The novel inhaled dual PDE3 and PDE4 inhibitor ensifentrine for the treatment of COPD: A systematic review and meta-analysis protocol on trough FEV1 and exacerbation according to PRISMA statement

The investigation of ensifentrine, an inhaled dual phosphodiesterase (PDE)3 and PDE4 inhibitor, for chronic obstructive pulmonary disease (COPD) maintenance therapy presents a significant clinical interest. Despite promising results from recent Phase III trials, a comprehensive synthesis of its therapeutic efficacy in COPD is lacking. This protocol outlines the first registered systematic review and meta-analysis in PROSPERO to assess the impact of ensifentrine on trough forced expiratory volume in the 1st second (FEV1) and acute exacerbations of COPD. By conducting a rigorous literature search and employing solid methodologies, this endeavour aims to provide robust evidence on the real efficacy of ensifentrine. Anticipated outcomes include a significant improvement in trough FEV1 and a reduction in AECOPD risk among ensifentrine-treated patients compared to controls, corroborating its bronchodilator and anti-inflammatory properties. The meta-analysis expects to reveal consistent results across different trials, enhancing confidence in the findings. Additionally, subgroup analyses may unveil factors influencing the efficacy of ensifentrine, guiding optimal therapeutic strategies. Overall, this protocol holds the potential to inform clinical practice and regulatory decisions, positioning ensifentrine as a valuable addition to COPD management.

According to its pharmacological characteristics, ensifentrine combines both bronchodilator and anti-inflammatory properties, distinguishing this molecule from any other COPD treatment that typically targets only one of these outcomes (Calzetta et al., 2024;Cazzola et al., 2023).Given the significant likelihood of its approval in the near future, ensifentrine could represent a major breakthrough.Notably, it has been over a decade since a drug with a novel mechanism of action was approved for the maintenance treatment of COPD (Calzetta et al., 2023(Calzetta et al., , 2024)).
The measurement of trough forced expiratory flow in the 1st second (FEV 1 ) is pivotal in COPD patients as it represents a global marker for pathophysiological changes, is strictly related to patient-reported outcomes, and is used by regulatory agencies for the drug approval process (Donohue et al., 2018;Westwood et al., 2011;Jones et al., 2011;Jones and Agusti, 2006).Nevertheless, trough FEV 1 has never been investigated as a primary endpoint in the RCTs constituting the pipeline development of ensifentrine in COPD (Pipeline -Verona Pharma).Generally, FEV 1 area under the curve at 0-12 h (AUC 0-12h ) has been identified as a primary outcome, relegating trough FEV 1 to a less important secondary outcome (NCT04542057, NCT04535986, NCT04027439, NCT04091360).
This represents an important issue for development strategy of ensifentrine, particularly given that the minimal clinically important difference (MCID) value, which regulatory authorities use to interpret drug efficacy in COPD trials, is available for trough FEV 1 but not for FEV 1 AUC 0-12h (Jones et al., 2014;Cazzola et al., 2008;Manzetti et al., 2023).
Therefore, considering the current background and scientific evidence, the primary aim of this paper is to provide a protocol for conducting a high-quality systematic review and meta-analysis on the effect of ensifentrine on trough FEV 1 in COPD patients.In line with the importance of pharmacological therapy for the prevention of AECOPD (MacLeod et al., 2021;Wedzicha et al., 2017), this systematic review and meta-analysis also examine this outcome.

Search strategy
The protocol of this systematic review and meta-analysis is registered in International Prospective Register of Systematic Reviews (PROS-PERO; registration number: CRD42023478153) in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols (PRISMA-P) (Moher et al., 2015).
Two reviewers perform a comprehensive literature search for RCTs evaluating the effects of ensifentrine in patients with COPD.The PICO (patient problem, intervention, comparison, and outcome framework) is used to develop the literature search strategy, as previously described (Schardt et al., 2007).Specifically, the patient problem includes subject affected by COPD; the intervention is ensifentrine, administered either

Study selection
RCTs involving patients with COPD treated with ensifentrine are included in this meta-analysis, while open-label studies are excluded.Two reviewers independently check the relevant studies identified from literature searches obtained from the previously mentioned databases.The titles and abstracts of all the records identified in initial research are reviewed, and then a list of full text articles to be assessed for selection is defined.RCTs reporting data concerning the impact of ensifentrine on patients suffering from COPD are selected, without limitations for study duration.Full text articles not reporting data from RCTs, and/or performed in non-COPD patients, and/or not reporting trough FEV 1 and/or acute AECOPD in the MEDLINE are excluded.The studies are selected in accordance with the previously mentioned criteria, and any difference in opinion about eligibility are resolved by consensus.

