Treatment with a neutrophil elastase inhibitor and ofloxacin reduces P. aeruginosa burden in a mouse model of chronic suppurative otitis media.

Chronic suppurative otitis media (CSOM) is a widespread, debilitating problem with poorly understood immunology. Here, we assess the host response to middle ear infection over the course of a month post-infection in a mouse model of CSOM and in human subjects with the disease. Using multiparameter flow cytometry and a binomial generalized linear machine learning model, we identified Ly6G, a surface marker of mature neutrophils, as the most informative factor of host response driving disease in the CSOM mouse model. Consistent with this, neutrophils were the most abundant cell type in infected mice and Ly6G expression tracked with the course of infection. Moreover, neutrophil-specific immunomodulatory treatment using the neutrophil elastase inhibitor GW 311616A significantly reduces bacterial burden relative to ofloxacin-only treated animals in this model. The levels of dsDNA in middle ear effusion samples are elevated in both humans and mice with CSOM and decreased during treatment, suggesting that dsDNA may serve as a molecular biomarker of treatment response. Together these data strongly implicate neutrophils in the ineffective immune response to P. aeruginosa infection in CSOM and suggest that immunomodulatory strategies may benefit drug-tolerant infections for chronic biofilm-mediated disease.

α1-Antitrypsin: Key Player or Bystander in Acute Respiratory Distress Syndrome?

Acute respiratory distress syndrome is characterized by hypoxemia, altered alveolar-capillary permeability, and neutrophil-dominated inflammatory pulmonary edema. Despite decades of research, an effective drug therapy for acute respiratory distress syndrome remains elusive. The ideal pharmacotherapy for acute respiratory distress syndrome should demonstrate antiprotease activity and target injurious inflammatory pathways while maintaining host defense against infection. Furthermore, a drug with a reputable safety profile, low possibility of off-target effects, and well-known pharmacokinetics would be desirable. The endogenous 52-kd serine protease α1-antitrypsin has the potential to be a novel treatment option for acute respiratory distress syndrome. The main function of α1-antitrypsin is as an antiprotease, targeting neutrophil elastase in particular. However, studies have also highlighted the role of α1-antitrypsin in the modulation of inflammation and bacterial clearance. In light of the current SARS-CoV-2 pandemic, the identification of a treatment for acute respiratory distress syndrome is even more pertinent, and α1-antitrypsin has been implicated in the inflammatory response to SARS-CoV-2 infection.

A Novel Supplementation Approach to Enhance Host Response to Sublingual Vaccination.

Sublingual immunization is emerging as an alternative to nasal immunization and induction of mucosal IgA responses. Using Bacillus anthracis edema toxin (EdTx) as an adjuvant, we previously showed that innate responses triggered after sublingual immunization could limit generation of IgA responses. We tested whether co-administration of a neutrophil elastase inhibitor (NEI) could rescue the ability of EdTx to induce broad antibody responses, including mucosal IgA. NEI supplementation of sublingual vaccines containing EdTx promoted antigen-specific serum IgA responses but also enhanced serum IgG1, and IgG2b responses. This enhancing effect of NEI did not extend to all antibody isotypes and IgG sublclasses, since NEI  reduced serum IgE responses and did not affect IgG2a/c and IgG3 responses. NEI supplementation also promoted anti-Bacillus anthracis protective antigen (PA) neutralizing antibodies and enhanced high affinity IgG1 and IgA antibodies. In addition to serum IgA, NEI supplementation stimulated antigen-specific mucosal IgA responses in the GI tract, and enhanced antigen-specific IgG responses in vaginal washes. Analysis of CD4+ T helper cell responses revealed that co-administration of NEI broadened the profile of cytokine responses, by stimulating Th1, Th2, Th17, and Tfh cytokines. We also noted that NEI had a higher stimulatory effect on IL-5, IL-10, IL-17 responses.

Kunitz type protease inhibitor EgKI-1 from the canine tapeworm Echinococcus granulosus as a promising therapeutic against breast cancer.

