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*Department of Intensive Care,
Department of Anesthesiology, and
First Department of Surgery, Graduate School of Medicine, Gunma University, Gunma, Japan
Address correspondence and reprint requests to Yuji Kadoi, MD, Department of Intensive Care, Gunma University, Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511, Japan. Address e-mail to kadoi{at}med.gunma-u.ac.jp
| Abstract |
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IMPLICATIONS: ONO-5046 decreased cytokine levels more rapidly in acute respiratory distress syndrome patients treated with sivelestat than in those who received placebo. However, these decreases did not appear to affect other clinical variables, such as the duration of mechanical ventilation or survival rates.
| Introduction |
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ONO-5046 (sivelestat; sodium N-[2-[4-(2,2-dimethyl-propionyloxy) phenylsulfonylaminobenzoyl]amino-acetate tetrahydrate]) is a specific inhibitor of neutrophil elastase (1012). Kawabata et al. (11) have reported that sivelestat prevents the development of lung injury induced by endotoxin inhalation in hamsters. Tamakura et al.1 compared the effect of large and small doses of sivelestat in Japanese patients whose lung injury was associated with systemic inflammatory response syndrome (SIRS). They found that although the time of discharge from the intensive care unit (ICU) (30 days) was significantly better for the large-dose (0.2 mg · kg1 · h1) than for the small-dose (0.004 mg · kg1 · h1) group, rates of removal from mechanical ventilation, remission of SIRS, PaO2/fraction of inspired oxygen (FIO2), and lung injury scores did not significantly differ. However, their study did not contain a control group or patients with ARDS. Zeiher et al. (10) suggested that additional clinical and preclinical studies of sivelestat were needed, not only to clarify the clinical potential of this intervention strategy, but also to define the activities of neutrophil elastase in inflammatory disorders such as acute lung injury (ALI) and multiple organ failure. Few clinical trials have examined the effectiveness of neutrophil elastase inhibitors for ARDS (2,10).1 This study examined the effects of a neutrophil elastase inhibitor (sivelestat) on plasma cytokine levels and oxygenation in patients with ARDS.
| Methods |
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Patients randomized within 12 h of meeting all inclusion or exclusion criteria (Table 1) received either a continuous infusion of sivelestat (Ono Pharmaceutical Co., Ltd., Osaka, Japan; 0.2 mg · kg1 · h1) or a placebo (same dose of saline) for 14 days in a double-blinded fashion according to a blocked randomization algorithm. To maintain the blind, the study drug was prepared for IV infusion by dilution to a total volume of 100 mL with normal saline. Neither the patients nor the medical personnel responsible for their care were aware of which treatment was being administered.
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Blood collected in sterile EDTA-treated tubes was immediately centrifuged at 1500g for 15 min at 4°C, and plasma was stored at 70°C. Neutrophil elastase activity was measured with an enzyme-linked immunosorbent assay kit (Diagnostica Merck, Darmstadt, Germany) as described by Fujita et al. (13), and IL-6 was measured by using commercially available enzyme-linked immunosorbent assay kits (R&D Systems, Tokyo, Japan). Neutrophil elastase activity and IL-6 were measured before and 24, 48, and 72 h after the administration of sivelestat or placebo.
SIRS was defined as the presence of 2 or more of the following: temperature
38°C or
36°C; heart rate
90 bpm; respiratory rate
20 breaths/min; PaCO2
32 mm Hg or requiring mechanical ventilation; white blood cell count
12,000/mm3 or
4,000/mm3; or
10% band forms. The primary end-point was the prevalence of ARDS occurring at any time during the 30 days after sivelestat or placebo administration. We considered that ARDS was reversed when the patient no longer met the criteria for ARDS on the basis of either arterial blood gas measurement or chest radiography and when the reversal lasted for at least 24 h.
The clinical outcome variable of secondary interest concerned the duration of mechanical ventilation, changes in oxygenation (PaO2/FIO2) from baseline, changes in cytokine levels (neutrophil elastase activity and IL-6) from baseline, number of patients alive at 30 days who did not need mechanical ventilation, and mortality rate at 30 days. Discontinuing mechanical ventilation was defined as extubation. The time to oxygenation criteria for extubation was defined as the first time that the patient maintained a PaO2 value of 60 mm Hg on an FIO2
0.4 with a positive end-expiratory pressure of
5 cm H2O.
