodman & Gilman's the pharmacological basis of therapeutics[END_REF]. However, when the bone marrow is producing granulocytes more quickly, it will also release immature granulocytes into the blood, which is not the case in our study population. Factors like stress, exercise, or infection could cause demargination of neutrophils close to the vessel wall, resulting in an increase in the neutrophil count in peripheral blood, but glucocorticoids are not likely to cause demargination [START_REF] Steele | Functional capacity of marginated and bone marrow reserve granulocytes[END_REF][START_REF] Hardman | Goodman & Gilman's the pharmacological basis of therapeutics[END_REF][START_REF] Ellis | G-CSF, but not corticosterone, mediates circulating neutrophilia induced by febrile-range hyperthermia[END_REF]. Glucocorticoids were found to inhibit neutrophil apoptosis [START_REF] Czock | Pharmacokinetics and pharmacodynamics of systemically administered glucocorticoids[END_REF][START_REF] Umland | Review of the molecular and cellular mechanisms of action of glucocorticoids for use in asthma[END_REF][START_REF] Zhang | Beclomethasone, budesonide and fluticasone propionate inhibit human neutrophil apoptosis[END_REF][START_REF] Liles | Glucocorticoids inhibit apoptosis of human neutrophils[END_REF][START_REF] Belvisi | Regulation of inflammatory cell function by corticosteroids[END_REF]. However, these findings were done in in-vitro experiments, and in-vivo studies only found short-term effects of glucocorticoids on the neutrophils [START_REF] Steele | Functional capacity of marginated and bone marrow reserve granulocytes[END_REF][START_REF] Chakraborty | Pharmacoimmunodynamic interactions of interleukin-10 and prednisone in healthy volunteers[END_REF]. Lastly, glucocorticoids could reduce the membrane CD11/CD18 appearance, causing weakened adhesion and less neutrophils leaving the blood [START_REF] Steele | Functional capacity of marginated and bone marrow reserve granulocytes[END_REF][START_REF] Shen | Magnolol inhibits Mac-1 (CD11b/CD18)-dependent neutrophil adhesion: relationship with its antioxidant effect[END_REF].

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In conclusion, the overall increased risk of an increase in neutrophil count in this study is the summed effect of several factors. The observed neutrophilia in users of systemic glucocorticoids is probably associated with underlying disease, rather than the use of glucocorticoids itself.

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 A C C E P T E D

19 Figure 1 .

 191 Figure 1. Study design. Overall analysis included all patients in the study, where subgroup

Figure 2 .

 2 Figure 2. Distribution of the difference of absolute neutrophil and eosinophil counts

Figure 3 .

 3 Figure 3. Effect of the period between the two blood samples on the change in absolute

Table 1 .

 1 Baseline characteristics of glucocorticoid users and non-users*

	Characteristic	GC users	Non-users	P-value
		(n=809)	(n=2,658)	(two-sided)
	Age, yr	57.4 ± 17.8	58.4 ± 18.3	0.171 a
	Sex					0.092 c
	Male	411 (50.8)	1,440 (54.2)
	Female	398 (49.2)	1,218 (45.8)
	Days between blood samples	6	(3-12)	4	(2-8)	<0.001 b
	Duration of hospitalization	10 (7-19)	10	(7-18)	0.332 b
	Death during hospitalization	54 (6.8)	157 (5.9)	0.360 c
	Absolute eosinophil count first blood sample (10 9 /l)	0.07 (0.03-0.17) 0.09 (0.04-0.18) < 0.001 b
	Absolute eosinophil count second blood sample (10 9 /l) 0.07 (0.03-0.15) 0.15 (0.06-0.27) < 0.001 b
	Absolute neutrophil count first blood sample (10 9 /l) Absolute neutrophil count second blood sample (10 9 /l) 8.5 ± 5.1 8.2 ± 4.7	8.0 ± 4.2 7.7 ± 4.1	0.214 a <0.001 a
	CRP first blood sample measured	311 (38.4)	1,196 (45.0)
	CRP (mg/l)	29.0 (7.0-75.0)	21.0 (7.0-68.8)	0.094 b
	CRP second blood sample measured	236 (29.2)	285 (10.7)
	CRP (mg/l)	23.5 (9.0-62.0)	33.0 (13.0-99.0)	0.001 b
	Diagnosis					<0.001 c
	Neoplasms	251 (31.0)	261 (9.8)
	Diseases of the circulatory system	93 (11.5)	1,023 (38.5)
	Diseases of the respiratory system	109 (13.5)	108 (4.1)
	Infectious and parasitic diseases	30 (3.7)	63 (2.4)
	Endocrine, nutritional and metabolic diseases, and	22 (2.7)	53 (2.0)
	immunity disorders
	Diseases of the nervous system and sense organs	30 (3.7)	65 (2.4)
	Diseases of the digestive system	44 (5.4)	198 (7.4)
	Diseases of the genitourinary system	73 (9.0)	95 (3.6)
	Diseases of the skin, subcutaneous tissue,	36 (4.4)	148 (5.6)
	musculoskeletal system, and connective tissue
	Other	121 (14.9)	644 (24.2)
	*Data are presented as n (%), or mean ± SD, or median (interquartile range)
	a Student t-test; b Mann-Whitney test; c χ 2 test

Table 2 .

