Reslizumab versus placebo for poorly controlled , severe eosinophilic asthma : meta-analysis Reslizumab u odnosu na placebo za neadekvatno kontrolisanu , tešku eozinofilnu astmu : meta-analiza

Background/Aim. Reslizumab is humanized monoclonal antibody produced by recombinant DNA technology which binds to circulating interleukin-5 (IL-5) and down-regulates the IL-5 signaling pathway. Reslizumab is indicated for the add-on maintenance treatment of patients 18 years and older with severe eosinophilic asthma phenotype whose symptoms were inadequately controlled with inhaled corticosteroids. The aim of this meta-analysis was to assess the efficacy and safety of reslizumab compared to placebo in patients suffering from inadequately controlled, moderateto-severe asthma with elevated blood eosinophil counts. Methods. Our meta-analysis was based on systematic search of literature and selection of high-quality evidence according to pre-set inclusion and exclusion criteria. The effects of reslizumab and placebo were summarized using Review Manager (RevMan) 5.3.5 and heterogeneity was assessed by the Cochrane Q test and I2 values. Several types of bias were assessed and publication bias shown by Funnel plot and Egger’s regression. Results. The meta-analysis included 5 randomized, placebo-controlled clinical trials. Reslizumab 3.0 mg/kg produced substantial improvements in forced expiratory volume in 1. second (FEV 1) (mean difference 0.15 [0.10, 0.21]) and in forced vital capacity (FVC) (mean difference 0.21 [0.09, 0.32]) over the 15 or 16-week treatment period, substantial decrease versus placebo in Asthma Control Questionnaire (ACQ) score (mean difference -0.28 [-0.41, -0.16]), and substantial increase vs. placebo from baseline in Asthma Quality of Life Questionnaire (AQLQ) total score (mean difference 0.24 [0.06, 0.43]). Also, reslizumab 3.0 mg/kg caused less adverse events versus placebo (OR 0.67 [0.51, 0.88]), especially asthma worsening (OR 0.53 [0.36, 0.77]) or bronchitis (OR 0.42 [0.24, 0.74]). Conclusion. On the basis of published clinical trials reslizumab could be considered as an effective and safe therapeutic option for severe, poorly controlled eosinophilic asthma for the time being.


Introduction
There are several phenotypes of bronchial asthma according to specific cellular mechanism and characteristics of patients 1,2 .Eosinophilic asthma involves subgroup of adult patients with late onset of disease, with hypereosinophilia in blood (> 1,000/mm 3 ) and in sputum (> 10%), with severe exacerbations which can be prevented by systematic and not by inhaled corticosteroids.Patients with this "endotype" of asthma have decreased level of athopy and their response on bronhodilatators is lower compared to subgroup of patients with allergic asthma [3][4][5] .This kind of inflammation pathway in asthma where eosinophiles dominate positively correlates with much more severe asthma exacerbations, higher rate of hospitalizations which contribute to increase burden of asthma 6 .Real prevalence of the eosinophilic asthma is not known, but it is estimated that 20% of patients with severe asthma would have this "endotype" of asthma 5 .Since eosinophiles mainly generate interleukin-5 (IL-5), blockade of pathways involving IL-5 can be effective therapeutic approach in patients with eosinophilic asthma 7 .
Eosinophils exhibit a substantial role in airway remodeling by promoting and sustaining airway inflammation, airway wall thickening, fibrosis and angiogenesis 8,9 .Therefore, suppressing the activity and number of eosinophils could be an important biological approach in the management of severe eosinophilic asthma 10 .IL-5 is a key cytokine for production, survival and maturation of eosinophils 11 .Due to a very specific effect on biology of eosinophils, IL-5 is considered to be an ideal molecular target for the treatment of severe eosinophilic asthma 10,12 .Inhibition of signaling mediated by IL-5 interrupts maturation and survival of eosinophils thus reducing eosinophilic inflammation 12 .Mepolizumab was the first anti-IL-5 antibody that was tested in randomized clinical trials on eosinophilic asthma 13 .Use of mepolizumab decreased exacerbation risk, improved quality of life, lowered eosinophil counts, improved asthma control and lung function in patients with severe eosinophilic asthma in several clinical studies 14 .
Reslizumab is humanized monoclonal antibody produced by recombinant DNA technology which binds to circulating IL-5 and down-regulates the IL-5 signaling pathway 12 .Reslizumab is indicated for the add-on maintenance treatment of patients 18 years and older with severe eosinophilic asthma phenotype whose symptoms were inadequately controlled with inhaled corticosteroids, with or without additional asthma controllers 15 .The recommended dosage regimen is 3 mg/kg once every 4 weeks administered by intravenous infusion over 20-50 min 16 .Although common adverse events of reslizumab are mild or moderate, like headache, nasopharyngitis and upper respiratory tract infection, reslizumab can also induce very serious adverse events as anaphylaxis 10 .Reslizumab is contraindicated in patients with known hypersensitivity to reslizumab or accompanying excipients 17 .
Although a few clinical trials and one meta-analysis with reslizumab for inadequately controlled asthma with elevated blood eosinophil counts were published, there are still some unresolved issues concerning all possible outcomes of treatment.Summarizing available evidence about all measures of efficacy and safety of reslizumab in this indication tested in clinical trials would be helpful for planning future studies with reslizumab in asthma.The aim of this metaanalysis was to assess the efficacy and safety of reslizumab compared to placebo in patients suffering from inadequately controlled, moderate-to-severe asthma with elevated blood eosinophil counts.