EgKI-1, a member of the Kunitz type protease inhibitor family, is highly expressed by the oncosphere of the canine tapeworm Echinococcus granulosus, the stage that is infectious to humans and ungulates, giving rise to a hydatid cyst localized to the liver and other organs. Larval protoscoleces, which develop within the hydatid cyst, have been shown to possess anti-cancer properties, although the precise molecules involved have not been identified. We show that recombinant EgKI-1 inhibits the growth and migration of a range of human cancers including breast, melanoma and cervical cancer cell lines in a dose-dependent manner in vitro without affecting normal cell growth. Furthermore, EgKI-1 treatment arrested the cancer cell growth by disrupting the cell cycle and induced apoptosis of cancer cells in vitro. An in vivo model of triple negative breast cancer (MDA-MB-231) in BALB/c nude mice showed significant tumor growth reduction in EgKI-1-treated mice compared with controls. These findings indicate that EgKI-1 shows promise for future development as an anti-cancer therapeutic.

Enhanced transdermal peptide delivery and stability by lipid conjugation: epidermal permeation, stereoselectivity and mechanistic insights.

PURPOSE: Efficient delivery of therapeutic peptides to the skin will facilitate better outcomes in dermatology. The tetrapeptide AAPV, an elastase inhibitor with potential utility in the management of psoriasis was coupled to short chain lipoamino acids (Laa: C6-C10) to enhance the peptide permeation into and through human epidermis. METHODS: AAPV was conjugated to Laas by solid phase synthesis. Peptide stability, skin distribution and permeation, elastase activity and surface activity were determined. RESULTS: Laas increased peptide permeation into the skin. The permeation lag time and amount of peptide remaining in the skin increased with the carbon chain length of the Laa conjugate. We also demonstrated stereoselective permeation enhancement in favour of the D-diastereomer. Importantly, the elastase inhibition activity of the peptide was largely retained after coupling to the Laa conjugates, showing potential therapeutic utility. The Laa-peptide structures were shown to be surface active, suggesting that this surfactant-like activity coupled with enhanced lipophilicity may contribute to their interaction with and permeation through the lipid domains of the stratum corneum. CONCLUSIONS: This study suggests that the Laa conjugation approach may be useful for enhancing the permeation of moderately sized peptide drugs with potential application in the treatment of skin disorders.

[Pharmacological therapies for ARDS].

Acute respiratory distress syndrome (ARDS) is a noncardiogenic pulmonary edema resulting from increased capillary permeability. Numerous pharmacologic therapies have been studied for prevention and treatment of ARDS. Although several pharmacological therapies could improve patient's respiratory function, there have been no controlled studies which clearly demonstrated the clinical benefit for ARDS-related mortality. The role of corticosteroids in ARDS remains controversial. Available evidence is against early administration of high-dose corticosteroids (methylprednisolon 120 mg x kg-1 x day - 1). In contrast, low-dose corticosteroid therapy (methylprednisolon 0.5-2.5mgg kg-1 xday-1)remains controversial. With regard to sivelestat sodium, a specific inhibitor of neutrophil elastase, although the effectiveness in decreasing mortality was not clarified, increases in lung oxygenation and ventilator-free days have consistently been revealed. Other probable pharmacologic therapies for ARDS include continuous infusion of cisatracurium. In conclusion, there are not established drugs for ARDS, and further studies are necessary to reveal the clinical effectiveness of the above mentioned and novel pharmacologic therapies.

Phase II study of a neutrophil elastase inhibitor (AZD9668) in patients with bronchiectasis.

UNLABELLED: Neutrophil elastase (NE) activity is increased in bronchiectasis and may play a role in this condition. We wished to determine the effect of AZD9668, a selective oral inhibitor of NE. Efficacy and safety of AZD9668 60 mg twice daily over 4 weeks were evaluated in a randomised, double-blind, placebo-controlled, parallel-group, Phase II, signal-searching study in patients with bronchiectasis. Outcome measures included: waking and post-waking sputum neutrophil counts; lung function tests; 24-h sputum weight; BronkoTest(®) diary card data; St George's Respiratory Questionnaire for COPD patients (SGRQ-C); sputum NE activity; inflammatory biomarker levels; desmosine levels; adverse events, safety haematology and biochemistry. AZD9668 levels in plasma and sputum were measured to confirm exposure. Thirty-eight patients were randomised: 16 to placebo and 22 to AZD9668. There was no change in sputum neutrophils with AZD9668. Forced expiratory volume in 1 s improved by 100 mL in the AZD9668 group compared with placebo (p = 0.006). Significant changes (defined a priori as p < 0.1) in favour of AZD9668 were also seen in slow vital capacity, plasma interleukin-8, and post-waking sputum interleukin-6 and Regulated on Activation, Normal T-cell Expressed and Secreted levels. Non-significant changes in favour of AZD9668 were seen in other lung function tests, sputum weight and the SGRQ-C. AZD9668 was well tolerated. In this small signal-searching study, 4 weeks' treatment with AZD9668 improved lung function in patients with bronchiectasis and there were trends for reductions in sputum inflammatory biomarkers. Larger studies of longer duration would be needed to confirm the potential benefits of this agent in bronchiectasis. REGISTRATION: NCT00769119.