 2 Association between glucocorticoid use and a change in absolute neutrophil count

		GC users	Non-users	Crude RR	Adjusted RR
		N (%)	N (%)	(95% CI)	(95% CI) a
		Overall analysis
		N=809	N=2,658
	No change	329 (40.7)	1127 (42.4)	Reference	Reference
	(-2.10 9 /l< change <2.10 9 /l)
	Increase (≥ 2.10 9 /l)	253 (31.3)	667 (25.1)	1.2 (1.0-1.4)	1.3 (1.1-1.5)
	Decrease (≤ -2.10 9 /l)	227 (28.1)	864 (32.5)	0.9 (0.8-1.1)	0.9 (0.8-1.1)
		Neoplasms
		N=251 (31.0)	N=261 (9.8)
	No change	114 (45.4)	119 (45.6)	Reference
	(-2.10 9 /l< change <2.10 9 /l)
	Increase (≥ 2.10 9 /l)	86 (34.3)	71 (27.2)	1.2 (0.8-1.6)
	Decrease (≤ -2.10 9 /l)	51 (27.2)	71 (27.2)	0.8 (0.6-1.2)
		Circulatory disease
		N=93 (11.5)	N=1,023 (38.5)
	No change	29 (31.2)	399 (39.0)	Reference
	(-2.10 9 /l< change <2.10 9 /l)
	Increase (≥ 2.10 9 /l)	38 (40.9)	376 (36.8)	1.2 (0.8-1.6)
	Decrease (≤ -2.10 9 /l)	26 (28.0)	248 (24.2)	1.2 (0.8-1.8)
		Respiratory disease
		N=109 (13.5)	N=108 (4.1)
	No change	47 (43.1)	37 (34.3)	Reference
	(-2.10 9 /l< change <2.10 9 /l)
	Increase (≥ 2.10 9 /l)	22 (20.2)	15 (13.9)	1.1 (0.6-2.1)
	Decrease (≤ -2.10 9 /l)	40 (36.7)	56 (51.9)	0.8 (0.5-1.1)
	GC = glucocorticoid
	a Adjusted for diagnosis (in case of overall analysis)

Table 3 .

 3 Dose effect among glucocorticoid users, stratified according to the dose used (in mg prednisone equivalents) before the first blood sample and the mean daily dose used between

	the two blood samples
	GC use before	Mean daily dose of	Increase	No change	Crude RR	Adjusted RR
	the first blood	GC use between	(≥ 2.10 9 /l)	(-2.10 9 /l<	(95% CI)	(95% CI) a
	sample	the two blood		change
		samples		<2.10 9 /l)
	0	< 15	40 (30.3)	51 (38.9)
		15 -40 ≥ 40	44 (33.3) 48 (36.4)	35 (26.7) 45 (34.4)	1.3 (0.8-1.9) 1.2 (0.8-1.8)	1.3 (0.8-2.0) 1.1 (0.7-1.8)
	0 -50	< 15	19 (51.4)	49 (56.3)
		15 -40 ≥ 40	13 (35.1) 5 (13.5)	27 (31.0) 11 (12.6)	1.2 (0.6-2.4) 1.1 (0.4-3.0)	1.8 (0.8-4.1) 1.6 (0.5-4.9)
	≥ 50	< 15	1 (1.2)	7 (6.3)
		15 -40	27 (32.1)	29 (26.1)	3.9 (0.5-28.4)	4.5 (0.6-34.2)
		≥ 40	56 (66.7)	75 (67.6)	3.4 (0.5-24.7)	3.6 (0.5-26.4)
	GC use before	Mean daily dose of	Decrease	No change	Crude OR (95%	Adjusted OR
	the first blood	GC use between	(≤-2.10 9 /l)	(-2.10 9 /l<	CI)	(95% CI) a
	sample	the two blood		change
		samples		<2.10 9 /l)
	0	< 15	34 (47.2)	51 (38.9)
		15 -40	21 (29.2)	35 (26.7)	0.9 (0.5-1.6)	1.0 (0.6-1.8)
		≥ 40	17 (23.6)	45 (34.4)	0.7 (0.4-1.2)	0.8 (0.5-1.6)
	0 -50	< 15	53 (57.0)	49 (56.3)
		15 -40	33 (35.5)	27 (31.0)	1.1 (0.7-1.6)	1.1 (0.7-1.8)
		≥ 40	7 (7.5)	11 (12.6)	0.7 (0.3-1.6)	0.8 (0.4-1.9)
	≥ 50	< 15	4 (6.5)	7 (6.3)
		15 -40	23 (37.1)	29 (26.1)	1.2 (0.4-3.5)	1.5 (0.5-4.6)
		≥ 40	35 (56.5)	75 (67.6)	0.9 (0.3-2.5)	1.0 (0.3-2.8)
	GC = glucocorticoid

a Adjusted for diagnosis

WWS has received unrestricted funding for pharmacoepidemiological research from

GlaxoSmithKline, Novo Nordisk, the private-public funded Top Institute Pharma (www.tipharma.nl, includes co-funding from universities, government, and industry), the Dutch Medicines Evaluation Board, and the Dutch Ministry of Health.