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Anesth Analg 2003;97:1751-1755
© 2003 International Anesthesia Research Society

CRITICAL CARE AND TRAUMA

ONO-1714, a Nitric Oxide Synthase Inhibitor, Attenuates Endotoxin-Induced Acute Lung Injury in Rabbits

Katsuya Mikawa, MD, Kahoru Nishina, MD, Yumiko Takao, MD, and Hidefumi Obara, MD

From the Department of Anesthesia & Perioperative Medicine, Faculty of Medical Sciences, Kobe University Graduate School of Medicine, Kobe, Japan

Address correspondence and reprint requests to Dr. K. Mikawa, Department of Anesthesia & Perioperative Medicine, Faculty of Medical Sciences, Kobe University Graduate School of Medicine, Kusunoki-cho 7, Chuo-ku, Kobe 650–0017, Japan. Address email to katzmikawa{at}yahoo.co.jp


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Overproduction of nitric oxide by inducible nitric oxide synthase (iNOS) expressed in the lung is thought to play a crucial role in the pathogenesis of endotoxin-induced acute lung injury (ALI). In this two-part study, we determined whether ONO-1714, a new selective iNOS inhibitor, attenuates endotoxin-induced ALI in rabbits. For Part I of the study, a control group received IV saline and ALI was induced by IV infusion of endotoxin 5 mg/kg over 30 min in 4 groups. Three groups received either 0.1, 0.03, or 0.01 mg/kg of ONO-1714 10 min before the start of endotoxin and the fourth group received saline. For Part II of the study, ALI was induced by endotoxin infusion in all 6 groups. One group was treated with saline. The other 5 groups received ONO-1714 0.1 mg/kg at various timings (10 min before or 1, 2, 3, or 4 h after ALI induction). The lungs were mechanically ventilated with 40% oxygen for 6 h after induction of ALI. In Part I, pretreatment with 0.1 mg/kg ONO-1714 mitigated endotoxin-induced ALI. In Part II, early posttreatment (within 2 h after the insult) with ONO-1714 was as effective as pretreatment in improving oxygenation, lung mechanics, lung leukosequestration, pulmonary edema, and histological change. However, lung damage was not improved in rabbits receiving the drug 3 or 4 h after endotoxin. These data suggest that the current study is a basis for future clinical trials to elucidate whether ONO-1714 can be a promising therapeutic approach in patients with acute respiratory distress syndrome induced by endotoxin/sepsis.

IMPLICATIONS: An excess of nitric oxide is thought to play a crucial role in the pathogenesis of acute organ injury in endotoxemia. Early posttreatment with ONO-1714, a nitric oxide synthase inhibitor, attenuated physiological, biochemical, and pathological changes in endotoxin-induced acute lung injury in rabbits.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Endotoxin causes multiple organ dysfunction, including acute lung injury (ALI) (1–4). It also overproduces nitric oxide (NO) through induction of inducible NO synthase (iNOS) in various organs (5). Increased proinflammatory cytokines often observed in endotoxemia also induce iNOS (6–8), leading to excessive generation of NO. A crucial role of large amounts of NO in the pathogenesis of endotoxin-induced ALI is evidenced by several previous experiments using iNOS knockout mice or nitric oxide synthase (NOS) inhibitors (aminoguanidine, S-methylisothiourea, and L-canavanine) (9–12). ONO-1714 (Ono, Osaka, Japan), a potent new NOS inhibitor, has recently been shown to reverse hypotension and lactic acidosis, with a tendency to prevent renal and hepatic dysfunction in a canine endotoxin shock model (13). ONO-1714 has an approximately twofold higher selectivity for iNOS than aminoguanidine (14). In the current study, therefore, we examined whether pre- or posttreatment with ONO-1714 attenuates endotoxin-induced ALI.


