Research progress on the effect of neutrophil elastase and its inhibitors in respiratory diseases

Abstract

Neutrophil elastase is a serine protease secreted by neutrophils that plays a crucial role in the onset and progression of various respiratory diseases. The imbalance between neutrophil elastase and its endogenous inhibitors can contribute to the onset and progression of respiratory diseases such as chronic obstructive pulmonary disease, cystic fibrosis, bronchiectasis, and acute respiratory distress syndrome. The excessive release of neutrophil elastase contributes to multiple pathophysiological processes, such as disruption of the alveolar–capillary barrier, oxidative stress and inflammatory responses, autophagy, and apoptosis. The use of neutrophil elastase as a biomarker in disease assessment and prognosis prediction as well as the potential of neutrophil elastase inhibitors in clinical treatment have shown broad application prospects. Neutrophil elastase inhibitors have certain limitations such as off-target effects, poor lung bioavailability, cost considerations, and variability in patient responses. This article reviews the literature on neutrophil elastase and its inhibitors in the field of respiratory diseases, aiming to provide new insights into the diagnosis and treatment of related respiratory diseases.

Introduction

Neutrophil elastase (NE) is a serine protease secreted by neutrophils that plays a crucial role in maintaining lung tissue homeostasis and defending against pathogen invasion. Under pathological conditions, a large amount of NE is released and accumulated, evading the regulation of various endogenous inhibitors. NE participates in multiple processes, including disruption of the alveolar–capillary barrier, oxidative stress and inflammatory responses, autophagy, and apoptosis. ¹ In recent years, with the elucidation of the biological functions and pathological roles of NE, its clinical significance in respiratory diseases has garnered increasing attention. This article reviews the pathophysiological role, current clinical applications, and future development directions of NE in respiratory diseases as well as the research progress and clinical application prospects of NE inhibitors to provide new insights into the diagnosis and treatment of related diseases. This narrative review was guided by the Scale for the Assessment of Narrative Review Articles (SANRA). ²

Sources and selection criteria of articles

We searched PubMed and Web of Science databases for peer reviewed articles from February 2010 to January 2025 to identify the most recent literature, using the following search terms: “Neutrophil elastase,” “Neutrophil elastase inhibitors,” “Endogenous neutrophil elastase inhibitors,” “Synthetic neutrophil elastase inhibitors,” and “respiratory diseases.” We prioritized systematic reviews, meta-analyses, randomized controlled trials, and large-scale studies from high impact general medicine and critical care journals.

Biological characteristics of NE

There are four types of serine proteases present in neutrophils—NE, proteinase 3, cathepsin G, and neutrophil serine protease 4—with NE being the most abundant. ³ NE is stored in the azurophilic granules of mature neutrophils. It consists of 218 residues and has a molecular mass of 29.5 kDa. ⁴ Under physiological conditions, NE participates in the regulation of inflammatory responses and promotes the phagocytic elimination of harmful pathogens, thus serving as an essential component of the body’s defense system. However, in certain diseases, the excessive release of NE can have detrimental effects. It can degrade elastin, collagen, fibronectin, and other extracellular matrix components of endothelial tissues. This degradation increases tissue permeability, which in turn promotes the extravasation of neutrophils and release of inflammatory factors.

Pathophysiological effects of NE

In respiratory diseases, the pathophysiological effects of NE are primarily manifested as the destruction of lung tissue structure and modulation of inflammatory responses. Moreover, NE plays a crucial role in immune function modulation. Specifically, it promotes mucus secretion, which in turn contributes to airway remodeling. Additionally, it creates a microenvironment conducive to the proliferation of tumor cells and may facilitate their metastasis under certain circumstances.

Direct destruction of lung tissue structure

NE is a potent protease capable of degrading various extracellular matrix components, such as elastin, collagen, fibronectin, and proteoglycans. These components are indispensable for the maintenance of the structural integrity and normal physiological function of lung tissue. Elastin is a major component of alveolar and vascular walls. Excessive activation of NE can lead to the degradation of elastin, which in turn causes alveolar wall destruction and emphysema. ⁵ Furthermore, collagen serves as the primary structural protein of lung tissue. Excessive activation of NE can also target collagen for degradation, leading to pulmonary fibrosis and a decline in lung function.

