© 2002 American Thoracic Society
A Novel Oral Neutrophil Elastase Inhibitor (ONO-6818) Inhibits Human Neutrophil Elastase-induced Emphysema in RatsDepartment of Respiratory Medicine, School of Medicine, Fukuoka University, Fukuoka, Japan Correspondence and requests for reprints should be addressed to Masayoshi Ishibashi, M.D., Respiratory Medicine, School of Medicine, Fukuoka University, 7-45-1 Nanakuma, Jonan-ku, Fukuoka, 814-0180 Japan. E-mail: kokyuki{at}minf.med.fukuoka-u.ac.jp
We investigated the effects of a novel oral neutrophil elastase inhibitor (ONO-6818) on acute lung injury and pulmonary emphysema induced by human neutrophil elastase (HNE). Young male Wistar rats were divided into four treatment groups: (1) control group (saline); (2) HNE group (HNE 200 U + 0.5% carboxymethyl-cellulose [solution for ONO-6818]); (3) low-dose ONO-6818 group (HNE 200 U + ONO-6818 10 mg/kg); and (4) high-dose ONO-6818 group (HNE 200 U + ONO-6818 100 mg/kg). Saline and HNE were applied via the trachea using a microsprayer. ONO-6818 was administered orally 1 hour before HNE application. Six hours after HNE application, neutrophil counts and hemoglobin concentration in bronchoalveolar lavage fluid and lung tissue myeloperoxidase activity were determined. Eight weeks after the application, FRC, TLC, lung compliance, and mean linear intercept were estimated. ONO-6818 attenuated dose-dependently HNE-induced increases in lung myeloperoxidase activity, hemoglobin, and neutrophil count in bronchoalveolar lavage fluid. Furthermore, it significantly attenuated HNE-induced increases in FRC, TLC, lung compliance, and mean linear intercept. ONO-6818 inhibited acute lung injury induced by HNE by minimizing lung hemorrhage and accumulation of neutrophils in the lung. ONO-6818 also inhibited the development of HNE-induced emphysematous changes including lung hyperinflation, degradation of elastic recoil, and airspace enlargement.
Key Words: human neutrophil elastase • acute lung injury • pulmonary emphysema • neutrophil elastase inhibitor
Emphysema is anatomically defined as a condition characterized by abnormal and permanent enlargement of the airspace distal to the terminal bronchiole, accompanied by destruction of their walls without obvious fibrosis (1). The proteases/antiprotease imbalance hypothesis as the basis for the pathogenesis of emphysema was advocated in the 1960s (2). However, more recent studies have also established the role of inflammatory processes and oxidative stress in the pathogenesis of emphysema, although the main pathogenic mechanism remains unknown.
In this regard, the major elastolytic enzyme in the proteases/antiprotease theory has not been confirmed. Matrix metalloproteinases (MMPs) degrade all components of the extracellular matrix of lung parenchyma. MMP-12 knockout mice do not develop cigarette smoke–induced emphysema (3), and high concentrations of MMPs have been described in bronchoalveolar lavage fluid (BALF) and lung tissues of patients with emphysema (4, 5). The latter studies suggested that MMPs are critical mediators in smoking-related emphysema. Neutrophil elastase, a neutral serine protease, is a major constituent of lung elastolytic activity and has various biologic activities such as potent stimulation of mucus secretion. Neutrophil elastase also induces the release of interleukin (IL)-8 from epithelial cells and may perpetuate the inflammatory process (6). Recently, a number of studies re-evaluated the role of neutrophil elastase in cigarette smoking–related emphysema. Cigarette smoke can induce acute connective tissue breakdown and this effect is mediated by neutrophil-derived serine proteases, most likely neutrophil elastase (7). High concentrations of neutrophil elastase- Protease inhibitors could be considered as therapeutically useful agents. Several human neutrophil elastase (HNE) inhibitors, both synthetic and natural, reversible or irreversible, have been evaluated biologically and found to inhibit HNE both in vitro and in vivo with varying degrees of success (9). In the present study, we used a diffuse emphysema model induced by spraying HNE onto the trachea, and investigated the effect of a novel oral neutrophil elastase inhibitor (ONO-6818) on pulmonary emphysema in rats.
