European Journal of Pharmacology, 217 (1992) 119-126 ~ 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
119
EJP 52523
The 5-1ipoxygenase inhibitor zileuton blocks antigen-induced late airway responses, inflammation and airway hyperresponsiveness in allergic sheep W i l l i a m M. A b r a h a m
a,
A s h f a q A h m e d a, A l e j a n d r o C o r t e s a, M a r e k W. Sielczak a, W e n d y H i n z b, J e n n i f e r B o u s k a b, C a r m i n e L a n n i b a n d R a n d y L. Bell b
a Harry Pearlman Biomedical Research Institute, Mount Sinai Medical Center, 4300Alton Road, Miami Beach, FL 33140, USA and h Immunosciences Research Area, Abbott Laboratories, Abbott Park, 1L 60064, USA Received 21 November 1991, revised MS received 26 February 1992, accepted 14 April 1992
Leukotrienes are thought to be involved in allergen-induced airway responses. To test this hypothesis we used a newly described 5-1ipoxygenase inhibitor, zileuton, and examined its effect on antigen-induced early and late bronchial responses, airway inflammation and airway hyperresponsiveness in allergic sheep. Early and late responses were determined by measuring specific lung resistance (SR e) before and serially for 8 h after antigen challenge. Airway inflammation was assessed by bronchoalveolar lavage performed before, 8 h after and 24 h after antigen challenge. Airway responsiveness was measured before and 24 h after challenge by determining the dose of inhaled carbachol that caused a 400% increase in SR L (PD400%). The sheep (n = 8) were challenged with A s c a r i s s u u m antigen once after vehicle treatment (methylcellulose) and once after treatment with zileuton (10 mg/kg in methylcellulose, p.o.) given 2 h before antigen challenge. Trials were separated by at least 21 days. Zileuton had no effect on the early bronchoconstrictor response to antigen but the drug inhibited the late bronchial response by 55% (P < 0.05). Unlike the control trial, there was no significant increase in bronchoalveolar lavage eosinophils at 8 h post challenge in the zileuton-treated sheep. Furthermore, zileuton treatment blocked (P < 0.05) the airway hyperresponsiveness seen 24 h after challenge. Ex vivo formation of leukotriene B 4 was inhibited over several hours after a single oral dose of zileuton, indicating that the compound was acting as a 5-1ipoxygenase inhibitor in vivo. These results suggest that 5-1ipoxygenase metabolites contribute to allergen-induced late responses, airway inflammation and airway hyperresponsiveness in this animal model of asthma. 5-Lipoxygenase; Leukotrienes; Asthma; Airway hyperresponsiveness; Inflammation; (Late responses)
1. Introduction Metabolism of arachidonic acid by 5-1ipoxygenase leads to the generation of leukotrienes (i.e. LTB a, LTC4, L T D 4 and LTE4), 5-hydroperoxyeicosatetraenoic acid ( 5 - H P E T E ) and 5-hydroxyeicosatetraenoic acid (5-HETE). The leukotrienes are thought to be involved in asthma because they can mimic many of the p h e n o m e n a that characterize the disease (Drazen and Austen, 1987). Thus, leukotrienes contract airway smooth muscle, slow mueociliary clearance (Russi et al., 1985), increase mucus secretion (Marom et al., 1982), increase smooth muscle responsiveness and can
Correspondence to: W.M. Abraham, Department of Research, Mount Sinai Medical Center, 4300 Alton Road, Miami Beach, FL 33140, USA. Tel. 1.305.674 2790, fax 1.305.674 2198.
act as chemotaxins for inflammatory leukocytes (Bray, 1986; Chan et al., 1990; Spada et al., 1988; FordHutchinson et al., 1980). Leukotrienes have been identified in body fluids (blood, urine and lung washings) from experimental animals (Abraham et al., 1991; Tagari et al., 1990; Okayama et al., 1989) and man (Creticos et al., 1984; Taylor et al., 1989; Manning et al., 1990; Wenzel et al., 1990; Diaz et al., 1989; Sladek et al., 1990) following allergen provocation and in some studies the severity of the bronchial responses to antigen is correlated with the concentration of leukotrienes found (Sladek et al., 1990; Tagari et al., 1990; Abraham et al., 1991). Animal studies have also shown that L T D a / L T E 4 antagonists reduce both the early and late bronchial responses to inhaled antigen (Abraham, 1988). More recent studies in man using potent and selective leukotriene antagonists have confirmed these animal experiments (Hendeles et al., 1990; Taylor et al., 1991).