Regulation of inflammatory responses

NE plays a dual role in inflammatory responses, both promoting the aggregation and activation of inflammatory cells and enhancing the inflammatory response by regulating inflammatory mediators. Specifically, NE can cleave chemokines (such as interleukin (IL)-8) and cytokine precursors (such as tumor necrosis factor-α precursor) to generate biologically active fragments, thereby promoting the aggregation and activation of inflammatory cells. Additionally, NE can enhance the inflammatory response by modulating the Toll-like receptor 4-nuclear factor kappa B (NFκB) inflammatory signaling pathway. Excessive activation of NE can lead to sustained activation of NFκB, which in turn causes chronic inflammation. ⁶

Dysfunction of immune defense

NE plays a pivotal role in the clearance of microorganisms and immune defense in the lungs. It serves as a crucial effector molecule in the host’s defense mechanism against pathogen invasion. However, its excessive activation may lead to tissue damage and chronic inflammation. A key mechanism by which excessive NE activity can compromise lung health is proteolytic inactivation. Peptides are essential components of the innate immune system, providing a first line of defense against a wide range of microorganisms. The inactivation of antimicrobial peptides by NE impairs the host’s overall defense capabilities, heightening susceptibility to lung infections. This can result in the persistence of infections and the development of chronic inflammatory states within the lungs.

Promotion of mucus secretion and airway remodeling

Excessive mucus expression and secretion are hallmarks of chronic inflammatory lung diseases. NE can stimulate goblet cells and mucus gland cells, augmenting mucus secretion. This increase in mucus production can lead to airway obstruction, a key characteristic of chronic bronchitis. Mucin 5AC is a major constituent of airway gel-forming mucin, with Munc13-4 protein serving as an important activator in this context. Notably, NE has been shown to stimulate the secretion of Munc13-4 protein. ⁷ NE participates in the formation of neutrophil extracellular traps after being released from azurophilic granules, which can cause airway remodeling, leading to airway stenosis and decreased lung function. The specific targeting of this pathway by relevant therapeutics may have considerable clinical benefit; however, current clinical trials and evidence are at a very early stage. ⁸

NE and respiratory diseases

Active research is being conducted on the role of NE in various respiratory diseases, and it is a promising biomarker for the diagnosis and assessment of the severity of respiratory diseases.

Chronic obstructive pulmonary disease (COPD)

COPD is a heterogeneous lung condition characterized by chronic respiratory symptoms (dyspnea, cough, expectoration, and/or exacerbations) due to abnormalities of the airways (bronchitis and bronchiolitis) and/or alveoli (emphysema) that cause persistent, often progressive, airflow obstruction. ⁹ In recent decades, no major therapeutic breakthroughs have been reported in the management of COPD. Consequently, early diagnosis and timely identification of acute exacerbations are particularly important in the clinical management of this complex disease. ¹⁰

The role of NE in COPD has been the subject of extensive investigation. NE serves as a key inflammatory mediator within the airways of patients with COPD. Furthermore, NE levels tend to increase in the peripheral blood and serum of patients with COPD. The measurement of NE mRNA expression levels holds promise as a potential predictive marker for acute exacerbations of COPD patients. ¹¹ Research has demonstrated that NE can trigger sustained airway inflammation in COPD patients through the cleavage of histone H3. ¹² This cleavage event activates macrophages, leading to the release of macrophage extracellular traps. This newly identified mechanism provides further insights into the complex role of NE in COPD pathophysiology.

Acute respiratory distress syndrome (ARDS)

ARDS is a grave life-threatening disease with increasing incidence in recent years. Although the associated mortality has decreased, it remains high at 30%–40%. ¹³ The current management of ARDS primarily revolves around supportive care measures. These include meticulous fluid management and implementation of protective mechanical ventilation strategies. However, there is a conspicuous absence of effective pharmacological treatment options for ARDS.

