The neutrophil elastase inhibitor, sivelestat, attenuates acute lung injury in patients with cardiopulmonary bypass

Background The sivelestat is a neutrophil elastase inhibitor thought to have an effect against acute lung injury (ALI) in patients after scheduled cardiac surgery. However, the beneficial effect of sivelestat in patients undergoing emergent cardiovascular surgery remains unclear. We aim to evaluate the effect of sivelestat on pulmonary protection in patients with ALI after emergent cardiovascular surgery. Methods Firstly, a case-control study in 665 patients undergoing emergent cardiovascular surgery from January 1st, 2020 to October 26th, 2022 was performed. 52 patients who received sivelestat (0.2mg/kg/h for 3 days) and 613 age- and sex-matched controls. Secondly, a propensity-score matched cohort (sivelestat vs control: 50 vs 50) was performed in these 665 patients. The primary outcome was a composite of adverse outcomes, including 30-day mortality, ECMO, continuous renal replacement therapy (CRRT) and IABP, etc. The secondary outcome included pneumonia, ventricular arrhythmias and mechanical ventilation time, etc. Results In propensity-matched patients, the 30-day mortality (16% vs 24%, P=0.32), stroke (2% vs 8%, P=0.17), ECMO(6% vs 10%, P=0.46), IABP(4% vs 8%, P=0.40) and CRRT(8% vs 20%, P=0.08) had no differences between sivelestat and control group; sivelestat could significantly decrease pneumonia (40% vs 62%, P=0.03), mechanical ventilation time (median: 96hours, IQR:72-120hours vs median:148hours, IQR:110-186hours, P<0.01), bilateral pulmonary infiltrates (P<0.01), oxygen index (P<0.01), interleukin-6(P=0.02), procalcitonin(P<0.01) and C-reactive protein(P<0.01). Conclusion Administration of sivelestat might improve postoperative outcomes in patients with ALI after emergent cardiovascular surgery. Our results show that sivelestat may be considered to protect pulmonary function against inflammatory injury by CPB. Registration http://www.chictr.org.cn/showproj.aspx?proj=166643, identifier ChiCTR2200059102.


Introduction
Cardiopulmonary bypass (CPB) is a necessary life support during open-heart surgery. Systemic inflammatory response syndrome (SIRS) caused by CPB has been well known to increase postoperative morbidity and mortality (1,2). Acute respiratory distress syndrome (ARDS) and acute lung injury (ALI), which are characterized by pulmonary edema associated with SIRS, are also induced after CPB and significantly contribute to postoperative morbidity and mortality (3)(4)(5)(6).
Documented components of the inflammatory reaction include the activation of complement, increased surface expression of adhesion molecules on leukocytes, and the presence of proinflammatory cytokines in the systemic circulation (7-12). Neutrophils which is a main part of leukocytes plays an important role in SIRS through the production of superoxide radicals and the release of chemical mediators (12,13). It has been proven that activated neutrophils is one of the most important initiating events of pulmonary dysfunction induced by CPB (14).
Sivelestat is a synthetic, specific, low molecular-weight neutrophil elastase inhibitor (15). It has been shown to reduce both neutrophil elastase levels and interleukin-6 production and to preserve neutrophil deformability during extracorporeal circulation (6,16,17). Several clinical studies have shown the beneficial effect of sivelestat for patients undergoing cardiovascular surgery with CPB (6,12). However, these studies evaluated only the scheduled cardiac surgery. The emergent cardiovascular surgery usually had more severe ALI compared with scheduled cardiac surgery (15,18). This agent may prevent the adverse reaction of SIRS and could be one of the best therapies to attenuate ALI in patients undergoing emergent cardiovascular surgery. We, therefore, designed this study to evaluate the effect of sivelestat on pulmonary protection in patients with ALI after emergent cardiovascular surgery.

Study population
This study is an investigator-initiated cohort study. It was initiated on January 1 st , 2020, and completed on October 26 th , 2022. This study was approval by the ethical committee of Nanjing Drum Tower Hospital (2022-102-01) and registered in the Chinese Clinical Trial Registry (ChiCTR2200059102). The study enrolled 665 patients with CPB from the department of cardiothoracic in Nanjing Drum Tower Hospital. Before enrollment, all patients had signed informed consent forms.
The inclusion criteria were as follows: adult patients undergoing on-pump emergent cardiovascular surgery; aged > 18 years old, oxygen partial pressure/fraction of inspired O 2 (PaO 2 /FiO 2 ) <150mmHg at the end of CPB.
Based on the retrospective review of our institution's database, 816 patients met the inclusion and exclusion criteria in our study. The sivelestat was routinely used in our hospital on the 1 st January 2022. Therefore, 52 patients who received sivelestat were assigned to the sivelestat group. To investigate whether sivelestat could improve outcomes, we selected 613 matched controls from the remaining 764 patients (control group). The control subjects were selected for each case and matched for sex and age (± 2 years).

Medical intervention
This section was available in the online supplement.