120
Both LTB 4 and 5-HETE are potent pro-inflammatory mediators (Barnes et al., 1988) up-regulating the functions neutrophils and eosinophils - two key effector ceils involved not only in the pathogenesis of the late response but also in the development of airway hyperresponsiveness that follows the late response (Larsen et al., 1987; Murphy et al., 1986; Gundel et al., 1990; Wegner et al., 1990). This post antigen-induced airway hyperresponsiveness is thought to be linked to factors which heighten the severity of asthma and so blocking this hyperresponsiveness may be important for reducing symptoms (O'Byrne et al., 1987). These observations indicate that it may be beneficial to inhibit the formation of all 5-1ipoxygenase products in designing therapies for asthma. Allergic sheep have many of the pathophysiological traits analogous to human asthma, including the development of both early and late airway responses after inhalation challenge with specific antigen (Abraham, 1989). The late response is associated with an influx of leukocytes into the lungs (Abraham et al., 1988a). The sheep that develop late responses also develop increased airway responsiveness to carbachol 24 h after challenge (Lanes et al., 1986). Furthermore, these antigen-induced responses are sensitive to pharmacological agents that are active in humans and so this animal model, in addition to its use for studying in vivo mechanisms of allergic airway disease, may be useful as a preclinical predictor for active drugs in the treatment of asthma. Zileuton (N(1-benzo[b]thien-2-ylethyl)-N-hydroxyurea) (Carter et al., 1991) is a specific orally active 5-1ipoxygenase inhibitor in both animals and man. Recently, the compound has shown efficacy against asthma induced by cold dry air (Israel et al., 1990) and allergen-induced nasal congestion in humans (Knapp, 1990). Whether or not zileuton would also be effective against allergen-induced bronchial responses is not yet known. Therefore, in this study, we determined the efficacy of zileuton on antigen-induced early and late responses and associated airway hyperresponsiveness in allergic sheep.
2. Materials and methods
2.1. In vitro studies
Eicosanoid synthesis in sheep whole blood: Dorset sheep were dosed by oral gavage with zileuton suspended in 0.1% methylcellulose at either 5 or 20 mg/kg. Venous blood (5 ml) was obtained 15 min prior to and at various times after dosing. A 0.5 ml aliquot was incubated at 37°C with 50 ~M calcium ionophore. A-23187 (Sigma Chemical Co., St. Louis, MO), to trigger LTB 4 biosynthesis. After 30 min, the blood was
rapidly cooled in an ice bath and centrifuged at 3°C for 10 rain at 2500 X g. The plasma was mixed with four volumes methanol at 3°C prior to centrifuging at 1800 X g for 30 min. LTB 4 concentrations were determined in 1:10 dilutions of the methanol extract by radioimmunoassay using a kit from Amersham. Average levels of LTB 4 were 3.2 ng/ml. 2.2. In rico studies 2.2.1. Animal preparation A total of eight sheep with a mean weight of 32 kg (range 24-38 kg) were used. All animals used developed both early and late airway responses to inhalation challenge with Ascaris suum antigen (Abraham et al., 1983). 2.2.2. Measurement of airway mechanics These methods have been described previously (Abraham et al., 1983). The unsedated sheep were restrained in a cart in the prone position with their heads immobilized. After topical anesthesia of the nasal passages with 2% lidocaine solution, a balloon catheter was advanced through one nostril into the lower esophagus. The animals were then intubated with a cuffed endotracheal tube through the other nostril using a flexible fiberoptic bronchoscope. Pleural pressure was estimated with the esophageal balloon catheter (filled with 1 ml of air) which was positioned 5-10 cm from the gastroesophageal junction. In this position the end expiratory pleural pressure ranged between - 2 and - 5 cm H20. Once the balloon was placed, it was secured so that it remained in position for the duration of the experiment. Lateral pressure in the trachea was measured with a sidehole catheter (inner dimension 2.5 mm) advanced through and positioned distal to the tip of the endotracheal tube. Transpulmonary pressure, the difference between tracheal and pleural pressure, was measured with a differential pressure transducer catheter system which showed no phase shift between pressure and flow to a frequency of 9 Hz. For the measurement of pulmonary resistance (RL), the proximal end of the endotracheal tube was connected to a pneumotachograph (Fleisch, Dyna Sciences, Blue Bell, PA). The signals of flow and transpulmonary pressure were recorded on a multichannel physiological recorder which was linked to a 386-DOS personal computer for on-line calculation of R L from transpulmonary pressure, respiratory volume (obtained by digital integration) and flow by the midvolume technique. Analysis of 5-10 breaths was used for the determination of R L. Immediately after the measurement of R L, thoracic gas volume (Vtg) was measured in a constant volume body plethysmograph to obtain specific lung resistance (SR L = RL • Vtg).
