Effect Ofa 5-Lipoxygenase Inhibitor on Leukotriene Generation and Airway Responses After Allergen Challenge in Asthmatic Patients 189

184 Thorax 1991;46:184-189

Effect of a 5-lipoxygenase inhibitor on leukotriene generation and airway responses after allergen Thorax: first published as 10.1136/thx.46.3.184 on 1 March 1991. Downloaded from challenge in asthmatic patients

Kok P Hui, Ian K Taylor, Graham W Taylor, Paul Rubin, James Kesterson, Neil C Barnes, Peter J Barnes

Abstract shown a slight reduction in the early asthmatic The effect ofa single oral dose (800 mg) of response to inhaled allergen" " and in cold air zileuton (A-64077), a specific 5-lipoxy- induced bronchoconstriction." The com- genase inhibitor, on the early and late pounds studied were relatively weak cysteinyl airway responses to inhaled allergen was leukotriene antagonists, however, and they studied in a randomised, double blind, would in addition leave the effects of the placebo controlled, and crossover trial dihydroxy acid LTB4 unopposed. An in nine subjects with atopic asthma. alternative approach is to inhibit the Leukotriene generation was also assessed 5-lipoxygenase enzyme to reduce synthesis of in vivo by measuring urinary leukotriene all the leukotrienes. Several different classes of (LT) E4 excretion, and ex vivo by 5-lipoxygenase inhibitors have been found to measuring calcium ionophore stimu- be effective in animal models ofasthma.14-16 No lated whole blood LTB4 production. significant reduction of allergen induced bron- Zileuton almost completely inhibited ex choconstriction was, however, proposed by vivo LTB4 production but reduced urin- piriprost" or nafazatrom'8 in two clinical ary excretion of LTE4 by only about half. studies in man-but nafazatrom was found There was a trend for the early asth- not to prevent ex vivo leukotriene production matic response to be less on the day of in man after oral dosing despite having zileuton treatment, but this did not 5-lipoxygenase inhibitory activity in vitro (the reach statistical significance (p = 0 08). activity of piriprost in man was not studied). The zileuton induced reduction in max- After exposure to inhaled allergen there is imum fall in FEVy in the early asthmatic an early and late bronchoconstrictor response