Methods
Our study was registered in the international prospective register of systematic reviews and meta-analyses (PRO-SPERO) under the number CRD42016041459 prior to commencement of the research.
The following criteria for considering studies for this review were used: 1) types of studies -randomized, doubleblind, placebo-controlled clinical trials; 2) types of participants -patients of both sex aged 12-75 years, with at least one blood eosinophil count of 400 cells per μL or higher during a 2-4 week period, or sputum eosinophils of 3% or more, with inadequately controlled asthma (Asthma Control Questionnaire-7 score ≥ 1.5), taking at least a medium dose of inhaled corticosteroids with or without another controller drug (including oral corticosteroids) and with airway reversibility [≥ 12% to short-acting beta-agonist (SABA)]; 3) types of interventions -intravenous infusion of reslizumab 3 mg/kg or of placebo (looking exactly the same as reslizumab) every 4 weeks, for 3 or more doses.Types of outcome measures used in our analysis were: change in forced expiratory volume in 1 second (FEV1) from baseline over 15 or 16 weeks of treatment, change in forced vital capacity (FVC) from baseline, change from baseline in asthma control questionnaire 7-point scale (ACQ-7) score, change from baseline in asthma symptom utility index (ASUI) score, rescue use of blood eosinophil count, asthma quality of life questionnaire (AQLQ) total score, immunogenicity and adverse events types and frequency.
Search methods for identification of studies primarily included electronic databases, and collection of journal articles and books of University Library, University of Kraguje-

inadequately[All Fields] AND controlled[All Fields] AND ("asthma"[MeSH Terms] OR "asthma"[All Fields]))
).There were no restrictions on publication date, format or language in the search strategy.The references of the retrieved articles were searched for further similar studies ("snowball search").The collection of journal articles and books of University Library, University of Kragujevac was hand searched for relevant studies by two authors independently (AP and MM).

Data collection and analysis
The data collection sheet was created and the articles included in review were assessed for: 1) study ID; 2) report ID; 3) review author initials; 4) citation and contact details; 5) eligibility for review; 6) study design; 7) total study duration; 8) risk of bias (randomization if any, sequence generation, allocation sequence concealment, blinding, other concerns about bias); 9) total number of patients; 10) age of patients; 11) sex of patients; 12) setting; 13) country; 14) frequency of asthma exacerbations during the last year prior to inclusion in the study; 15) frequency of clinical asthma exa-cerbations per patient during the study treatment period; 16) mean change in FEV1 from baseline over 16 weeks of treatment; 17) mean change in FVC from baseline; 18) mean change in forced expiratory flow (FEF) at 25% to 75% of FVC (FEF25-75%) from baseline; 19) mean change from baseline in ACQ-7 score; 20) mean change from baseline in ASUI score; 21) frequency of rescue use of short-acting βagonist per patient; 22) mean blood eosinophil count after 16 weeks of treatment; 23) mean change from baseline in AQLQ total score; 24) percentage of patients developing anti-reslizumab antibodies during the treatment course; 25) number of different intervention groups (reslizumab, placebo); 26) route of administration; 27) dose regimen; 28) duration of administration; 29) incidence of adverse events; 30) treatment discontinuation due to side effects.Values provided as percentages were converted into actual patient numbers for analysis as well as standard errors into standard deviations using number of patients, when reported as such.

Selection of studies
Based on the searching strategy, all titles and abstracts retrieved were independently scanned by five authors (AP, MM, MK, VO and JM).Eligibility of the retrieved articles was assessed at first from the title and the abstract, and if it was not possible, the full text of the articles was retrieved and searched.An article was included for review if all authors (AP, MK, JM, VO and MM) agreed that eligibility criteria had been met.In case that the reviewers had different opinions about eligibility of a study for inclusion, the matter was resolved by the corresponding author (SJ).