Inhaled neutrophil elastase inhibitor reduces oleic acid-induced acute lung injury in rats.

RATIONALE: Neutrophil elastases (NE) play an important role in the pathogenesis of acute lung injury (ALI). NE activities are significantly increased in serums and lungs of patients or animals with ALI. Intravenous infusion (IV) of Sivelestat, an NE inhibitor, can reduce ALI. Through inhalation, drugs reach lungs directly and in high concentration. We hypothesized that inhaled Sivelestat would alleviate oleic acid (OA)-induced ALI in rats. METHODS: Rats were anesthetized and mechanically ventilated, and then ALI was induced by OA injection. One hour later, the animals were randomized to receive either Sivelestat (3 mg/kg/h) or saline inhalation. The effect of Sivelestat IV (3 mg/kg/h) was also investigated. All animals were ventilated and observed for 6 h. RESULTS: OA injection increased NE activities in lung tissues and serums. The increase of NE activities in lung tissues and serums markedly reduced by 77%, and 29%, respectively, by the inhalation of Sivelestat; and 53.8%, and 80%, respectively, by Sivelestat IV. Additionally, inhaled Sivelestat resulted in ameliorated lung injury by reducing edema and infiltration of neutrophils in the lung, improved oxygenation and survival. CONCLUSIONS: An over increased NE activity in lungs may play a vital effect in the pathogenesis of OA-induced ALI in rats. Topical application of nebulized Sivelestat, an NE inhibitor, may reduce OA-induced ALI in rats. Sivelestat inhalation can be developed as a novel treatment for ALI.

Early administration of sivelestat, the neutrophil elastase inhibitor, in adults for acute lung injury following gastric aspiration.

Gastric aspiration is the major cause of acute lung injury (ALI) and acute respiratory distress syndrome. Aspiration-induced ALI is believed to be, at least in part, facilitated by neutrophil-derived mediators and toxic molecules. We conducted a prospective cohort study based on the hypothesis that sivelestat, a specific neutrophil elastase inhibitor, is effective for treating ALI following gastric aspiration. Forty-four ALI patients who showed evidence of aspiration were observed within 12 h before intensive care unit admission and who had been mechanically ventilated within 12 h after admission were included in this study. Lung injury score (LIS) and PAO2/FiO2 (P/F) ratio on day 7 were defined as the primary outcomes of the study. Twenty-three patients were assigned to the sivelestat group and 21 to the control group. In univariate analyses, the proportions of patients with LIS lower than 1.0 on day 7 and a P/F greater than 300 on day 7 were significantly higher in the sivelestat group than in the control group (60.9% vs. 26.3%, P = 0.03; 87.0% vs. 36.8%, P = 0.001). In the logistic regression model, the use of sivelestat was an independent predictor for LIS lower than 1.0 on day 7 (relative risk, 7.4; 95% confidence interval [CI], 1.51-36.48) and for a P/F ratio higher than 300 on day 7 (relative risk, 18.5; 95% CI, 2.72-126.46). In the Cox proportional hazards model, the use of sivelestat was associated with a lower cumulative proportion of patients who received mechanical ventilation during the initial 14 days (hazard ratio, 2.6; 95% CI, 1.17-5.55).

Optimal period for the prophylactic administration of neutrophil elastase inhibitor for patients with esophageal cancer undergoing esophagectomy.