response was, however, significantly in asthmatic subjects and an increase in airway http://thorax.bmj.com/ related to the reduction in urinary LTE4 responsiveness. Several mediators, including excretion (r = 0 8), but not to the reduc- the leukotrienes, are generated during antigen tion in LTB4 generation ex vivo. There challenge and may be important in mediating was no significant change in the allergen the airway responses. Zileuton, a hydroxamic induced late asthmatic response, or in acid 5-lipoxygenase inhibitor,'9 has been the increase in airway responsiveness to shown to inhibit calcium ionophore methacholine following antigen. The stimulated human neutrophil production of results provide some support for the LTB4 in man ex vivo after oral dosing,'0 and is hypothesis that the cysteinyl leuko- effective in inhibiting allergen induced con- on September 25, 2021 by guest. Protected copyright. trienes have a role in the allergen traction of tracheal smooth muscle in the induced early asthmatic response. More guinea pig.2' We have investigated the efficacy Department of complete in vivo inhibition of 5-lipoxy- of zileuton (800 mg) on the generation of Thoracic Medicine, genase may be needed to produce a sig- cysteinyl leukotriene in vivo (assessed by National Heart and nificant reduction in airway response to urine LTE4 concentration) induced by Lung Institute, London SW3 6LY allergen challenge. inhaled allergen and on the generation of K P Hui LTB4 ex vivo (by calcium ionophore P J Barnes stimulated whole blood) in asthmatic subjects. Department of The leukotrienes, products of the 5-lipoxy- We assessed the inhaled allergen induced air- Clinical Pharmacology, Royal genase pathway of arachidonic acid metab- way responses of the early and late asthmatic Postgraduate Medical olism, are thought to be important mediators response and changes in airway responsive- School, London in the pathogenesis of asthma because their ness. W12 ONN that are I K Taylor biological activities produce changes G W Taylor similar to those seen in asthma.'-3 The cys- Department of teinyl leukotrienes, LTC4, LTD4, and LTE4, Methods Thoracic Medicine, are potent bronchoconstrictor agents in nor- SUBJECTS London Chest mal and asthmatic subjects,' and LTE4 has Eleven non-smoking men with atopic asthma Hospital, London E2 9JX been reported to increase airway hyper- (mean age 28, range 19-44 years) with an N C Barnes responsiveness.7 LTB4 is a potent chemotactic FEV1 above 70% predicted were recruited Abbott Laboratories, agent for leucocytes,8 and may be important in (table). Subjects were having inhaled sal- Abbott Park, Illinois mediating the inflammatory process in asth- butamol only, apart from one who was also 600064, USA matic can increase mucus P Rubin airways. LTD4 inhaling steroids and taking oral theo- J Kesterson production in human airway preparations9 phyllines. All subjects had been shown to Reprint requests to: and airway microvascular leakage in animals.'0 have a dual asthmatic response to allergen on Dr N C Barnes Several clinical studies with cysteinyl leuko- screening. None had had an upper respiratory Accepted 3 December 1990 triene antagonists in asthmatic subjects have tract infection or exacerbation of asthma Effect of a 5-lipoxygenase inhibitor on leukotriene generation and airway responses after allergen challenge in asthmatic patients 185 within six weeks of starting the study. Non- every 10 minutes for the first hour after steroidal anti-inflammatory drugs were not challenge, and then hourly for the next seven allowed during the study period. The study hours. At least two weeks later subjects re- was approved by the ethics committee of the turned for the second study to continue with Thorax: first published as 10.1136/thx.46.3.184 on 1 March 1991. Downloaded from National Heart and Lung Hospitals, and fully an identical protocol provided that the informed written consent was obtained from baseline FEV, was within 15% of that on the each subject. first study day. If not, they were asked to Subjects had to be healthy at the physical return for another study day. examination and to have normal results in FEV, was measured with a dry bellow biochemical and haematological tests and a spirometer (Vitalograph, Buckingham) with normal electrocardiogram before entry into the subject sitting upright. The mean of three the study. These were repeated during the good expiratory efforts was taken. Before each study and at the end to assess the safety and test subjects rested in a warm room for at least tolerability of zileuton. 