121
2.2.3. Aerosol delivery systems All aerosols were generated using a disposable medical nebulizer (Raindrop ®, Puritan Bennett, Lenexa, KS) that provided an aerosol with a mass median aerodynamic diameter of 3.2 p.m (geometric S.D. 1.9) as determined by an Andersen cascade impactor. The nebulizer was connected to a dosimeter system, consisting of a solenoid valve and a source of compressed air (20 psi). The output of the nebulizer was directed into a plastic T-piece, one end of which was connected to the inspiratory port of a Harvard respirator. The solenoid valve was activated for 1 s at the beginning of the inspiratory cycle of the respirator. Aerosols were delivered at a tidal volume of 500 ml and a rate of 20 breaths per rain.
2.3. Agents 2.3.1. Ascaris suum extract Ascaris suum extract (Greer Diagnostics, Lenoir, NC) was diluted with phosphate-buffered saline to a concentration of 82000 protein nitrogen u n i t s / m l and delivered as an aerosol (20 b r e a t h s / r a i n x 20 min). Carbamylcholine (Carbachol, Sigma Chemical Co., St. Louis, MO) was dissolved in buffered saline at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0% w / v and delivered as an aerosol. Zileuton was suspended in methylcellulose at a dose of 10 m g / k g and delivered via a nasogastric tube. Methylcellulose alone was used for control experiments. 2.4. Experimental protocol
2.2.4. Determination of airway responsiveness Airway responsiveness was determined as previously described for this model. We performed cumulative dose response curves to carbachol by measuring SR L immediately after inhalation of buffer and after each consecutive administration of 10 breaths of increasing concentrations of carbachol (0.25, 0.5, 1.0, 2.0 and 4.0% w / v buffered saline). The provocation test was discontinued when SRL increased over 400% from the post-saline value or after the highest carbacho! concentration had been administered. Airway responsiveness was estimated by determining the cumulative carbachol breath units that increased SR L by 400% over the post-saline value (PD400%) by interpolation from the dose response curve. One breath unit was defined as 1 breath of an aerosol solution containing 1% w / v carbachol (Soler et al., 1991).
Baseline dose-response curves to aerosol carbachol (to measure PD400%) were obtained before the start of the study. Then, on occasions 3 weeks apart, in a randomized fashion, baseline values of specific lung resistance (SR L) were obtained 1.5 h after drug or placebo administration. The baseline measurements of SR c were followed by bronchoalveolar lavage. Then the sheep were challenged with Ascaris suum antigen. Measurements of SR L were obtained immediately after challenge, hourly from 1 to 6 h after challenge and on the half-hour from 6.5 to 8 h after challenge. Bronchoalveolar lavage was performed again at 8 h after the last SR e measurement. Measurements of SR L were obtained 24 h after challenge followed by the 24 h post challenge determination of PD400o~ and a third bronchoalveolar lavage.
2.5. Statistical analysis 2.2.5. Bronchoalveolar lavage The distal tip of a specially designed 80 cm fiberoptic bronchoscope was wedged into a randomly selected subsegmental bronchus. Lung lavage was performed by slow infusion and gentle aspiration of 3 × 30 ml aliquots of phosphate-buffered saline (pH 7.4) at 39°C, using 30 ml syringes attached to the working channel of the bronchoscope (Lanes et al., 1986; Abraham et al., 1988a). The cell pellet was resuspended in buffered saline, and an aliquot of this resuspension was transferred to a hemocytometer chamber to estimate total cells. Total viable cells were estimated by trypan blue exclusion. A second aliquot of the cell suspension was spun in a cytospin (600 rpm for 10 min) and stained by Wright-Gimesa to identify cell populations. Five hundred cells per slide were identified to establish the differential cell count (100 X ; oil objective). Cell categories included epithelial cells, macrophages, lymphocytes, neutrophils, basophils, eosinophils, and monocytes; unidentifiable cells were grouped into a category termed 'others'.