Human sputum elastase (SE563; Elastin Products, Owensville, MO) was used without further purification (10). A novel oral neutrophil inhibitor (ONO-6818: C23H28N6O4, molecular weight: 452.51) was obtained from Minase Research Institute of ONO Pharmaceutical Co. (Osaka, Japan) (11). The inhibitory activity of ONO-6818 (Ki value) against neutrophil elastases from humans is 12.16 nmol/L. Studies were performed in young male Wistar rats weighing 228 ± 15 g (mean ± SD). This study was approved by the animal experimental guidance committee of Fukuoka University.
The Rat Emphysema Model
Effects of Neutrophil Elastase Inhibitor
Lung Hemorrhage and Neutrophil Accumulation in the Lung
Lung Volumes and Pulmonary Mechanics
Mean Linear Intercept
Statistical Analysis
Rat Model of Diffuse Emphysema Tracheal application of HNE induced emphysematous changes in all lung lobes, including lobus cranials, lobus medius, lobes caudalis, lobus accessorius, and left lung. Table 1 shows morphologic changes consistent with emphysema in HNE-treated groups. The mean Lm value of all lobes increased significantly from 61.6 ± 3.5 µm in the control group to 79.3 ± 8.0 µm in the HNE 200 U group and 86.5 ± 14.0 µm in the HNE 400 U group. In each treatment group, the mean value of Lm for each lobe was similar across the five lobes (61.3–62.5 µm in the control group, 75.3–83.0 µm in the HNE 200 U group, and 84.1–89.0 µm in the HNE 400 U group). Because HNE 200 U treatment induced emphysematous changes in all lobes, we used this dose in the remaining experiments.
Effects of ONO-6818 on HNE-induced Acute Lung Injury Lung hemorrhage. Tracheal application of HNE resulted in severe lung hemorrhage, as demonstrated by increased hemoglobin (Hb) concentration in BALF. Hb concentration was not detected in the control group, but significantly increased to 0.068 ± 0.019 g/dl in the HNE group. At a dose of 10 mg/kg, ONO-6818 significantly reduced Hb in BALF to 0.030 ± 0.014 g/dl (p < 0.01), and the reduction was even larger when ONO-6818 was administered at 100 mg/kg (0.022 ± 0.008 g/dl, p < 0.01). These results showed that orally administered ONO-6818 at 1 hour before elastase application significantly and dose-dependently inhibited lung hemorrhage (Figure 1) .
Neutrophil accumulation in lungs. Neutrophil count in BALF was significantly higher in the HNE group (650 ± 290/mm3) compared with the control group (190 ± 80/mm3, p < 0.01). Such increase was significantly suppressed by administration of 100 mg/kg ONO-6818 (10 mg/kg: 310 ± 170, p = 0.057; 100 mg/kg: 160 ± 70/mm3, p < 0.01; Figure 2A) . Furthermore, HNE significantly increased MPO concentrations in the lung tissue from a control level of 18.2 ± 2.6 to 33.3 ± 4.5 U (p < 0.01; Figure 2B). However, such increase was significantly abrogated by the ONO-6818 group (10 mg/kg: 23.4 ± 8.5, p = 0.076; 100 mg/kg: 18.3 ± 5.0 U, p < 0.01; Figure 2B). There were no significant differences in neutrophil counts and MPO levels in control rats and those treated with 100 mg/kg ONO-6818.