BACKGROUND: The present study was designed to determine the optimal period for the prophylactic administration of the neutrophil elastase inhibitor, Sivelestat, in patients undergoing transthoracic esophagectomy. Sivelestat is reported to be effective in patients who undergo esophagectomy by providing an increased oxygenation ability and suppressing the serum inflammatory cytokines in the postoperative period. However, the optimal period for the prophylactic administration of Sivelestat remains to be elucidated. METHODS: The 30 patients who underwent esophagectomy for thoracic esophageal cancer were enrolled in one of two groups. The initial 15 patients were assigned to group A and received intravenous infusion of Sivelestat sodium hydrate until postoperative day (POD) 2, and the subsequent 15 patients were assigned to group B and received Sivelestat until POD 5. Historical controls without Sivelestat administration were used. The postoperative courses and serum inflammatory cytokines were evaluated. RESULTS: Sivelestat improved oxygenation in the postoperative period; however, there were no differences between the two groups in terms of duration of mechanical ventilation, intensive care unit stay, systemic inflammatory response syndrome, and postoperative change of oxygenation. In addition, there were no differences in the postoperative changes in the serum interleukin (IL)-6 and high mobility group box chromosomal protein 1. Although the serum IL-8 on POD 3 was lower in group B than in group A, the neutrophil elastase showed no difference between these groups. None of the patients in either group suffered respiratory complications. CONCLUSIONS: The two-day administration of Sivelestat initiated immediately after intrathoracic manipulation was found to be sufficient for prophylactic use to prevent pulmonary complications by suppressing hypercytokinemia after esophagectomy.

Effects of a synthetic protease inhibitor (gabexate mesilate) and a neutrophil elastase inhibitor (sivelestat sodium) on acid-induced lung injury in rats.

The present study was designed to examine the combined effects of a synthetic protease inhibitor, gabexate mesilate, with a specific neutrophil elastase inhibitor, sivelestat sodium, on acid-induced lung injury. Adult male Sprague-Dawley rats weighing 300-350 g were anaesthetised intraperitoneally with pentobarbitone sodium and the right jugular vein was cannulated. Following tracheostomy, rats were ventilated mechanically and underwent intratracheal instillation of hydrochloric acid (HCl, 0.1N 1.5 ml/kg) or normal saline. Gabexate mesilate (10mg/kg, i.p.) and/or sivelestat sodium (10mg/kg/h, i.v.) were administered 30 min before HCl instillation. Bronchoalveolar lavage fluid samples were obtained 5h after HCl instillation. In bronchoalveolar lavage fluid, the HCl-induced increases in total nucleated cell counts, neutrophil counts, optical density at 412 nm as an index of pulmonary haemorrhage, concentrations of albumin and cytokine-induced neutrophil chemoattractant (CINC) were significantly attenuated by either gabexate mesilate or sivelestat sodium treatment. Gabexate mesilate or sivelestat sodium treatment also significantly attenuated the wet to dry weight ratio induced by HCl. However, combined treatment with both gabexate mesilate and sivelestat sodium did not show additive effects on HCl-induced lung injury, compared with single treatments. These findings suggested that gabexate mesilate and sivelestat sodium each exhibited protective effects on acid-induced lung injury, but that synergistic effects of both agents are limited in this acid-induced lung injury model.

Effect of a neutrophil elastase inhibitor on acute lung injury after cardiopulmonary bypass.

Cardiopulmonary bypass (CPB) has been implicated as a cause of acute lung injury (ALI) in cardiac surgical patients. We used a bronchoscopic microsampling (BMS) probe to examine alveolar biochemical constituents and evaluated the effect of sivelestat sodium hydrate, a novel synthesized polymorphonuclear (PMN) neutrophil elastase inhibitor, on ALI induced by CPB. Twelve patients undergoing aortic valve replacement were treated with either sivelestat 0.2 mg/kg/h (sivelestat group, n=6) or 0.9% saline (control group, n=6) from the start of surgery. Samples were collected by the BMS probe at three time points: after tracheal intubation, 1 h after CPB introduction, and 3 h after CPB termination. Pulmonary function was assessed perioperatively. There were no differences in baseline characteristics. The concentration of PMN elastase was significantly suppressed in the sivelestat group, compared with the control group (P=0.001). The sivelestat group also had lower levels of interleukin-6 and interleukin-8. Alveolar-arterial oxygen difference markedly increased, and a worsening of the PaO(2)/FiO(2) ratio indicated severe impairment after CPB. However, sivelestat attenuated the pattern of physiological deterioration of gas exchange. Sivelestat may attenuate neutrophil elastase or proinflammatory cytokines, and improve pulmonary dysfunction in patients undergoing CPB.