The peak immediate and peak late increases in S R L (i.e. the largest increase in S R L between 6-8 h irrespective of when the increase occurred) and the area under the curve (AUC) for the immediate (0-4 h) and the late response (4-8 h) between the control and drug trial were compared with a paired t-test. The difference in logarithms of PD400% before and after antigen challenge in the control and drug trial was used to determine the effect of zileuton on airway responsiveness. A logarithmic difference (i.e. baseline PD400%post challenge PD400%) of zero indicates that airway responsiveness is unchanged, whereas an increase in the logarithmic difference represents an increased responsiveness. Analysis of these differences was also made with a paired t-test. The Wilcoxon test was used to compare lavage data between control and drug trials at a specific time, while Friedman's analysis of variance followed by a post-hoc comparison was used to determine differences over time within each group. For all statistical tests, significance was accepted if P < 0.05
122 120Z
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Fig. 1. Zileuton was given by oral gavage as a suspension in 0.1% methyl cellulose to six sheep, three each at 5 and 20 mg/kg. Prior to dosing and at several times thereafter blood was drawn and stimulated to synthesize LTB 4 by stimulation with 50 /~M calcium ionophore A23187. Data are plotted as the means+S.E.M, in six sheep.
using a two-tailed analysis. All values in the text, tables and figures are expressed as means + S.E.M.
3. Results
3.1. Ex uiuo studies Ex vivo inhibition of leukotriene formation has proven to be valuable tool in studying the effects of 5-1ipoxygenase inhibitors (Rubin et al., in press); and, in fact, has been used to choose dosage levels for clinical trials. As a first look at leukotriene inhibition in the sheep, six sheep were dosed at two doses by oral gavage. As shown in fig. 1, both 5 and 20 m g / k g oral doses of zileuton resulted in > 80% inhibition of ex vivo LTB 4 production through 9 h. Nearly complete inhibition through 24 h was seen at the 20 m g / k g dose and in two of the three animals dosed at 5 mg/kg. The 5 and 20 m g / k g doses were chosen to bracket the clinical doses used in phase I clinical trials of zileuton. A dose of 10 m g / k g was chosen for further studies. This is approximately equivalent to the maximum clinical dose used thus far for zileuton (800 rag).
3.2. In uiuo studies
.~
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Fig. 2. Time course of the changes in specific lung resistance (SR L) before and after antigen challenge in allergic sheep when treated with zileuton (10 m g / k g p.o.) and when the sheep were given placebo (control). Zileuton had no effect on the early response but significantly inhibited the late response (4-8 h). Values are means_+ S.E.M. for eight sheep.
in these sheep, but did not affect the immediate response to allergen. In the drug trial, SR L increased 258 _+ 54% from a baseline value of 1.02 + 0.03 L × cm H 2 0 / 1 per second. SR L then returned towards baseline and remained low for the duration of the experiment. The peak late increase in SR L in the zileuton trial was only 80 +_ 24%. AUC analysis of the immediate (0-4 h) and late (4-8 h) responses yielded similar results. Zileuton had no significant effect on the immediate response, but inhibited the late A U C by 59% (P < 0.05) (fig. 3). Plasma levels of zileuton were determined in seven of the eight sheep studied. Mean plasma concentrations at 3, 6 and 9 h after drug administration were 5.5 ___1.2, 6.4 + 1.8 and 5.0_+ 1.1 /~M, respectively. These concentrations are approximately 5- to 10-fold higher than the ICs0 of the compound against LTB 4 formation in human whole blood.