Statistical analysis
The SPSS statistical software (version 24) was used for analysis. The mean ± SD or median (interquartile range) were used to present continuous variables. Discrete variables are depicted as frequencies (n, %). The Student's t-test was used to evaluate normally distributed continuous variables. The Mann-Whitney U nonparametric method was used for non-normally distributed continuous variables. Continuous variables were determined to be normally distributed by the Shapiro-Wilk test. The chi-Square test or Fisher's exact test was used to compare categorical data. Differences between the two groups were also analyzed by repeated measures ANOVA considering the repeated measurements from POD1 to POD5. R software (version:4.2.2) was used for univariate analysis. In univariate analysis, the study cohort (n=665) was divided into the adverse events group and the control group. The adverse events include 30day mortality, ECMO, CRRT, IABP, and stroke. It is the same as the definition of primary outcomes. Covariates reaching statistical significance (P ≤ 0.10) in the univariate analysis were entered into a forward selection multivariable logistic regression model. Collinearity diagnostics were performed using tolerance estimates for individual variables in a linear regression model.
The possibility of the existence of bias may affect our findings. To avoid bias, we used a propensity score to adjust the study cohort. This methodology permitted the comparison of patients who received sivelestat against control with a similar risk profile. Propensity score 1-to-1 matching was utilized with the nearest neighbor algorithm without replacement and a 0.01 caliper set (23). Age, gender, body mass index (BMI), left ventricular ejection fraction (LVEF), type of cardiac surgery, CPB, DHCA, and diabetes were put into a logistic regression model to estimate the propensity score. The standardized mean differences (absolute SMD < 10%) are used to assess pre-match imbalances and post-match balance. A P value of < 0.05 was considered statistically significant.

Results
After propensity matching, a total of 100 patients were enrolled in our study (Figure 1, 50 in the sivelestat group and 50 in the control group). Before and after propensity-score matching, the SMD of age, gender, BMI, LVEF, type of cardiac surgery, CPB, DHCA, and diabetes were shown in eFigure 1 (online supplementary). The absolute SMDs of these variables were <10%. Therefore, these variables were well balanced after propensity matching.
In our study cohort, the baseline and demographic variables were not different between the two groups ( Table 2). There were no gastrointestinal disturbances observed in the two groups. There were 65 patients who underwent emergent on-pump coronary artery bypass grafting (CABG), 383 patients who underwent aortic arch replacement because of aortic dissection, 26 patients who underwent emergent left atrial myxoma resection, 4 patients who underwent emergent repair of ruptured sinus of valsalva aneurysm, and 187 patients who underwent emergent valvular replacement because of acute heart failure or left atrial thrombus. Before propensity matching, adverse events occurred in 38 patients FIGURE 1 Enrollment flow chart.    Figure 2 which applied log2 transformation.
The baseline (admission to ICU) of oxygen index (PaO 2 /FiO 2 ) was a well balance between the two groups ( Table 4). The postoperative inflammatory biomarkers, including WBC count, neutrophil, CRP, IL-6, and PCT, were significantly different between the sivelestat and control group (P<0.05). Compared with the control group, the sivelestat group showed a significantly decreased level of IL-6 (P<0.01), CRP(P<0.01) and PCT (P<0.01) on the POD3. Moreover, the sivelestat group also had lower levels of CRP, PCT, and IL-6 on POD2 (P<0.05). When analyzing with repeated measures ANOVA test to take repeated measurements into account, the sivelestat group still had a lower level of PaO 2 / FiO 2 ( Figure Table 4.