   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
After institutional approval, we conducted a pretreatment study (Part I) to determine the optimal dose of ONO-1714 and a posttreatment study (Part II) to assess the therapeutic effects. Male Japanese white rabbits (2.2–2.5 kg) underwent tracheotomy and controlled ventilation (Sechrist, Anaheim, CA) (initial tidal volume, 10 mL/kg; initial PaCO2, 32–38 mm Hg) with 40% oxygen and 2 cm H2O of positive end-expiratory pressure, arterial and central venous pressure monitoring, and lactated Ringer’s solution (8 mL · kg-1 · h-1) under anesthesia with infusion of ketamine (10 mg · kg-1 · h-1). There were no differences in body temperatures of the animals (37.5°–40.2°C) among the groups. For Part I (n = 8 in each group), a control group received IV saline and ALI was induced by IV infusion of endotoxin (E. coli, 055:B5 from the same lot) (Difco, Detroit, MI) 5 mg/kg over 30 min in 4 groups. Three groups received either 0.1, 0.03, or 0.01 mg/kg of ONO-1714 10 min before the start of endotoxin and the fourth group received saline. The rationale for the use of ONO-1714 at 0.01–0.1 mg/kg was based on previous data (15,16). Part I revealed that ONO-1714–0.1 mg/kg was the most effective in attenuating lung damage without any lethality (see Results). For Part II (n = 8 in each group), ALI was induced by endotoxin infusion in all 6 groups. One group was treated with saline. The other 5 groups received ONO-1714 0.1 mg/kg at various timings (10 min before, or 1, 2, 3, or 4 h after ALI induction). ALI has been assessed in detail elsewhere (17). The main outcome measures included hemodynamics, oxygenation (PaO2 determined by Radiometer ABL2), lung compliance and resistance, peripheral blood cell counts (determined by an automated blood cell counter), and plasma nitrite plus nitrate (NOx) levels (indicator of NO production as determined by an automatic analyzer). Lung mechanics were measured by the passive expiratory flow-volume technique (18) using a Flemish 00 pneumotachograph, a differential pressure transducer, and a computer interface. The animals were killed by overdose of thiamylal 6 h after the induction of ALI. The lungs were excised for determination of wet/dry (W/D) lung weight ratio (index of lung edema), bronchoalveolar lavage fluid (BALF) analysis, and pathological examination. BALF was obtained from the right lung using saline (35 mL for 3 times), and analyzed for cell counts (Sysmex K-1000) and differentiation (Diff-Quick, Harleco, Gibbstown, NJ). The cell-free supernatant of BALF was analyzed for albumin, 11-dehydro-thromboxane B2 (11-DTxB2, a stable metabolite of thromboxane A2), leukotriene B4 (LTB4), activated complement C3a, interleukin (IL)-8, and NOx. These were determined by immune-nephelometry (albumin), radioimmunoassay (11-DTxB2, LTB4, and C3a), enzyme-linked immunosorbent assay (IL-8), or an automatic analyzer using the Griess reaction (NOx). The left lower lobe was fixed by 10% formaldehyde solution at 20 cm H2O followed by embedding in paraffin. The specimens were stained with hematoxylin and eosin for light microscopic evaluation. ALI was scored as follows: a) alveolar congestion, b) hemorrhage, c) infiltration or aggregation of neutrophils in airspace or vessel wall, and d) thickness of alveolar wall/hyaline membrane formation. Each item was scored on a 5-point scale as follows: 0 = minimal damage, 1+ = mild damage, 2+ = moderate damage, 3+ = severe damage, and 4+ = maximal damage. Repeated-measures data were statistically analyzed using repeated-measures analysis of variance (ANOVA). The data of BALF and W/D weight ratio were analyzed by one-way ANOVA followed by the Tukey-Kramer post hoc test. The ALI scores were analyzed using the Kruskal-Wallis rank test followed by the Dunnett test. P < 0.05 was deemed statistically significant.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Part I
ONO-1714 0.1 mg/kg attenuated deterioration of oxygenation and lung mechanics (Fig. 1A). Hemodynamics did not differ among the 6 groups. ONO-1714 0.1 mg/kg attenuated leukopenia and thrombocytopenia in endotoxemia, although not significantly (see Supplementary Table online at http://www.anesth-analg.org). This dose of ONO-1714 blunted endotoxin-induced acidosis (Supplementary Table) and the increase in plasma and BALF NOx concentrations (Fig. 2A). ONO-1714 0.1 mg/kg suppressed the increases in leukocyte counts and percentage of neutrophils (%PMN) in BALF (Table 1). ONO-1714 0.1 mg/kg alleviated the increase of BALF albumin levels and W/D lung weight ratio (Table 1). The drug reduced the release of 11-DTxB2, LTB4, C3a, and IL-8, although not significantly (Table 1). Endotoxin-induced morphological changes were less pronounced with ONO-1714 0.1 mg/kg (Fig. 3 and Table 1).



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  		Figure 1. Changes in PaO2 and lung mechanics. A) Part I, B) Part II. Values are expressed as mean ± SD. Some SD bars are omitted for clarity. E/S: infusion of endotoxin (5 mg/kg) or saline. Arrowhead: administration of saline or ONO-1714.

 


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  		Figure 2. NOx (nitrite + nitrate) changes in plasma and bronchoalveolar lavage fluid (BALF). (A) Part I, (B) Part II. Values are expressed as mean ± SD. Some SD bars are omitted for clarity. E/S: infusion of endotoxin (5 mg/kg) or saline. Arrowhead: administration of saline or ONO-1714.