15 minutes. Non-specific airway responsiveness was STUDY PROTOCOL assessed according to the method of Cockcroft Allergen challenge et al2" on the day before and after each aller- On a screening day the atopy of each subject gen test day, at the same time of the day for was confirmed by obtaining a positive skin- each subject. The provocative dose of metha- prick test response to either Dermatophagoides choline needed to reduce FEV, by 20% of the pteronyssinus or mixed grass pollen allergen post-diluent value (PC20 FEV,) was deter- extracts (Pharmacia, Milton Keynes) dis- mined. Methacholine was inhaled in the same solved in normal saline. Subjects first inhaled way as allergen. the diluent, aerosolised with a Wright nebu- liser (volume 2 ml, flow rate 7 1/min, output Assessment of5-lipoxygenase inhibition 0-2 ml/min), through a face mask with open 5-Lipoxygenase activity after allergen mouthed tidal breathing for two minutes, and challenge was assessed by measuring urinary FEV, was then measured for 15 minutes to LTE4 excretion.23 Immediately before exclude a bronchoconstrictor response to challenge subjects emptied their bladder and diluent. Cumulative doses of allergen were urine was then collected for four hours. Urine then inhaled in the same way to identify volume and pH were measured and aliquots of subjects with a dual asthmatic response. The 30 ml were stored at -70°C. LTE4 was initial allergen concentration used for inhala- extracted from the urine by reverse phase high tion was that which caused a 2-3 mm dia- performance liquid chromatography, tritiated meter weal in the skinprick test. Doubling LTE4 being used as an internal stan- concentrations of allergen were inhaled until dard,24 and measured by radioimmunoassay http://thorax.bmj.com/ an early asthmatic response (fall in FEV, of at (Amersham International, Amersham). The least 20% of the post-diluent value) was seen. efficacy of zileuton was also assessed by cal- No further allergen was then given, FEVy was cium ionophore stimulated whole blood LTB4 measured up to eight hours to detect any late production ex vivo. Three and a half, four, asthmatic response, which was defined as a fall five, seven, nine, and 10 hours after it was in FEV, of at least 15% of post-diluent value. given 5 ml of whole blood was incubated with If a dual response was seen, the final allergen 5 pl of 50 mM calcium ionophore for 15 min- dose reached was used on the two allergen utes and centrifuged, and the supernatant was on September 25, 2021 by guest. Protected copyright. challenge study days. Of the 17 subjects stored at - 70°C. LTB4 was extracted by high screened, six subjects with an isolated early performance liquid chromatography, and asthmatic response were not enrolled into the assayed by radioimmunoassay. main study. Two to three weeks later subjects returned Drug level for the first allergen challenge study. Zileuton Plasma level of drug was measured at 3 5, 4 (800 mg) or matching placebo was ingested in and 5 hours post-dose on both placebo and the morning, and allergen challenge was per- active days. Zileuton was extracted by high formed three hours later. FEVy was measured performance liquid chromatography and measured by ultraviolet absorbance as developed by Abbott Laboratories. Characteristics of the asthmatic subjects ANALYSIS PC20 PC20 values were log transformed before Subject Age FEV, methacholine* No (Y) I (% pred) (mg/ml) Drugst analysis. FEV, and PC20 values were com- pared by analysis of variance. Paired data 1 22 49 (98) 0-44 Salbutamol were analysed by the Wilcoxon sign rank test 2 33 3-0 (81) 0 19 Salbutamol and the relation 3 21 4 9 (117) 0-2 Salbutamol between two variables by 4 22 4-1 (98) 0-26 Salbutamol regression analysis. Results are presented as 5 24 4-8 (117) 3 4 Salbutamol means with the standard errors of the mean in 6 27 4-3 (100) 0 15 Salbutamol parentheses unless otherwise stated. p < 0 05 7 27 4-6 (105) 1-18 Salbutamol 8 23 26 (84) 0 57 Salbutamol was accepted as significant. 9 29 4-6 (112) 124 Salbutamol, beclomethasone, theophylline Results *The provocative dose of inhaled methacholine needed to cause a 20% fall in baseline FEV,. SUBJECTS tInhaled, apart from the theophylline (oral) taken by subject 9. Of the 11 subjects who showed a late response 186 Hui, Taylor, Taylor, Rubin, Kesterson, Barnes, Barnes