All data are expressed as means ± SD. An unpaired Students t-test or Fishers exact test was used to analyze demographics and continuous variables between groups. After the confirmation of equal variance among groups by the Bartlett test, two-way repeated-measures analysis of variance was used to perform multiple comparisons. When the F value was significant, the Bonferroni method was used to perform multiple comparisons. To eliminate a Type II error, each individual P value was adjusted. The Kaplan-Meier product-limit estimation compared ventilator-free days between treatment groups. The duration of mechanical ventilation was written down, and the duration was set to 30 days for patients who died during the study.
Statistical significance was established at P < 0.05. All calculations were performed by using a Macintosh computer with SPSS (SPSS Inc., Chicago, IL) and StatView 5.0 (Abacus Concepts, Inc., Berkeley, CA).
| Results |
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Table 3 shows the time course of changes in PaO2/FIO2 ratio, neutrophil elastase activity, and IL-6 levels. Neutrophil elastase activity significantly differed between groups at 72 h after treatment. Levels of IL-6 were lower in the sivelestat than in the control group at 48 h after treatment.
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| Discussion |
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Neutrophil elastase can also potentiate the inflammatory response by increasing IL-8 expression and release (7). In addition, Donnelly et al. (8) reported that the early appearance of IL-8 in BALF from patients at risk for ARDS might be an important prognostic indicator for its development. Nakamura et al. (7) found that neutrophil elastase released by neutrophils induces the bronchial epithelium to secrete IL-8, which in turn recruits additional neutrophils to the bronchial surface. These reports imply that early inhibition of increased plasma neutrophil elastase levels could prevent an increase in cytokine levels and thus hinder the development of ARDS. Therefore, the effects of neutrophil elastase inhibitors should be understood in patients with ARDS.
ONO-5046 (sivelestat) is a novel, specific neutrophil elastase inhibitor with a 50% inhibiting concentration of 0.044 ± 0.003 µM (Ki, 0.20 ± 0.02 µM; suc-Ala-Pro-Ala pNa substrate) (11,15) that inhibits leukocyte elastase obtained from rabbits, rats, hamsters, and mice. The inhibition is competitive, as determined by Lineweaver-Burk analyses. The binding of ONO-5046 to neutrophil elastase is highly specific. Sivelestat acts in the microenvironment between neutrophils and tissues, whereas
1-protease inhibitor incompletely blocks neutrophil-mediated tissue destruction. The inhibition of neutrophil elastase is useful in models of lung injury (16).
Results of animal studies support the notion that the neutrophil elastase inhibitor ONO-5046 (sivelestat) can improve an animals condition after lung injury (10,12,16,17). Miyazaki et al. (17) examined the effect of ONO-5046 in a model of ALI induced by tumor necrosis factor-
in isolated perfused rabbit lungs. ONO-5046 attenuated ALI by inhibiting the alveolar epithelial and vascular endothelial damage triggered by activated neutrophils. Kawabata et al. (11) examined the relationship between the progression of ALI and neutrophil elastase activity in BALF and the effects of sivelestat on ALI in hamsters caused by endotoxin inhalation. They concluded that delayed inhibition of neutrophil activity with sivelestat prevents the subsequent progression of ALI in hamsters. Kaneko et al. (16) reported that ONO-5046 attenuates the increased permeability of the endothelium induced by acid instillation. Nishina et al. (12) reported that IV ONO-5046 attenuated subsequent endotoxin-induced lung injury in rabbits by reducing the levels of the mediators that activate neutrophils. These findings from animal studies indicate that ONO-5046 (sivelestat) can prevent the progression of ALI in humans. However, few clinical trials have examined the effect of neutrophil elastase inhibitors on patients with ARDS (2,10). Tamakura et al.1 examined the effect of sivelestat on 230 Japanese patients with lung injury associated with SIRS in a multicenter double-blinded Phase III clinical study compared with a small-dose group. The patients were randomized to either large (0.2 mg · kg1 · h1) or small (0.004 mg · kg1 · h1) doses of sivelestat for up to 14 days.