Quality of studies, risk of bias, and evidence profile
The summary of the risk of bias for each included RCT is analysed via the Jadad score (Halpern and Douglas, 2005) and Cochrane Risk of Bias 2 (RoB2) (Guyatt et al., 2011).The Jadad score, with a scale of 1-5 (score of 5 being the best quality), is used to assess the quality of the RCTs regarding the likelihood of biases related to randomization, double blinding, withdrawals, and dropouts (Jadad et al., 1996).Studies are considered of low quality at Jadad score <3, of medium quality at Jadad score = 3, and of high quality at Jadad score >3 (Halpern and Douglas, 2005).The weighted assessment of the risk of bias is analysed via the Cochrane RoB 2 (Guyatt et al., 2011).
Funnel plot and Egger's test are performed to assess the origin and risk of publication bias for the primary endpoint if ≥ 10 studies are included in the meta-analysis (Page et al., 2019).For Egger's test, the following regression equation is applied: SND = a + b × precision, where SND represents the standard normal deviation (treatment effect divided by its standard error [SE]), and precision represents the reciprocal of the SE.Evidence of asymmetry from Egger's test is considered to be significant for P < 0.01 and the graphical representation of 90% confidence bands is reported (Sterne et al., 2000;Sterne and Egger, 2001;Egger et al., 1997).
The quality of the evidence is assessed for the primary endpoint in accordance with the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) system, indicating ++++ for high quality of evidence, +++ for moderate quality of evidence, ++ for low quality of evidence, and + for very low quality of evidence (Guyatt et al., 2011).Two reviewers independently assess the quality of individual studies and any difference in opinion is resolved by consensus.

Data extraction
The data sourced from RCTs are extracted and verified through a comprehensive examination of published papers.The extraction process involves several studies and patients characteristics, including the study duration, inhaler device, age, gender, smoking habits, FEV 1 , AECOPD, and items to calculate the Jadad score.Data extraction is conducted independently by two reviewers and any and any discrepancy or inconsistency encountered during the extraction process is resolved by consensus.Due to the complexity of this meta-analysis, data are extracted in accordance with the recommendations provided by the Cochrane Handbook for Systematic Reviews of Interventions (Higgins and Green, 2011).

Endpoints
The primary endpoint is the effect of ensifentrine on the change in trough FEV 1 in COPD patients; the secondary endpoint is the effect of ensifentrine on the risk of moderate or severe AECOPD.

Data analysis
A pairwise meta-analysis is performed to quantify the effect of ensifentrine on the primary and secondary endpoints.The obtained results are expressed as mean difference (MD) or relative risk (RR), according to the analysed variables, with 95% confidence interval (95% CI).
Generally, data from RCTs are extracted from a series of studies performed by researchers operating independently, and therefore a common effect size cannot be assumed.Thus, the binary derSimonian-Laird random-effects model is used to balance the study weights and correctly assess the effect estimates and relative 95%CI (Calzetta et al., 2016).
The test for heterogeneity (I 2 ) is performed to quantify the betweenstudy dissimilarity, as previously reported (Wallace et al., 2012), and sensitivity analysis is carried out to identify the studies that introduce substantial and significant levels of heterogeneity (I 2 >50%, P < 0.05) in the quantitative synthesis (Higgins et al., 2003).If necessary, meta-regression analysis is performed for the primary endpoint to identify potential effect modifier that could alter the efficacy of ensifentrine on trough FEV 1 (Cazzola et al., 2017).
Subgroup analyses are carried out with respect to the specific maintenance COPD therapies to which ensifentrine is added and the dose of ensifentrine under evaluation by the FDA.

Software and statistical significance
GraphReader is used to extract data from the figures, when necessary (http://www.graphreader.com/);OpenMeta-Analyst (version 12.11.14,Wallace et al., Tufts University, Boston, MA, USA) (Wallace et al., 2012) software is used to perform the pairwise meta-analysis; GraphPad Prism (version 7.0a, GraphPad Software Inc., San Diego, CA, USA) software is used to graph the data; GRADEpro GDT software (online version available from gradepro.org,McMaster University and Evidence Prime Inc., Hamilton, ON, Canada) is used to assess the quality of evidence (Guyatt et al., 2011), and the robvis visualization software (online version available from mcguinlu.shinyapps.io/robvis/,McGuinness et al., University of Bristol, Bristol, UK) is used to apply the RoB 2 tool (Sterne et al., 2019;McGuinness and Higgins, 2021).The statistical significance of the effect estimates resulting from the pairwise meta-analysis is assessed for P < 0.05.

Expected results
To date, there exists no published systematic review or meta-analysis offering a quantitative synthesis of the therapeutic efficacy of ensifentrine in COPD, making this protocol the first formal endeavour registered in PROSPERO to specifically investigate trough FEV 1 and AECOPD.
This protocol allows performing a high-quality systematic review and meta-analysis on the effect of enisfentrine on trough FEV 1 and AECOPD in COPD patients.The primary endpoint is expected to demonstrate a significant improvement in trough FEV 1 among COPD patients treated with ensifentrine, administered either as a single agent or added on top of any maintenance COPD therapy, compared to those receiving PCB.This improvement should provide robust evidence of the efficacy of ensifentrine as a bronchodilator, potentially meeting or exceeding the MCID value for trough FEV 1 .Additionally, the secondary

Table 1
Pharmacological characteristics of ensifentrine in human inflammatory cells and human medium and small airways.asingle agent or added on top of any maintenance COPD therapy; the comparison includes placebo (PCB); the outcomes are trough FEV 1 and acute exacerbation of COPD (AECOPD).The terms "ensifentrine" OR "RPL554" are searched in ClinicalTrials.gov, Cochrane Central Register of Controlled Trials (CENTRAL), EMBASE, EU Clinical Trials Register, and MEDLINE via PubMed without language restrictions.