In the initial phases of ARDS, inflammatory chemokines play a pivotal role in promoting neutrophil migration and facilitating the release of NE. Once NE is released, it plays a regulatory role in the infiltration of inflammatory cells as well as the expression and secretion of cytokines and chemokines. Excessive activation of NE is widely regarded as a crucial factor in disrupting the alveolar–capillary barrier, ultimately leading to the development of pulmonary edema. Numerous studies have demonstrated a correlation between the plasma levels of NE and the severity of lung injury, both in animal models and humans with ARDS.^14,15 Furthermore, 11C-NES is a novel positron emission tomography (PET) tracer specific to NE. A study involving four patients with coronavirus disease 2019 (COVID-19)–induced hypoxia and two healthy controls revealed that 11C-NES selectively accumulates in the shadow areas of COVID-19 lesions on PET, indicating elevated levels of NE in these regions. ¹⁶ Consequently, NE is closely associated with severe pulmonary inflammation in COVID-19 patients. This suggests that NE inhibitors hold the potential to alleviate lung inflammation and improve patient outcomes. Exosome-associated NE may represent a novel and promising therapeutic target. The efficacy of NE inhibitors against exosome-associated forms of NE is an important consideration for the treatment of endothelial damage in lung diseases. ¹⁷ A report on 47 ARDS patients with noninvasive ventilation showed that NE inhibitors can improve the lung injury scores and 28-day survival ratio of patients with mild and moderate ARDS (initial oxygenation index (PaO₂/FiO₂) >150 mmHg). ¹⁸

Cystic fibrosis (CF)

CF is a genetic disorder that predominantly impacts the respiratory and digestive systems, affecting approximately 70,000 individuals worldwide. ¹⁹ The disease burden and survival outcomes associated with CF are primarily attributed to the progressive deterioration of lung function, which ultimately progresses to respiratory failure.

The primary pulmonary manifestation of CF is abnormal mucus secretion, leading to airway obstruction, with dysfunction of the protease–antiprotease balance playing a critical role. Significantly elevated NE activity is observed in the airways of CF patients, contributing to chronic infection, inflammation, and lung tissue destruction. A study on pediatric CF patients reported increased NE release from airway neutrophils in all patients, with cellular measurements being correlated with early lung damage. ²⁰ The correlation between sputum NE activity and the severity of lung disease in CF patients suggests that it may serve as a novel biomarker for CF lung disease. ²¹ In the context of mucus-obstructed airways, NE and matrix metalloproteinase (MMP)-12, which are released from activated neutrophils and macrophages, play a crucial role in the initiation and progression of CF pathogenesis. ²² These factors may thus represent potential therapeutic targets for the prevention and/or delay of irreversible structural lung damage in CF patients.

Bronchiectasis (BE)

BE is a chronic airway disease characterized by neutrophilic inflammation, persistent bacterial infection, and structural damage to the bronchi. Moreover, in recent years, the prevalence of BE has increased considerably. NE plays a crucial role in disease progression due to its close association with the key pathological processes of the disease, making NE one of the most promising biomarkers.

NE-activated MMPs can promote the early progression of BE. Consequently, targeted inhibition of NE holds the potential to impede the early development of BE. ²³ A study on pediatric BE patients reported that sputum NE can be considered a good biomarker for the severity of BE, with sputum NE levels correlating with an increased risk of acute exacerbation, higher hospitalization rates, and lower severity scores in BE patients. ²⁴ Currently, bedside assays for measuring NE activity have been implemented in clinical practice. ²⁵ These assays are utilized to identify airway infections in BE patients and predict which patients are at high risk of experiencing an acute exacerbation within the subsequent 12 months, which can help clinicians make more informed treatment decisions.

Other respiratory diseases

Asthma is a chronic inflammatory disorder of the airways, and neutrophil-activated NE plays a key role in the pathological processes of asthma. A study involving 42 children with severe asthma in the pediatric intensive care unit reported a significant increase in NE release in children with severe asthma. ²⁶ This finding indicates that detecting NE levels in asthma patients can be a valuable diagnostic tool for identifying acute asthma exacerbations. Overactivation of NE can promote the occurrence of lung tumors. Animal experiments have shown that NE directly induces the proliferation of lung tumor cells in murine models by entering lung tumor cells and degrading insulin receptor substrate-1. ²⁷ Furthermore, NE plays a crucial role in promoting tumor invasion and metastasis by degrading the extracellular matrix. This degradation disrupts the normal tissue architecture, thereby facilitating the spread of tumor cells. In addition to its direct effects on tumor cells, NE can modulate the tumor microenvironment, thereby influencing the efficacy of immunotherapy.