FEV, 120 (I) Thorax: first published as 10.1136/thx.46.3.184 on 1 March 1991. Downloaded from 4.5 r 100 F Placebo 4.0 E 80 I-- 60 h 3.5 -j 40 1 3.0 20 1 O 2.5 zileuton I I I I I1 0 2 4 6 8 10 0 60 120 180 240 300 360 420 480 Time (h) Time (min) Figure 3 Time course ofeffect of zileuton on calcium Figure I Effect of zileuton (800 mg orally) on early and late asthmatic responses after ionophore stimulated whole blood leukotriene (L T) B4 allergen challenge. * Zileuton; C placebo. Values are means with 1 SEM. production. Values are means with 1 SEM. *p < 0-005.

to inhaled allergen, two were withdrawn. One was found by two way analysis according the developed an exacerbation of asthma following the method of Hills and Armitage.25 There was a chest infection after the first study day a trend for the fall in FEV1 during the early (placebo). The other subject who had an FEV, asthmatic response to be less with zileuton, but of just over 70% predicted was withdrawn this did not reach statistical significance at any when his FEV, fell to below 70% predicted point (maximum fall in FEVI: placebo 108 before allergen challenge. The remaining nine (0 25)1, zileuton 0-83 (0 21)1; p = 0 18). The subjects completed the study, and all results maximum difference between placebo and were included in the analysis. Zileuton was well zileuton was 40 minutes after allergen tolerated and there were no important bio- challenge, which is after the nadir of the fall in http://thorax.bmj.com/ chemical, haematological, or electrocardio- FEV1 (fig 1; difference in FEVI-0 33 (0 16) 1; graphic changes after treatment. p = 008). There was no difference between placebo and zileuton in the late asthmatic AIRWAY RESPONSES response (maximum fall in FEV1: placebo 1 16 Baseline values before allergen challenge on (0-24) 1, zileuton 0-92 (0 27)1; p = 0 22). placebo and treatment days were closely Therewereno significantdifferences intheareas matched (fig 1). No period or treatment effect under the curve during the early or late phases. on September 25, 2021 by guest. Protected copyright.

PC20 FEV, methacholine (mg/ml) (a) (b) 10- 80- 200 0 m

. : 4) 1 - 0-... C- =

-.c 0- - o o- . +0m~ E,o o ob 0.1 - o -* a] 100. E: ., 4) _ Z5 >0 zileuto 5o 0 ._ r 0.01- -J 0 0 Pre Post Pre Post

Figure 5 Scatter diagrams showing the (a) (b) li?iear regression r = 0.8, p<0.01 r = 0.04 relationships between Thorax: first published as 10.1136/thx.46.3.184 on 1 March 1991. Downloaded from zileuton induced change in 2- r. maxilnum fall in FEV, .,-j during the early asthmatic -4 1-4 response (EAR) and 40. <. reduction In urinary W (U leukotrienie (LT) E4 4 production. 0

E - . 't-*j '" r. O x . -, CZ ;-, *-0 0 0 E 111:3 rl -, 0 .-4 -.4 (U m - z C,3 4 W u ;4 -1 0 10 20 30 Change in urinary excretion Maximum inhibition (%baseline) of LTE4 (ng/mmol creatinine) of ex vivo LTB4 production

Baseline airway responses to inhaled meta- and substantially reduced calcium ionophore choline before allergen challenge were very stimulated whole blood LTB4 production ex similar for placebo and zileuton (geometric vivo. There was a trend towards a reduction in mean PC20 FEV, = 0 41 (95% confidence the fall in FEV, during the early asthmatic interval 0 20-0 84) and 0 44 mg/ml (0 15- response with zileuton, but neither this nor the 0 78). The increase in airway responsiveness late asthmatic response or increase in airway after allergen challenge did not differ significan- responsiveness differed significantly between tly after placebo and zileuton treatment zileuton and placebo. The change in the early (before/after challenge PC20 FEV, metacholine: asthmatic response induced by zileuton placebo 2 13, zileuton 1-89; fig 2). correlated with the change in urinary LTE4 concentrations but not with the change in ex EFFECTS ON IN VIVO AND EX VIVO LEUKOTRIENE vivo LTB4 production. GENERATION In vivo activation of the 5-lipoxygenase

Zileuton substantially inhibited calcium iono- pathway in asthmatic patients during an asth- http://thorax.bmj.com/ phore stimulated whole blood LTB4 produc- matic attack has been shown by the increased tion with maximum inhibition (931% of urinary excretion of LTE4,23 and by increased baseline; p < 0-005) four hours post-dose; concentrations of cysteinyl leukotrienes in after the dose had been given significant inhi- bronchoalveolar lavage fluid recovered by bition was still present at 10 hours after the fibreoptic bronchoscopy during the early26 and dose (63-5% of baseline; p < 0-005; fig 3). late27 responses to inhaled allergen challenge. Mean urinary LTE4 excretion after allergen In the present study the activation of 5-