Data extraction and management
The data were extracted from eligible studies using the data collection sheet described previously (under the "data collection and analysis" subheading).The data collection sheet was made in electronic form, using an Excel 2007 worksheet.The data were extracted by four investigators independently (AP, MK, VO and MM) and then collating of the four tables was done by another investigator (JM), who produced the final extraction table.

Assessment of risk of bias in included studies
Risk of bias was assessed by two investigators independently (MK and JM), and collating the assessments was done by the corresponding investigator (SJ).The following sources of bias were assessed: 1) randomization if any; 2) sequence generation; 3) allocation sequence concealment; 4) blinding; 5) performance bias; 6) detection bias; 7) attrition bias; and 8) reporting bias.None of the studies had high risk of bias, so none was excluded from further analysis.

Measure of treatment effect
The following outcomes used in the studies were continuous: frequency of clinical asthma exacerbations per patient during the study treatment period, change in FEV1 from baseline over 16 weeks of treatment, change in FVC from baseline, change in FEF at 25% to 75% of FVC [FEF25-75%]) from baseline, change from baseline in ACQ-7 score, change from baseline in ASUI score, rescue use of short-acting βagonist, blood eosinophil count and AQLQ total score.For these outcomes the treatment effect was measured by mean difference, since the outcomes were measured on the same scale in all studies.However, two of the continuous outcomes could not be summarized because they were reported in only one of the included studies: frequency of clinical asthma exacerbations per patient during the study treatment period and change in FEF at 25% to 75% of FVC [FEF25-75%]) from baseline.The following outcomes were dichotomous: immunogenicity (whether antibodies are present or not) and adverse events frequency.For these outcomes the treatment effect was measured by odds ratio (OR).

Unit of analysis issues
Unit of analysis in the clinical trials that were included in this meta-analysis were individual patients.Individual participants were randomized to one of two parallel intervention groups, and a single measurement for each outcome from each participant was collected and analyzed.

Dealing with missing data
Missing data were requested directly from the original investigators, however they did not respond to our requests except with courtesy.The missing data were then retrieved from the results presented on ClinicalTrials.gov,when available.Finally, the potential impact of missing data on the findings of the meta-analysis will be addressed in the Discussion section.

Assessment of heterogeneity
Between-study heterogeneity was assessed with the Cochrane Q test using a χ 2 function (p values < 0.10 were considered significant).I² values were calculated to quantify inconsistency across studies.I² values of 30% or less may represent low heterogeneity, values from 30% to 50% may represent moderate heterogeneity, values from 50% to 90% substantial heterogeneity and values of 90% or more may represent considerably heterogeneity.An I² value > 30% was considered significant in this meta-analysis.

Assessment of reporting biases
The possibility of within-study selective outcome reporting was examined for each study included in this metaanalysis.First, by constructing matrix of the outcomes for all studies, we identified studies and specific outcomes that were not reported.Then we searched for published protocols of such studies at ClinicalTrials.gov and other forms of publications of the same studies, in order to find the missing outco-mes.Finally, the authors were contacted with a request to provide the missing data, but they did not send us the data.The possibility of between-study publication bias was examined by construction of funnel plots for continuous outcomes and by Egger's regression for discrete outcomes 18 .Klein's number was also calculated for all outcomes 19 .

Data synthesis
The random effects model (which includes both withinstudy and between-study variations in calculation of the weighted average) was used to combine the results from the studies.The Mantel-Haenszel method (fixed effect model) was also used to estimate how our conclusions could be influenced by assumptions about the model and by the study heterogeneity.Since significant heterogeneity of the studies was not found, subgroup analysis was not performed.All calculations were done by Review Manager (RevMan) software version 5.3.5 20 .

Sensitivity analysis
Sensitivity analysis was performed by excluding individual trials one at a time and recalculating the pooled odds ratio and mean difference estimates for the remaining studies.In this way we got insight how each of the included studies influenced our conclusions.