3.2.2. Bronchoalveolar lauage There were no differences between the control and drug trials in the total number of cells or total number 600
b.l > (lC
D o
3.2.1. Airway response In the control trial, antigen challenge resulted in immediate and late increases in SR L. Immediately after antigen challenge SR L increased 306 _+ 53% from a baseline value of 0.88 _+ L × cm H 2 0 / I per second. SR L returned towards baseline over the next 5 h, but then the animals developed a late response (fig. 2). The peak increase in SR L during the 6 - 8 h period (irrespective of when the increase occurred) was 176 + 23%. Zileuton inhibited the late response by 55% (P < 0.05)
~
TIME (hr) AFTER ANTIGEN
[C~ CONTROL m I ZILEUTON
__L 400
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200 tY
119
EJP 52523
The 5-1ipoxygenase inhibitor zileuton blocks antigen-induced late airway responses, inflammation and airway hyperresponsiveness in allergic sheep W i l l i a m M. A b r a h a m
a,
A s h f a q A h m e d a, A l e j a n d r o C o r t e s a, M a r e k W. Sielczak a, W e n d y H i n z b, J e n n i f e r B o u s k a b, C a r m i n e L a n n i b a n d R a n d y L. Bell b
a Harry Pearlman Biomedical Research Institute, Mount Sinai Medical Center, 4300Alton Road, Miami Beach, FL 33140, USA and h Immunosciences Research Area, Abbott Laboratories, Abbott Park, 1L 60064, USA Received 21 November 1991, revised MS received 26 February 1992, accepted 14 April 1992
Leukotrienes are thought to be involved in allergen-induced airway responses. To test this hypothesis we used a newly described 5-1ipoxygenase inhibitor, zileuton, and examined its effect on antigen-induced early and late bronchial responses, airway inflammation and airway hyperresponsiveness in allergic sheep. Early and late responses were determined by measuring specific lung resistance (SR e) before and serially for 8 h after antigen challenge. Airway inflammation was assessed by bronchoalveolar lavage performed before, 8 h after and 24 h after antigen challenge. Airway responsiveness was measured before and 24 h after challenge by determining the dose of inhaled carbachol that caused a 400% increase in SR L (PD400%). The sheep (n = 8) were challenged with A s c a r i s s u u m antigen once after vehicle treatment (methylcellulose) and once after treatment with zileuton (10 mg/kg in methylcellulose, p.o.) given 2 h before antigen challenge. Trials were separated by at least 21 days. Zileuton had no effect on the early bronchoconstrictor response to antigen but the drug inhibited the late bronchial response by 55% (P < 0.05). Unlike the control trial, there was no significant increase in bronchoalveolar lavage eosinophils at 8 h post challenge in the zileuton-treated sheep. Furthermore, zileuton treatment blocked (P < 0.05) the airway hyperresponsiveness seen 24 h after challenge. Ex vivo formation of leukotriene B 4 was inhibited over several hours after a single oral dose of zileuton, indicating that the compound was acting as a 5-1ipoxygenase inhibitor in vivo. These results suggest that 5-1ipoxygenase metabolites contribute to allergen-induced late responses, airway inflammation and airway hyperresponsiveness in this animal model of asthma. 5-Lipoxygenase; Leukotrienes; Asthma; Airway hyperresponsiveness; Inflammation; (Late responses)
1. Introduction Metabolism of arachidonic acid by 5-1ipoxygenase leads to the generation of leukotrienes (i.e. LTB a, LTC4, L T D 4 and LTE4), 5-hydroperoxyeicosatetraenoic acid ( 5 - H P E T E ) and 5-hydroxyeicosatetraenoic acid (5-HETE). The leukotrienes are thought to be involved in asthma because they can mimic many of the p h e n o m e n a that characterize the disease (Drazen and Austen, 1987). Thus, leukotrienes contract airway smooth muscle, slow mueociliary clearance (Russi et al., 1985), increase mucus secretion (Marom et al., 1982), increase smooth muscle responsiveness and can
Correspondence to: W.M. Abraham, Department of Research, Mount Sinai Medical Center, 4300 Alton Road, Miami Beach, FL 33140, USA. Tel. 1.305.674 2790, fax 1.305.674 2198.
act as chemotaxins for inflammatory leukocytes (Bray, 1986; Chan et al., 1990; Spada et al., 1988; FordHutchinson et al., 1980). Leukotrienes have been identified in body fluids (blood, urine and lung washings) from experimental animals (Abraham et al., 1991; Tagari et al., 1990; Okayama et al., 1989) and man (Creticos et al., 1984; Taylor et al., 1989; Manning et al., 1990; Wenzel et al., 1990; Diaz et al., 1989; Sladek et al., 1990) following allergen provocation and in some studies the severity of the bronchial responses to antigen is correlated with the concentration of leukotrienes found (Sladek et al., 1990; Tagari et al., 1990; Abraham et al., 1991). Animal studies have also shown that L T D a / L T E 4 antagonists reduce both the early and late bronchial responses to inhaled antigen (Abraham, 1988). More recent studies in man using potent and selective leukotriene antagonists have confirmed these animal experiments (Hendeles et al., 1990; Taylor et al., 1991).