Of the 230 enrolled patients, 221 were considered assessable according to the primary end-point of the investigators assessment of global improvement on Day 14. The assessment showed that 71.7% (81 of 113 patients) were moderately or significantly improved in the large-dose group, compared with 55.5% (60 of 108 patients) in the small-dose group. The survival rates did not differ between groups, but the large-dose group was discharged from the ICU sooner than the small-dose group. The large-dose group tended toward a reduced duration of mechanical ventilation compared with the small-dose group (P = 0.0636). However, the study did not compare the group given sivelestat with a control group or with ARDS patients.
We found that sivelestat rapidly decreased neutrophil elastase activity and levels of cytokines such as IL-6 in patients with ARDS. These findings are consistent with those of Nishina et al. (12) and Tamakura et al.1 Because increased levels of proinflammatory cytokines play pivotal roles in the development of ARDS (1,2), a decrease in such levels should be important for improving this condition.
The mechanism through which sivelestat decreased the cytokine levels remains a matter of speculation. Cai and Wright (18) reported that neutrophil elastase is an endogenous ligand for integrin complement receptor-3 and that neutrophil elastase expression on the neutrophil surface allows neutrophil migration by eluting immobilized ligands such as intercellular adhesion molecule-1. Neutrophil elastase might indirectly facilitate the migration of neutrophils via the production of chemotactic factors such as IL-8 (11,19,20).
Some evidence indicates that serine proteases might be inhibited only by multiple inhibitors, because the various types of these enzymes, along with oxidants, might inactivate the inhibitors before they can act (15,21). However, several animal studies have uncovered a direct relationship between decreased neutrophil elastase activity and sivelestat (12,16,17).
We examined the time course of plasma cytokine levels during ARDS. Some reports have suggested that plasma cytokine levels do not reflect lung levels (2,3). Donnelly et al. (6) found that plasma neutrophil elastase levels were significantly increased in patients who progressed to ARDS. Moreover, Chollet-Martin et al. (9) described interactions between neutrophils and cytokines in blood alveolar spaces during ARDS.
We administered a continuous infusion of sivelestat 0.2 mg · kg1 · h1 as recommended by the production pharmacy (Ono Pharmaceutical Co.). In addition, Sakura et al. (15) and Kawabata et al. (11) showed that sivelestat 0.2 mg · kg1 · h1 could prevent neutrophil elastase activities in animals. In addition, Tamakura et al.1 recommended that sivelestat be administered at 0.2 mg · kg1 · h1 for 14 days in the clinical setting.
Why the rapid decrease in neutrophil elastase activity and IL-6 in the sivelestat group did not correlate with either a decreased requirement for ventilation or decreased mortality remains unclear. The small patient cohort, heterogeneous target group, and selection bias might be factors. Further studies are required to clarify the effects of sivelestat in patients with ARDS.
Although neutrophil elastase might be deleterious during the early course of ALI, it may play a crucial immunomodulatory or reparative role thereafter. Neutrophil elastase might also exert a beneficial bacterial effect. The investigations of Belaaouaj et al. (22) in neutrophil elastase knockout mice revealed increased susceptibility to infection with Gram-negative Klebsiella pneumoniae and Escherichia coli, but not to Gram-positive Staphylococcus aureus.
In conclusion, we examined the effects of a neutrophil elastase inhibitor (sivelestat) on plasma cytokine levels and oxygenation in patients with ARDS. The cytokine levels decreased more rapidly in patients treated with sivelestat than in those who received placebo. However, these decreases apparently did not affect cytokine levels or other clinical variables, such as the duration of mechanical ventilation or survival.
| Acknowledgments |
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The authors thank Forte Inc. (Tokyo, Japan) for assistance with the manuscript.
| Footnotes |
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| References |
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) and activated neutrophils in isolated perfused rabbit lungs. Am J Respir Crit Care Med 1998; 157: 8994.
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