 

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  		Table 1. Analysis of Bronchoalveolar Lavage Fluid, Wet-to-Dry Weight Ratio, and Histological Examination, Part I
 


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  		Figure 3. Light micrograph (hematoxylin and eosin staining, original magnification x 100) in Part I. (A) Group S-S, (B) Group E-S, (C) Group E-ONO0.1.

 
Part II
ONO-1714 given 1 h or 2 h after endotoxin attenuated the deterioration of oxygenation and lung mechanics, although the drug given 3 h or 4 h after endotoxin failed to do so (Fig. 1B). Hemodynamics did not differ among the 6 groups. There were no differences in peripheral leukocyte or platelet counts (online Supplementary Table) or hemodynamics among the 6 groups. ONO-1714 given before or 1 or 2 h after endotoxin similarly attenuated the endotoxin-induced NOx increase in plasma and BALF (Fig. 2B). ONO-1714 given 1 or 2 h after endotoxin blunted acidosis, the increase in leukocyte counts, %PMN, and albumin levels in BALF (Table 2). Endotoxin-induced release of the mediators was not reduced by posttreatment with ONO-1714 (Table 1). ONO-1714 given 1 or 2 h after endotoxin attenuated the increase in W/D weight ratio (Table 2). In rabbits receiving the drug 1 or 2 h after endotoxin, less severe pathological damage was observed (Fig. 4). The ALI scores were lower in these groups (Table 2).


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  		Table 2. Analysis of Bronchoalveolar Lavage Fluid, Wet-to-Dry Weight Ratio, and Histological Examination, Part II
 


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  		Figure 4. Light micrograph (hematoxylin and eosin staining, original magnification x 100) in Part II. (A) Group saline, (B) Group ONO-pre, (C) Group ONO-1, (D) Group ONO-2, (E) Group ONO-3, (F) Group ONO-4.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Pretreatment or early posttreatment with ONO-1714 (0.1 mg/kg) successfully alleviated endotoxin-induced physiological (oxygenation, lung mechanics, and W/D weight ratio), biochemical (BALF albumin as an index of hyperpermeability), and morphological changes as well as systemic and local production of NO. Degrees of attenuation were similar across these variables. ONO-1714 may have reduced pulmonary edema associated with hyperpermeability by protecting endothelium/epithelium from the toxic effects of NO. However, one drawback of the current study is a lack of minute hemodynamic measurement. Rapid regional pulmonary blood flow probably exacerbates ALI (19). Endotoxin often causes cardiac insufficiency (20) and left cardiac dysfunction worsens pulmonary edema (21). Because we did not measure pulmonary capillary or left atrial pressure, we cannot deny that pulmonary edema may have been induced by heart failure in our experimental model. We must emphasize that ONO-1714 may modify lung edema by alteration of pulmonary blood flow, cardiac output, or capillary perfusion pattern. Use of larger species (e.g., sheep, swine), in which detailed hemodynamics can be measured, may have provided instructive new findings. Metabolic acidosis, a common occurrence in sepsis, can attenuate ALI (22). The beneficial effect of ONO-1714 on endotoxin-induced ALI may be spoiled by attenuation of metabolic acidosis. Recruitment of neutrophils to the lung was significantly attenuated by ONO-1714 given within 2 hrs after endotoxin, although the drug failed to inhibit release of representative chemotaxins. Breaking the vicious cycle constituting ALI may be responsible for attenuation of neutrophil sequestration, although the precise mechanism remains unknown.

Because pharmacokinetics, pharmacodynamics, and activation of ONO-1714 may be different in rodents and humans, we are unable to extrapolate our data directly or simply to clinical settings. Proliferation of lung fibroblasts in response to inflammatory stimuli is associated with upregulation of the iNOS gene (23), and anti-iNOS activity is greater in ONO-1714 than in aminoguanidine or S-methylisothiourea (14). Thus, further studies are required to determine whether ONO-1714 can inhibit fibroproliferative changes in endotoxin-induced ALI and to compare the efficacy with the well-characterized NOS inhibitors.


   Acknowledgments
 
We would like to express our gratitude to Ono Pharmaceutical Company for the generous supply of ONO-1714.


   Footnotes
 
Supplemental material available at www.anestheisa-analgesia.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Wu CC, Ruetten H, Thiemermann C. Comparison of the effects of aminoguanidine and N omega-nitro-L-arginine methyl ester on the multiple organ dysfunction caused by endotoxaemia in the rat. Eur J Pharmacol 1996; 300: 99–104.[Web of Science][Medline]
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Accepted for publication June 30, 2003.




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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins and Stanford University Libraries' HighWire Press®. Copyright 2003 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press