120
Both LTB 4 and 5-HETE are potent pro-inflammatory mediators (Barnes et al., 1988) up-regulating the functions neutrophils and eosinophils - two key effector ceils involved not only in the pathogenesis of the late response but also in the development of airway hyperresponsiveness that follows the late response (Larsen et al., 1987; Murphy et al., 1986; Gundel et al., 1990; Wegner et al., 1990). This post antigen-induced airway hyperresponsiveness is thought to be linked to factors which heighten the severity of asthma and so blocking this hyperresponsiveness may be important for reducing symptoms (O'Byrne et al., 1987). These observations indicate that it may be beneficial to inhibit the formation of all 5-1ipoxygenase products in designing therapies for asthma. Allergic sheep have many of the pathophysiological traits analogous to human asthma, including the development of both early and late airway responses after inhalation challenge with specific antigen (Abraham, 1989). The late response is associated with an influx of leukocytes into the lungs (Abraham et al., 1988a). The sheep that develop late responses also develop increased airway responsiveness to carbachol 24 h after challenge (Lanes et al., 1986). Furthermore, these antigen-induced responses are sensitive to pharmacological agents that are active in humans and so this animal model, in addition to its use for studying in vivo mechanisms of allergic airway disease, may be useful as a preclinical predictor for active drugs in the treatment of asthma. Zileuton (N(1-benzo[b]thien-2-ylethyl)-N-hydroxyurea) (Carter et al., 1991) is a specific orally active 5-1ipoxygenase inhibitor in both animals and man. Recently, the compound has shown efficacy against asthma induced by cold dry air (Israel et al., 1990) and allergen-induced nasal congestion in humans (Knapp, 1990). Whether or not zileuton would also be effective against allergen-induced bronchial responses is not yet known. Therefore, in this study, we determined the efficacy of zileuton on antigen-induced early and late responses and associated airway hyperresponsiveness in allergic sheep.
2. Materials and methods
2.1. In vitro studies
Eicosanoid synthesis in sheep whole blood: Dorset sheep were dosed by oral gavage with zileuton suspended in 0.1% methylcellulose at either 5 or 20 mg/kg. Venous blood (5 ml) was obtained 15 min prior to and at various times after dosing. A 0.5 ml aliquot was incubated at 37°C with 50 ~M calcium ionophore. A-23187 (Sigma Chemical Co., St. Louis, MO), to trigger LTB 4 biosynthesis. After 30 min, the blood was
rapidly cooled in an ice bath and centrifuged at 3°C for 10 rain at 2500 X g. The plasma was mixed with four volumes methanol at 3°C prior to centrifuging at 1800 X g for 30 min. LTB 4 concentrations were determined in 1:10 dilutions of the methanol extract by radioimmunoassay using a kit from Amersham. Average levels of LTB 4 were 3.2 ng/ml. 2.2. In rico studies 2.2.1. Animal preparation A total of eight sheep with a mean weight of 32 kg (range 24-38 kg) were used. All animals used developed both early and late airway responses to inhalation challenge with Ascaris suum antigen (Abraham et al., 1983). 2.2.2. Measurement of airway mechanics These methods have been described previously (Abraham et al., 1983). The unsedated sheep were restrained in a cart in the prone position with their heads immobilized. After topical anesthesia of the nasal passages with 2% lidocaine solution, a balloon catheter was advanced through one nostril into the lower esophagus. The animals were then intubated with a cuffed endotracheal tube through the other nostril using a flexible fiberoptic bronchoscope. Pleural pressure was estimated with the esophageal balloon catheter (filled with 1 ml of air) which was positioned 5-10 cm from the gastroesophageal junction. In this position the end expiratory pleural pressure ranged between - 2 and - 5 cm H20. Once the balloon was placed, it was secured so that it remained in position for the duration of the experiment. Lateral pressure in the trachea was measured with a sidehole catheter (inner dimension 2.5 mm) advanced through and positioned distal to the tip of the endotracheal tube. Transpulmonary pressure, the difference between tracheal and pleural pressure, was measured with a differential pressure transducer catheter system which showed no phase shift between pressure and flow to a frequency of 9 Hz. For the measurement of pulmonary resistance (RL), the proximal end of the endotracheal tube was connected to a pneumotachograph (Fleisch, Dyna Sciences, Blue Bell, PA). The signals of flow and transpulmonary pressure were recorded on a multichannel physiological recorder which was linked to a 386-DOS personal computer for on-line calculation of R L from transpulmonary pressure, respiratory volume (obtained by digital integration) and flow by the midvolume technique. Analysis of 5-10 breaths was used for the determination of R L. Immediately after the measurement of R L, thoracic gas volume (Vtg) was measured in a constant volume body plethysmograph to obtain specific lung resistance (SR L = RL • Vtg).