challenge was reduced by about half by lipoxygenase in airways was assessed by on September 25, 2021 by guest. Protected copyright. zileuton treatment (placebo 111-5 (23 5), urinary excretion of LTE4 as this is simple and zileuton 58 2 (14 8) ng/mmol creatinine; non-invasive and did not interfere with p < 001; fig 4). measurements of lung function. Urinary LTE4 The zileuton induced change in maximum excretion after allergen challenge is likely to fall in FEV, for the early asthmatic response reflect airway leukotriene generation, as there correlated with the reduction in urinary LTE4 was no evidence of systemic effects in any production (r = 0 8, p < 0 01; fig 5) but not subjects. Zileuton reduced the urinary with the inhibition of calcium ionophore excretion of LTE4 by about half, though not stimulated whole blood LTB4 production down to the range found in non-asthmatic (r = 0 04, p > 0 5). subjects in this laboratory.23 This suggests that 5-lipoxygenase activity was only partially PLASMA ZILEUTON CONCENTRATIONS inhibited, and may explain the non-significant Mean plasma zileuton concentrations three and reduction in bronchoconstriction during the a half, four, and five hours after ingestion were early asthmatic response. A significant 3-37 (0 36), 3 03 (0-39) and 2 6 (0-27) Mg/ml reduction ofairway response to allergen may be respectively on the days of zileuton treatment; seen only if more complete inhibition of no drug was detected on the placebo day. There leukotrienes generation can be achieved, as the was no relation between the peak drug concen- leukotrienes are very potent biological agents: trations and change in FEV, during the early the cysteinyl leukotrienes are thousands of and late asthmatic responses, reduction in times more potent than histamine in causing urinary LTE4 excretion, or calcium ionophore bronchoconstriction,28 and LTB4 is one of the stimulated whole blood LTB4 production. most potent chemotactic agents known.29 The early bronchoconstrictor response after allergen challenge is thought to be due to Discussion release of mediators, such as histamine and We found that zileuton at the dose used in the leukotrienes, from inflammatory cells via IgE study partially reduced urinary LTE4 excretion mediated mechanisms. Although not statis- 188 Hui, Taylor, Taylor, Rubin, Kesterson, Barnes, Barnes tically significant, the maximum reduction of zileutonwas only partially effective in inhibiting the fall in FEV, produced by zileuton was after activation of 5-lipoxygenase in vivo by inhaled