Results
Results of the literature search are shown in Figure 1.Only five clinical trials [21][22][23][24] fulfilled all inclusion and missed all exclusion criteria which were set prior the study commencement (two of the trials were published in the same publication.Characteristics of the included studies with risk of bias are shown in detail in Table 1. Summaries of differences in effects of reslizumab vs. placebo for the main outcomes (using random effects model) were as following: reslizumab 3.0 mg/kg produced substantial improvements in FEV1 (mean difference 0.15) and in FVC (mean difference 0.21) over the 15 or 16-week treatment period; substantial decrease versus placebo in ACQ score (mean difference -0.28), substantial increase vs. placebo from baseline in AQLQ total score (mean difference 0.24) rescue inhaler use (mean difference -0.33) and blood eosinophil count after 15 or 16 weeks of treatment (mean difference -478.17) was observed with reslizumab 3.0 mg/kg; reslizumab 3.0 mg/kg caused less adverse events versus placebo (odds ratio 0.67), especially asthma worsening (odds ratio 0.53) or bronchitis (odds ratio 0.42) while there was no significant difference for nasopharyngitis (odds ratio 0.97) or upper respiratory tract infection (odds ratio 0.86).Details of the summaries of differences in effects are shown in Tables 2  and 3. Sensitivity analysis did not show significant changes with exclusion of single trials.Summaries of differences in effects of reslizumab and placebo for the most important outcomes (improvements in FEV 1 and decrease in ACQ score) with heterogeneity estimates are shown by Forest plots (Figures 2 and 3).
The reporting bias was assessed by Funnel Plot, using "trim and fill" method for continuous outcomes.The central symmetry axis of Funnel Plots for all tested continuous outcomes did not change place significantly after "trim and fill" exercise.In Figures 4 and 5  For discrete outcomes (frequencies of adverse effects) the reporting bias was assessed by Klein's number and Egger's regression.Klein's number for total incidence of adverse effects was 4.52 and the Egger's regression showed minimal correction of the summary effect estimate: from OR = 0.67 to OR = 0.42 (Figure 6).For percent of patients developing anti-reslizumab antibodies the Klein's number was 6.79, and OR was corrected by the Egger's regression from 19.66 to 42.23.Klein's number for incidence of asthma worsening was 16.92 and the Egger's regression corrected the summary effect estimate: from OR = 0.53 to OR = 2.25.For incidence of nasopharyngitis, incidence of upper respiratory tract infection and incidence of bronchitis the Klein's numbers were -3.96, -3.45 and 5.48, respectively, and OR was corrected by the Egger's regression from 0.97 to 2.47, from 0.86 to -1.16, and from 0.42 to -0.83, respectively.

Discussion
Results of our study showed that reslizumab is significantly more efficient than placebo in the treatment of severe, poorly controlled eosinophilic asthma.Our results indicated that reslizumab led to significantly greater increase in FEV1 and FVC compared to placebo.On the other hand, reslizumab led to greater reduction of ACQ score and blood eosinophil counts compared to placebo, which also suggested that administration of reslizumab in patients with eosinophilic asthma had significant benefits.These results were in agreement with results of the recently published metaanalysis of reslizumab efficacy and safety 25 .However, in this meta-analysis authors did not analyze impact of reslizumab to AQLQ score, as it was done in another meta-analysis which compared effects of mepolizumab (also monoclonal antibody against IL-5) with placebo in the same clinical entity 26 .After we summarized results of clinical trials with reslizumab in our meta-analysis, it turned out that it improved the AQLQ score much more than placebo.Therefore, reslizumab not only had beneficial effects on clinical outcomes of severe, poorly controlled eosinophilic asthma, but it also markedly improved quality of life of these severely ill patients.Heterogeneity of the included studies was low and publication bias small, so effects were consistent from study to study.
Our meta-analysis indicated that reslizumab use was associated with significantly lower overall incidence of adverse events, asthma worsening and bronchitis compared to placebo.In addition, there was no significant difference in incidence of nasopharyngitis and upper respiratory tract infections in general between reslizumab and placebo.Our results are in agreement with results of the recently published metaanalysis of reslizumab efficacy and safety which has reported that there was no difference in proportion of individuals who withdrew due to adverse events as well as in incidence of upper respiratory adverse events 25 .Although reslizumab use can be associated with development of anti-reslizumab antibodies, they did not appear to have impact on its efficacy and safety and their appearance is transient 21,27 .All these imply that reslizumab is generally well tolerated, although longerterm safety still needs to be assessed.The risk of anaphylaxis, which was reported in 0.3% of the patients, remains the main reason why reslizumab should be administered in a health care setting where patient can be observed and managed properly in the case of allergy 28 .
Our results should be taken with certain reserve, since some of the important clinical outcomes were reported in only one of the included studies [frequency of clinical asthma exacerbations per patient during the study treatment period and change in forced expiratory flow from 25% to 75% of FVC (FEF25-75%) from baseline], and overall number of the included studies was low.Since several clinical trials with reslizumab are ongoing, new meta-analysis should be made in close future to challenge our results.

Conclusion
On the basis of published clinical trials reslizumab could be considered as effective and safe therapeutic option for severe, poorly controlled eosinophilic asthma for the time being.Future studies which would include ongoing clinical trials are necessary to confirm this conclusion.

Fig. 3 -
Fig. 3 -Summary of differences in forced expiratory volume in 1 second (FEV1) with reslizumab vs. placebo from baseline over 16 weeks of treatment.SD -standard deviation; CI -confidence interval.