121
2.2.3. Aerosol delivery systems All aerosols were generated using a disposable medical nebulizer (Raindrop ®, Puritan Bennett, Lenexa, KS) that provided an aerosol with a mass median aerodynamic diameter of 3.2 p.m (geometric S.D. 1.9) as determined by an Andersen cascade impactor. The nebulizer was connected to a dosimeter system, consisting of a solenoid valve and a source of compressed air (20 psi). The output of the nebulizer was directed into a plastic T-piece, one end of which was connected to the inspiratory port of a Harvard respirator. The solenoid valve was activated for 1 s at the beginning of the inspiratory cycle of the respirator. Aerosols were delivered at a tidal volume of 500 ml and a rate of 20 breaths per rain.
2.3. Agents 2.3.1. Ascaris suum extract Ascaris suum extract (Greer Diagnostics, Lenoir, NC) was diluted with phosphate-buffered saline to a concentration of 82000 protein nitrogen u n i t s / m l and delivered as an aerosol (20 b r e a t h s / r a i n x 20 min). Carbamylcholine (Carbachol, Sigma Chemical Co., St. Louis, MO) was dissolved in buffered saline at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0% w / v and delivered as an aerosol. Zileuton was suspended in methylcellulose at a dose of 10 m g / k g and delivered via a nasogastric tube. Methylcellulose alone was used for control experiments. 2.4. Experimental protocol
2.2.4. Determination of airway responsiveness Airway responsiveness was determined as previously described for this model. We performed cumulative dose response curves to carbachol by measuring SR L immediately after inhalation of buffer and after each consecutive administration of 10 breaths of increasing concentrations of carbachol (0.25, 0.5, 1.0, 2.0 and 4.0% w / v buffered saline). The provocation test was discontinued when SRL increased over 400% from the post-saline value or after the highest carbacho! concentration had been administered. Airway responsiveness was estimated by determining the cumulative carbachol breath units that increased SR L by 400% over the post-saline value (PD400%) by interpolation from the dose response curve. One breath unit was defined as 1 breath of an aerosol solution containing 1% w / v carbachol (Soler et al., 1991).
Baseline dose-response curves to aerosol carbachol (to measure PD400%) were obtained before the start of the study. Then, on occasions 3 weeks apart, in a randomized fashion, baseline values of specific lung resistance (SR L) were obtained 1.5 h after drug or placebo administration. The baseline measurements of SR c were followed by bronchoalveolar lavage. Then the sheep were challenged with Ascaris suum antigen. Measurements of SR L were obtained immediately after challenge, hourly from 1 to 6 h after challenge and on the half-hour from 6.5 to 8 h after challenge. Bronchoalveolar lavage was performed again at 8 h after the last SR e measurement. Measurements of SR L were obtained 24 h after challenge followed by the 24 h post challenge determination of PD400o~ and a third bronchoalveolar lavage.
2.5. Statistical analysis 2.2.5. Bronchoalveolar lavage The distal tip of a specially designed 80 cm fiberoptic bronchoscope was wedged into a randomly selected subsegmental bronchus. Lung lavage was performed by slow infusion and gentle aspiration of 3 × 30 ml aliquots of phosphate-buffered saline (pH 7.4) at 39°C, using 30 ml syringes attached to the working channel of the bronchoscope (Lanes et al., 1986; Abraham et al., 1988a). The cell pellet was resuspended in buffered saline, and an aliquot of this resuspension was transferred to a hemocytometer chamber to estimate total cells. Total viable cells were estimated by trypan blue exclusion. A second aliquot of the cell suspension was spun in a cytospin (600 rpm for 10 min) and stained by Wright-Gimesa to identify cell populations. Five hundred cells per slide were identified to establish the differential cell count (100 X ; oil objective). Cell categories included epithelial cells, macrophages, lymphocytes, neutrophils, basophils, eosinophils, and monocytes; unidentifiable cells were grouped into a category termed 'others'.