Discussion
Acute lung injury after CPB, which is one of the most serious inflammatory reactions induced by CPB, has significantly increased morbidity and mortality (3)(4)(5). Neutrophil activation is an important initiating event of this phenomenon (14). An imbalance between neutrophil elastase and its endogenous protease inhibitors has been considered to be a possible mechanism by which neutrophil elastase causes lung tissue destruction (14). The sivelestat which is a neutrophil elastase inhibitor has been proven to be useful for anti-ALI in scheduled cardiac surgery (6,12). However, it is still unknown what is the effect of sivelestat on emergent cardiovascular surgery. Our study showed that the postoperative adverse outcomes were acceptable (5.71%) in patients who underwent emergent cardiovascular surgery; the pre-operative LVEF<35%, postoperative pneumonia, and the BPI on chest X-ray were independent risk factors for postoperative adverse outcomes; the administration of sivelestat might improve postoperative outcomes in patients with ALI after emergent cardiovascular surgery. According to our findings, sivelestat may be considered to protect pulmonary function against inflammatory injury by CPB.
Activated neutrophils adhere to pulmonary vascular endothelial cells by expression of adhesion molecules and damage endothelial cells (5,(7)(8)(9). Moreover, activated neutrophils stimulate the production of elastase, superoxide, and cytokines (5,(7)(8)(9)11). It has resulted in hyperpermeability of pulmonary capillaries and interstitial edema of the lung (13,14). The ALI/ARDS are serious complications after open heart surgery. They could lead to 15% to 28% mortality in patients with CPB. Therefore, an agent which could inhibit neutrophil or neutrophil elastase may be useful and helpful to prevent ALIrelated poor outcomes. Several previous studies have reported that the neutrophil elastase inhibitor, sivelestat, could significantly improve postoperative outcomes in patients with CPB (6,12,15,18). However, their study populations contained congenital heart patients (6,12) or a small sample (only enrolled 14 patients) (18). Morimoto et al. reported (15) that prophylactic administration of sivelestat at the initiation of CPB results in better postoperative pulmonary function, leading to earlier extubation time in patients who underwent total arch replacement. However, they excluded patients who underwent emergent surgery. It is well known that emergent operation has more serious SIRS compared with scheduled surgery. Our work found that sivelestat could improve the outcomes in ALI patients after CPB. It is filled a gap in emergent cardiac surgery.
The PaO 2 /FiO 2 ratio is well established as a parameter that quantifies impaired respiratory function (24). The moderate ALI/ ARDS was defined by PaO 2 /FiO 2 ratio ≤ 200mmHg and > 100mmHg; the severe ALI/ARDS was defined by PaO 2 /FiO 2 ratio ≤ 100mmHg (24). Our study cohort enrolled the patients who had PaO 2 / FiO 2 ratio < 150mmHg at the end of CPB. It could ensure that the patients with ALI after CPB were enrolled in the study population. And patients with fluid overload and cardiogenic shock were excluded. Meanwhile, we used methods, including case-control and propensity score, to ensure the maximized homogeneity between the two groups. Furthermore, in our study, the inflammatory biomarkers (PCT, CRP, and IL-6) were decreased in the sivelestat group. It strengthened the evidence for the conclusion that anti-neutrophil therapies could reduce the severity of ALI. Finally, we found that WBC count and neutrophil had no difference among the two groups. The sivelestat is an inhibitor of neutrophil elastase. It does not work on the absolute value of leukocytes or neutrophils. It may be the reason why no differences in WBC count and neutrophil among the two groups.
Liu et al. reported that patients with acute type A aortic dissection (ATAAD) usually had severe inflammatory reactions, which resulted in poor short-term outcomes (25). Thourani et al. reported that emergent status on mitral valve replacement surgery and coronary artery bypass grafting (CABG) significantly increased morbidity and mortality (26). Because emergent cardiovascular surgery easily leads to severe inflammation (27). Sivelestat, which is a potential antiinflammatory drug, may have positive effects on organ protection in patients undergoing emergent cardiovascular surgery. Our study added evidence to this hypothesis. However, our finding, which needs to be confirmed in a larger sample random control trial to assess clinical endpoints, suggests a potential role of sivelestat in the alleviation of postoperative inflammation.
In conclusion, the administration of sivelestat could improve postoperative outcomes in patients with ALI after emergent cardiovascular surgery. Our results show that sivelestat may be considered to protect pulmonary function against inflammatory injury by CPB.

Study limitation
Our study design involves a single center's experiences with the inherent disadvantages of a retrospective study, which is highly prone to bias. This observational study could be influenced by potential biases. We used propensity score matching to avoid these biases. However, factors that affect assignment to treatment and outcomes but that cannot be observed cannot be accounted for in the matching procedure. Any hidden bias due to latent variables might remain after matching, which could lead to some statistical faults. Moreover, with this analysis, we remove a large number of patients from the analysis but may have elevated statistical errors. Thus, the implementation of the prognostic value of sivelestat should be assessed in future studies. Furthermore, to our best knowledge, no published clinical trials reported positive or negative effects on gastrointestinal function. Gastrointestinal disturbances were also not observed in our study. It may need to confirm in the next prospective clinical trial. Furthermore, the pre-operative lung function is necessary to submit. However, lung function is not routinely implemented in our hospital. The lack of lung-function data may lead to some errors. Finally, our study found that pre-operative ACEi/ARB was an independent risk factor for postoperative adverse outcomes. ACEi/ ARB are well-known drugs that could result in the greatest mortality reduction in patients with heart failure (28). Patients with heart failure with reduced ejection fraction were usually suggested to administrate ACEi/ARB (28). The pre-operative LVEF<35% was also proven to be an independent risk factor for postoperative adverse outcomes in our study. In another word, ACEi/ARB might be a confounding factor. A high-quality clinical trial might be needed to be implemented if we wanted to conclude the negative effect of ACEi/ARB on patients who underwent emergent cardiovascular surgery. These limitations of this assessment are important and should be acknowledged.

Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics statement
The studies involving human participants were reviewed and approved by the ethical committee of Nanjing Drum Tower Hospital. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

Author contributions
TP, TT, and XC carried out the study, participated in the statistical analysis, and drafted the manuscript. C-YJ and Y-FZ participated in the design of the study. Z-SL and X-YJ participated in the sequence alignment. H-TZ and HZ performed the statistical analysis. Y-PW, WC, L-CL, and MG participated in the data collection. Y-QC, D-JW, and QZ conceived the study, participated in its design and coordination, and helped to draft the manuscript. All authors contributed to the article and approved the submitted version.

Funding
The clinical medical center of Nanjing cardiovascular diseases (CZLB506-2020).