Effects of ONO-6818 on HNE-induced Emphysematous Changes Lung volume and mechanics. Eight weeks after administration of HNE, the mean body weight was 456 ± 41 g. There were no significant differences in body weight among the four groups. The mean FRC in the control group was 3.6 ± 1.1 ml and increased to 6.0 ± 1.6 ml in the HNE group. The increase in FRC was suppressed significantly to 4.7 ± 1.0 ml by low-dose ONO-6818 and to 3.2 ± 0.5 ml by 100 mg/kg ONO-6818. TLC25 was 10.5 ± 1.8 ml in the control group and increased to 14.0 ± 2.7 ml in the HNE group. The increase in TLC25 was suppressed significantly to 11.6 ± 1.7 ml by low-dose ONO-6818 and further suppressed to 9.7 ± 1.3 ml at 100 mg/kg (Figures 3A and 3B) . The increase in Cst was also inhibited by 10 mg/kg and 100 mg/kg. Cst was 0.47 ± 0.13 ml/cm H2O in the control group, was increased to 0.61 ± 0.08 ml/cm H2O in the HNE group, but was suppressed significantly to 0.43 ± 0.11 ml/cm H2O at 10 mg/kg and 0.43 ± 0.12 ml/cm H2O at 100 mg/kg of ONO-6818 (Figure 3C). These findings indicate that ONO-6818 dose-dependently inhibited hyperinflation and degradation of elastic recoil in HNE-treated lungs. These increases in FRC, TLC25, and Cst in the HNE group produced an upward and left shift in the pressure–volume curve of the lung. ONO-6818 resulted in recovery of the pressure–volume curve by downward and right shift.
Histopathologic findings. Histopathologic examination showed that oral administration of ONO-6818 inhibited the development of elastase-induced airspace enlargement and breakdown of alveoli. At the dose of 10 mg/kg ONO-6818, emphysematous changes were significantly decreased but focal emphysema lesions were present. At higher doses of 100 mg/kg ONO-6818, breakdown of alveolar walls was rarely found and the findings were similar to those in the control group (Figure 4) .
Mean Lm. The mean Lm increased from 58.0 ± 1.8 µm in the control group to 73.46 ± 2.8 µm in the HNE group. The latter increase was abrogated by ONO-6818 (10 mg/kg: 63.6 ± 2.1 µm, p < 0.01; 100 mg/kg: 58.9 ± 2.1 µm, p < 0.01; Figure 5) . These results indicated that tracheal application of HNE at 200 U induced a distinct increase of Lm, whereas ONO-6818 prevented the increase in Lm dose-dependently. Furthermore, measurement of Lm showed that ONO-6818 prevented airspace enlargement induced by HNE.
The main findings of the present study were the following: (1) HNE caused lung hemorrhage and neutrophil accumulation into the lungs during the acute phase; (2) HNE caused lung hyperinflation, degradation of the elastic recoil, and airspace enlargement during the chronic phase; and (3) pretreatment with ONO-6818 inhibited both acute lung injury and emphysematous changes induced by HNE, and these effects were dose-dependent, with more potent effects noted at 100 mg/kg.
To prevent proteolysis, plasma and interstitial fluid contain potent proteinase inhibitors such as ONO-6818, a low–molecular weight compound (MW: 452), is a specific, reversible, and nonpeptide neutrophil elastase inhibitor (11). This agent competitively inhibits neutrophil elastase in humans, rats, and hamsters, but does not inhibit other proteases such as porcine pancreatic elastase, trypsin, proteinase 3, cathepsin G, or MMPs (11). Thus, ONO-6818 might attenuate lung injury by directly inhibiting the decomposition of elastin or collagen by neutrophil elastase. However, the compound itself has no direct effect on elastin or collagen (Dr. Kazuyuki Ohmoto, Minase Research Institute of ONO Pharmaceutical Co., Osaka, Japan, personal communication). Various animal models of protease-induced emphysema have been reported previously (26). In these models, emphysema could be induced by papain, pancreatic elastase, or HNE (26). Recently, many low–molecular weight synthetic inhibitors of HNE have been reported, and their in vivo effects have often been evaluated by assessing the severity of lung hemorrhage or the extent of emphysematous changes in hamsters after intratracheal instillation of HNE. However, animal models of HNE-induced emphysema reported earlier showed only slight changes in pulmonary function, and the dose required to induce emphysematous changes was lethal due to severe hemorrhage (20). Furthermore, intratracheal instillation with gravity resulted in increased lung volume and airspace enlargement, but these changes were noted only on the dependent side (27). In the present study, we used a microspray to apply HNE to the airway, and this technique induced emphysematous changes that were evident histopathologically in all lobes (Table 1), functionally (e.g., FRC and TLC25), and mechanically (Cst), at a dose that was not associated with any mortality.