The peak immediate and peak late increases in S R L (i.e. the largest increase in S R L between 6-8 h irrespective of when the increase occurred) and the area under the curve (AUC) for the immediate (0-4 h) and the late response (4-8 h) between the control and drug trial were compared with a paired t-test. The difference in logarithms of PD400% before and after antigen challenge in the control and drug trial was used to determine the effect of zileuton on airway responsiveness. A logarithmic difference (i.e. baseline PD400%post challenge PD400%) of zero indicates that airway responsiveness is unchanged, whereas an increase in the logarithmic difference represents an increased responsiveness. Analysis of these differences was also made with a paired t-test. The Wilcoxon test was used to compare lavage data between control and drug trials at a specific time, while Friedman's analysis of variance followed by a post-hoc comparison was used to determine differences over time within each group. For all statistical tests, significance was accepted if P < 0.05
122 120Z
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Fig. 1. Zileuton was given by oral gavage as a suspension in 0.1% methyl cellulose to six sheep, three each at 5 and 20 mg/kg. Prior to dosing and at several times thereafter blood was drawn and stimulated to synthesize LTB 4 by stimulation with 50 /~M calcium ionophore A23187. Data are plotted as the means+S.E.M, in six sheep.
using a two-tailed analysis. All values in the text, tables and figures are expressed as means + S.E.M.
3. Results
3.1. Ex uiuo studies Ex vivo inhibition of leukotriene formation has proven to be valuable tool in studying the effects of 5-1ipoxygenase inhibitors (Rubin et al., in press); and, in fact, has been used to choose dosage levels for clinical trials. As a first look at leukotriene inhibition in the sheep, six sheep were dosed at two doses by oral gavage. As shown in fig. 1, both 5 and 20 m g / k g oral doses of zileuton resulted in > 80% inhibition of ex vivo LTB 4 production through 9 h. Nearly complete inhibition through 24 h was seen at the 20 m g / k g dose and in two of the three animals dosed at 5 mg/kg. The 5 and 20 m g / k g doses were chosen to bracket the clinical doses used in phase I clinical trials of zileuton. A dose of 10 m g / k g was chosen for further studies. This is approximately equivalent to the maximum clinical dose used thus far for zileuton (800 rag).
3.2. In uiuo studies
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Fig. 2. Time course of the changes in specific lung resistance (SR L) before and after antigen challenge in allergic sheep when treated with zileuton (10 m g / k g p.o.) and when the sheep were given placebo (control). Zileuton had no effect on the early response but significantly inhibited the late response (4-8 h). Values are means_+ S.E.M. for eight sheep.
in these sheep, but did not affect the immediate response to allergen. In the drug trial, SR L increased 258 _+ 54% from a baseline value of 1.02 + 0.03 L × cm H 2 0 / 1 per second. SR L then returned towards baseline and remained low for the duration of the experiment. The peak late increase in SR L in the zileuton trial was only 80 +_ 24%. AUC analysis of the immediate (0-4 h) and late (4-8 h) responses yielded similar results. Zileuton had no significant effect on the immediate response, but inhibited the late A U C by 59% (P < 0.05) (fig. 3). Plasma levels of zileuton were determined in seven of the eight sheep studied. Mean plasma concentrations at 3, 6 and 9 h after drug administration were 5.5 ___1.2, 6.4 + 1.8 and 5.0_+ 1.1 /~M, respectively. These concentrations are approximately 5- to 10-fold higher than the ICs0 of the compound against LTB 4 formation in human whole blood.
3.2.2. Bronchoalveolar lauage There were no differences between the control and drug trials in the total number of cells or total number 600
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3.2.1. Airway response In the control trial, antigen challenge resulted in immediate and late increases in SR L. Immediately after antigen challenge SR L increased 306 _+ 53% from a baseline value of 0.88 _+ L × cm H 2 0 / I per second. SR L returned towards baseline over the next 5 h, but then the animals developed a late response (fig. 2). The peak increase in SR L during the 6 - 8 h period (irrespective of when the increase occurred) was 176 + 23%. Zileuton inhibited the late response by 55% (P < 0.05)
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