NE inhibitors

NE inhibitors can be classified as endogenous, synthetic, and natural. Currently, the clinical application of NE inhibitors is mainly focused on diseases such as COPD, ARDS, CF, and BE. Beyond respiratory diseases, NE inhibitors are being explored for other conditions, including psoriasis, rheumatoid arthritis, inflammatory bowel disease, and cancer.

Endogenous NE inhibitors

NE and its endogenous inhibitors work in concert to maintain a delicate equilibrium within the human body, thereby safeguarding tissues from the potentially detrimental effects of hydrolysis. This balance is of utmost importance in preserving the integrity and normal physiological function of various tissues. However, once this balance is disrupted, activated neutrophils can counteract the inhibitory effects of endogenous NE inhibitors, resulting in an imbalance. Endogenous NE inhibitors in the human body include α1-antitrypsin (AAT), secretory leukocyte protease inhibitor, α2-macroglobulin, serine protease inhibitor B1 (Serpin B1), and elafin. These endogenous inhibitors play crucial roles in maintaining the balance with NE.

AAT is the most important endogenous NE inhibitor in the human body, synthesized by alveolar macrophages, neutrophils, and endothelial cells. AAT plays a crucial role in preventing proteolytic tissue damage by inhibiting NE via a suicide substrate mechanism. In COPD treatment, AAT replacement therapy has been approved for patients with AAT deficiency. This therapeutic approach capitalizes on AAT’s capacity to bind to NE, thereby forming the AAT–NE complex. Consequently, this complex effectively reduces NE activity, mitigating its detrimental effects on lung tissue. Consequently, the AAT–NE complex can be regarded as a biomarker of pulmonary inflammation, which is critical for preventing the irreversible stages of the disease. ²⁸ However, in the context of COVID-19 treatment, a study revealed that supplementation with exogenous AAT for the treatment of COVID-19 patients may not be successful, as AAT can be oxidized and inactivated by the highly inflammatory microenvironment, thus preventing the formation of the AAT–NE complex. ²⁹

Synthetic NE inhibitors

Recent research on NE inhibitors has primarily focused on synthetic NE inhibitors. In addition to conducting clinical efficacy studies on the commercially available synthetic NE inhibitor sivelestat, researchers have identified a substantial number of small-molecule compounds that exhibit high selectivity and potent inhibitory properties. These compounds encompass nitrogen-containing heterocyclic compounds and phosphonic acid compounds.

Sivelestat

Sivelestat is a selective NE inhibitor with various beneficial effects in several pulmonary and systemic conditions. It can reverse neutrophil-mediated increases in vascular permeability, thereby mitigating alveolar epithelial and vascular endothelial damage and alleviating the symptoms of acute lung injury (ALI)/ARDS. ³⁰ Moreover, research has shown that sivelestat significantly attenuates lipopolysaccharide-induced ALI/ARDS in mice during the recovery from neutropenia. ³¹ Another animal experiment reported that sivelestat treatment significantly improved the survival rate of mice with gefitinib-induced severe acute pneumonia. ³² In the context of COVID-19-related respiratory complications, studies on COVID-19-induced ARDS patients have provided compelling evidence regarding sivelestat’s efficacy. Patients treated with sivelestat exhibited improved oxygenation, reduced Murray lung injury scores, shorter intensive care unit stays, and enhanced survival outcomes. ³³ Similarly, a study involving 102 patients with COVID-19–induced ALI/ARDS revealed that sivelestat significantly decreased the levels of several cytokines, such as interferon alpha, IL-1 beta, and IL-2, along with a reduction in mortality. ³⁴ Regarding sepsis-induced ALI, sivelestat has shown differential effectiveness. It is more efficacious in patients with an oxygenation index (PaO₂/FiO₂) ≥140 mmHg and initial serum procalcitonin levels ≥0.5 ng/mL, resulting in improved survival rates. ³⁵ In pediatric ARDS patients, a study demonstrated that the treatment group showed significant improvements in the oxygenation index (PaO₂/FiO₂) at 48 and 72 h. Additionally, the plateau pressure levels at 24, 48, and 72 h were significantly lower in the treatment group than in the control group, with a concomitant reduction in mortality rates. ³⁶ Among patients undergoing lobectomy, a study revealed that NE activity in pulmonary venous blood at the end of surgery was significantly higher than that at the beginning of surgery (during thoracotomy). ³⁷ In patients with ALI after lung resection, sivelestat has been shown to improve lung function, shorten the duration of mechanical ventilation, and reduce mortality. ³⁸ Neutrophil infiltration plays a key role in the pathogenesis of ALI after liver transplantation. An animal experiment demonstrated that sivelestat could reduce pulmonary neutrophil infiltration and oxidative stress in the lungs of rats undergoing liver transplantation, ultimately restoring the function of the pulmonary barrier. ³⁹ Sivelestat also holds therapeutic potential during the fibroproliferative phase of ALI/ARDS. A report on an animal experiment showed that inhibiting NE activity with sivelestat can prevent the development of pulmonary fibrosis after ALI in mice. ¹⁵ Inhalation of sivelestat can be used as a new method for treating ALI. A study found that excessive increase in pulmonary NE activity may play a crucial role in the pathogenesis of oleic acid–induced ALI in rats, and nebulized sivelestat can reduce oleic acid–induced ALI in rats. ⁴⁰