the nadir in the fall in FEV, following allergen allergen despite near complete ex vivo Thorax: first published as 10.1136/thx.46.3.184 on 1 March 1991. Downloaded from challenge. This time course of action is con- inhibitory activity. There was a trend towards a sistent with the known action of leukotrienes reduction in the early asthmatic broncho- and histamine, inhaled cysteinyl leukotrienes constrictor response but this was not causing maximum bronchoconstriction at significant. Measurement of leukotriene about 15 minutes,4- and histamine somewhat production in vivo rather than ex vivo may be earlier. Leukotrienes are not stored by resting more useful in future studies of 5-lipoxygenase cells (unlike histamine), and are generated only inhibitors in man. Our results would fit a role after stimulation, so they would be expected to for the leukotrienes in the early asthmatic contribute more to the latter part of the early response to inhaled allergen, particularly if bronchoconstrictor response after allergen inhibition of leukotriene related airway challenge. In a study by Britton and coworkers, responses requires near complete inhibition of in which a leukotriene antagonist, L649923, the 5-lipoxygenase enzyme in vivo. Further reduced the early response to allergen, the studies with higher or repeated doses of maximum effect was seen after the nadir of the zileuton or with more potent 5-lipoxygenase early asthmatic response.'" In another study an inhibitors in man are needed. antihistamine was effective in the first 15 minutes of the early asthmatic response after allergen challenge.0 The significant correlation between inhibition of in vivo LTE4 generation Samuelsson B. Leukotrienes: mediators of immediate hyper- and reduction in early asthmatic response also sensitivity reactions and inflammation. Science 1983; supports the role of the leukotrienes as 220:568-75. 2 Hannarstrom S. Leukotrienes. Ann Rev Biochem 1983; mediators in the early asthmatic response. 52:355-77. Although the pattern of change in the early 3 Drazen JM, Austen KF. Leukotrienes and airway responses. Am Rev Respir Dis 1987;136:985-8. asthmatic response is similar to that in the 4 Barnes NC, Piper PJ, Costello JF. Comparative effects of study of Britton et al," the lack of statistical inhaled leukotriene C4, leukotriene D4, and histamine in significance in our study may be due to a type II normal human subjects. Thorax 1984;39:500-4. 5 Adelroth E, Morris MM, Hargreave FE, O'Byrne PM. error. Airway responsiveness to leukotrienes C4 and D4 and to Although leukotriene production has been methacholine in patients with asthma and normal controls. N Engl J Med 1986;315:480-4. shown during the late asthmatic response 6 Davidson AB, Lee TH, Scanlon PD, et al. Broncho- following allergen challenge,27 zileuton did not constrictor effects of leukotriene E4 in normal and reduce the late fall in significantly in the asthmatic subjects. Am Rev Respir Dis 1987;135:333-7. FEV, 7 Arm JA, Spur BW, Lee TH. The effects of inhaled leuk- present study. This may be because the activity otriene E4 on the in airway responsiveness to histamine http://thorax.bmj.com/ of zileuton in the single dose of 800 mg used in subjects with asthma and normnal subjects. J AllergyClin Immunol 1988;82:654-60. this study may have been insufficient by that 8 Ford-Hutchinson AW, Bray MA, Doig MV, Shipley ME, time, particularly when it was only partially Smith MJH. Leukotriene B, a potent chemokinetic and as by aggregating substance from polymorphonuclear leuko- effective during the early phase measured cytes. Nature 1980;286:264-5. urinary LTE4 levels. In two patients where 9 MaromS, Shelhamer JH, Bach MK, Morton DR, Kaliner serum drug concentrations seven hours after M. Slow reacting substances leukotrienes C4 and D4 increase the release of mucus from human airways in vitro. the dose were available little zileuton was Am Rev Resp Dis 1982;126:449-51. detected. 10 Evans TW, Rogers DF, Aursudkig B, Chung KF, Barnes Previous studies of 5-lipoxygenase inhibitors PJ. Regional and time-dependent effects of inflammatory on September 25, 2021 by guest. Protected copyright. mediators on airway microvascular permeability in the in man have depended on ex vivo methods of guinea pig. Cli Sci 1989;76:161-6. assessing its activity. In the present study 11 BrittonJR, Hanley SP, Tattersfield AE. The effect of an oral leukotriene D4 antagonist L649,923 on the response to zileuton was more effective in inhibiting ex vivo inhaled antigen in asthma. J Allergy Clin Immunol calcium ionophore stimulated whole blood 1987;79:81 1-6. 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Alternatively, inhibitor of leukotriene synthesis (U-66,858) in the rhesus different inflammatory cells may have different monkey Ascaris reactor.Int Arch Allergy Appl Immunol to the effect of zileuton, such 1988;87:204-7. susceptibilities 16 Ku EC, Raychaudhuri A, Ghai G,et al. Characterisation of that the mast cells, macrophages, or eosinophils CGS 8515 as a selective 5-lipoxygenase inhibitor using in in the airways may not have been affected as vitro and in vivo models. Biochim Biophys Acta 1988; much as neutrophils in the peripheral blood. In 959:332-42. 17 MannJS, Holgate ST. Effect of piriprost (U-60257), a novel addition, the cellular generation of the cys- lipoxygenase inhibitor, on allergen and exercise induced teinyl leukotrienes may be less affected than that asthma.-Thorax 1986;41:746-52. 18 Fuller RW, Maltby N, Richmond R, etal. Oral nafazatrom of LTB,. The lack of relation between the in man: effect on inhaled antigen challenge. 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