The underlying mechanisms of emphysema include inflammatory processes in the lung and airways. Cigarette smoke and other irritants activate macrophages and airway epithelial cells in the respiratory tract, which release neutrophil chemotactic factors, including IL-8 and leukotriene (LT) B4. Neutrophils and macrophages then release proteases that break down the connective tissue in the lung parenchyma, resulting in emphysema (28). In animal models of HNE-induced emphysema, development of emphysema depends on the inflammatory process induced by HNE itself in addition to the elastolytic activity of the above mediators. Furthermore, HNE also has certain important biologic activities that induce IL-8, other proteases like cathepsins (29), and HNE-
To prevent tissue degradation in subjects with emphysema (with the exception of those with Our results demonstrated that ONO-6818 administered orally 1 hour before HNE treatment inhibited lung hemorrhage and neutrophil accumulation in the lung during the acute phase. These results suggest that ONO-6818 suppressed HNE-induced elastolysis and neutrophil migration. The long-term effects described in our study also reflected suppression of emphysematous changes in the lung by ONO-6818. These findings suggest that inhibition of elastolysis and neutrophil migration into the lung during the acute phase prevented the subsequent development of emphysema without direct inhibition of cathepsins and MMPs. In our study, lung hemorrhage was not completely blocked by a high dose of ONO-6818. ONO-6818 is a specific inhibitor of HNE and therefore might be independent of the inflammatory process that involves cathepsins and MMPs (29). Other studies have reported the effects of other low–molecular weight elastase inhibitors. FK706, ONO5046, and FR901277 have been used for lung hemorrhage induced by intratracheal administration of HNE (20, 24, 25). Pretreatment with such agents administered intratracheally or intravenously completely inhibited lung hemorrhage in hamster models. However, oral administration of FK706 and FR901277 before HNE administration significantly but weakly inhibited lung hemorrhage (20, 25). Only ICI200.880 was reported to inhibit white blood cell accumulation induced by intratracheal administration of HNE, and pretreatment with this agent prevented increases in total cell count in BALF (21). A few reports have investigated the long-term effects of HNE on pulmonary emphysema (20, 21). ICI 200,800, a specific inhibitor of HNE, reduced high Lm levels in hamsters when administered intratracheally 8 weeks after instillation of HNE (21). Pretreatment with FR901277, a nonspecific serine protease inhibitor, administered intratracheally significantly but incompletely inhibited PPE-induced increases in VC and Cst (20). To our knowledge, there are no reports that have previously shown the effects of low molecular elastase inhibitors on FRC, TLC, Cst, or Lm in the HNE-induced emphysema model. In the present study, pretreatment with ONO-6818 attenuated the loss of elastic recoil in low doses, and inhibited both changes of mechanics and airspace enlargement in high doses (Figure 6) .
It is still unknown whether neutrophil elastase is the major elastolytic enzyme in smoke-related emphysema. In mice exposed to whole cigarette smoke, acute connective tissue breakdown is prevented by human  1-antitrypsin, and mediated by neutrophil-derived serine proteases, most likely neutrophil elastase (7). Further studies using ONO-6818 should explain the role of HNE in cigarette smoke–related emphysema and its importance clinically. In summary, we demonstrated in the present study that a novel oral neutrophil elastase inhibitor, ONO-6818, could inhibit lung hemorrhage and accumulation of neutrophils in the lung during the acute phase of lung injury induced by HNE. Long-term studies showed that administration of the same inhibitor at 8 weeks after HNE prevented HNE-induced emphysema, which was evident by minimization of lung hyperinflation, correction of elastic recoil, and prevention of airspace enlargement. Received in original form March 27, 2001; accepted in final form May 20, 2002
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