In countries such as China and Japan, sivelestat has been approved for clinical use in the treatment of ARDS. It is important to note that sivelestat causes irreversible inhibition of NE. The N-sulfonyl group of sivelestat sodium forms an irreversible covalent bond with the serine-195 residue of NE, resulting in the formation of an acyl enzyme complex. This mechanism may underlie the side effects associated with sivelestat, including liver damage.

Azd9668

AZD9668 is a novel, orally administered selective NE inhibitor. In a phase II clinical trial involving patients with BE, it was reported that oral AZD9668 (60 mg twice daily for 4 weeks) improved lung function and reduced the levels of sputum inflammatory biomarkers compared with placebo. ⁴¹ Another study involving CF patients demonstrated a significant reduction in the levels of sputum inflammatory biomarkers within the AZD9668 treatment group. Statistically significant changes were observed in IL-6; regulated upon activation, normal T-cell expressed and secreted (RANTES); and urinary tenascin levels. However, no significant impact on lung function or clinical outcomes was observed. ⁴² A study involving COPD patients demonstrated that oral AZD9668 (60 mg twice daily for 12 weeks) improved the forced expiratory volume in 1 s (FEV1); Exacerbations of Chronic pulmonary disease Tool (EXACT) scores; and Breathlessness, Cough, and Sputum Scale (BCSS) cough and sputum scores compared with placebo; however, computed tomography assessment showed no improvement in airway structure after treatment. ⁴³ Two additional studies have indicated that AZD9668 showed no efficacy in the treatment of COPD patients.^44,45 Despite the variability in the results of the aforementioned studies, which necessitate further investigation, all of them reported that AZD9668 was well tolerated by the patients.

Other synthetic NE inhibitors

POL6014 is a novel selective NE inhibitor administered via inhalation. Studies have indicated that inhaled POL6014 can safely achieve high concentrations within the lungs, significantly inhibiting NE in the sputum of CF patients after a single dose. ⁴⁶ Sirtinol possesses NE inhibitory activity, which is concentration-dependent and does not affect neutrophil function. Animal experiments have shown that sirtinol can inhibit NE-induced pulmonary edema and reduce the lung wet/dry weight ratio in a lipopolysaccharide-induced ALI mouse model. ⁴⁷ Therefore, sirtinol, a novel NE inhibitor, has potential clinical application value in the treatment of inflammatory lung diseases.

Conclusion

NE plays a pivotal role in the onset and progression of various respiratory diseases. Through in-depth research into the biological characteristics and pathophysiological effects of NE, we have not only enhanced our understanding of the pathogenesis of pulmonary diseases but also provided novel insights into the diagnosis and treatment of related conditions. It remains unclear why NE inhibitors, despite having high in vitro affinities, do not produce the expected clinical results. We considered that it may be due to their insufficient local concentration in vivo. Therefore, the challenge is not selectivity but achievement of sufficient local concentration without completely inhibiting all NE with the associated known risks. Despite their significant potential in the treatment of respiratory diseases, NE inhibitors have limitations such as off-target effects, poor lung bioavailability, cost considerations, and variability in patient responses. Future research should focus on exploring the specific mechanisms of NE in different pulmonary diseases and developing more efficient and selective NE inhibitors.