Neutrophil elastase inhibitor attenuates hippocampal neuronal damage after transient forebrain ischemia in rats.

Inflammatory responses have been known to contribute to the development of neuronal damage after brain ischemia in experimental animals. Also, neutrophil elastase activity in the plasma has been elevated in the patients with acute cerebral infarction. In order to clarify whether neutrophil elastase distributes into the brain parenchyma and exacerbates neuronal damage following ischemia, we examined the effects of specific neutrophil elastase inhibitor, ONO-5046, on hippocampal CA1 neuronal death in relation to neutrophil elastase activity in the plasma and its distribution in the brain and to caspase-3/7 activity. ONO-5046 (5 and 10 mg/kg) or saline (control group) was administrated after 8 min of forebrain ischemia in rats. Ratio of surviving neurons (median, [range]) in hippocampal CA1 seven days after ischemia was significantly higher in the ONO-5046 5 mg/kg (31% [12-57%]), and 10 mg/kg groups (69% [39-76%]) than in the control group (3.2% [0-10%]). Plasma neutrophil elastase activity in the ONO-5046 10 mg/kg group was significantly lower than in the control group (14 [11-25] vs. 41 [35-68] nmol/ml). Neutrophil elastase distributed in the extracellular space in the hippocampal CA1 neuronal layer in the control group, while, in the ONO-5046 10 mg/kg group, trace of neutrophil elastase was detected only in the endothelium. Caspase-3/7 activity was elevated after ischemia over 8 h in the control group, while, in the ONO-5046 10 mg/kg group, no elevation was observed. The results suggest that neutrophil elastase may contribute to neuronal death in hippocampal CA1 following forebrain ischemia and that neutrophil elastase inhibitor attenuates neuronal death.

[Acute respiratory failure due to pneumocystis pneumonia successfully treated with combined use of sivelestat sodium hydrate].

A 22-year-old man was admitted to our hospital with fever, cough and dyspnea. His chest radiograph showed diffuse ground-glass attenuation in both lung fields. Arterial blood gas analysis showed hypoxemia (PaO2 28.7 Torr breathing room air) and he required mechanical ventilation within 6 hours after admission. Gomori methenamine silver (GMS) stain of the bronchoalveolar lavage (BAL) fluid smear showed round and indented organisms, and polymerase chain reaction revealed pneumocystis jirovecii in the BAL fluid. The HIV antibody was positive and peripheral blood CD4-positive lymphocytes decreased to 4.0%. Pneumocystis pneumonia complicated with acquired immunodeficiency syndrome (AIDS) was diagnosed. There was no four-fold rise in screen viral titers. We treated him with antibiotics, trimethoprim-sulfamethoxazole, ganciclovir, fos-fluconazole, steroid pulse therapy and sivelestat sodium hydrate. Respiratory failure was relieved within 5 days following treatment. The percentage of neutrophils in the BAL fluid was elevated (44.6%). Neutrophil elastase on admission was increased and improved to the normal range after treatment. Sivelestat sodium hydrate is an anti-neutrophil elastase inhibitor and may be one of the treatment options for acute respiratory failure due to pneumocystis pneumonia in AIDS patients.

[Effect of sivelestat sodium on acute lung injury after acute aortic dissection].

Acute lung injury is a frequent and serious complication in patients with acute aortic dissection (AAD). Elevated neutrophil elastase has been reported to be one of the major determinants occurring in AAD. On admission, we administered sivelestat sodium hydrate, a neutrophil elastase inhibitor, to 11 patients with AAD who were medically treated to prevent lung injury. We compared their clinical course with that of 12 patients of control group in which sivelestat was not used prophylacticaly. Although there were 5 patients (42%) who suffered from respiratory failure and needed mechanical ventilation in the control group, no one needed intubation in the sivelestat group. Our study suggested that sivelestat sodium hydrate could be effective in preventing intubation due to respiratory failure. Further prospective study is necessary to evaluate prophylactic administration of sivelestat sodium hydrate in AAD.