The effect of GR32191 (a thromboxane receptor antagonist) on airway responsiveness in asthma

Pulmonary Pharmacology (1992) 5, 199-202 The Effect of GR32191 (a Thromboxane Receptor Antagonist) on Airway Responsiveness in Asthma S . C. Stenton, C . A . Young, A. Harris*, J . B. D. Palmer*, D . J. Hendrick, E . H. Walters Chest Unit, Newcastle General Hospital, University ofNewcastle upon Tyne and *Glaxo Group Research, Greenford, Middlesex, UK SUMMARY: Airway responsiveness to methacholine was measured in nine subjects (22-53 years, seven male) with chronic stable asthma. All subjects were taking inhaled beclomethasone (< 1000 pg daily) . The mean baseline FEV, was 2.841(77% of predicted) and the geometric mean PD WFEV, was 31 pg. After a run-in period, the subjects were randomly allocated to two treatment periods with the specific thromboxane receptor antagonist GR32191, 40 mg four times daily for 3 weeks, and identical placebo capsules . A double-blind, placebo-controlled, cross-over design was employed with 4 weeks between the two treatment periods . Treatment with GR32191 did not result in any significant improvement in mean FEV, (2 .941 after placebo and 2.861 after GR32191 ; F,,.,,=1 .02, P>0.1) or PDJEV, (24.3 pg after placebo and 38 .5 pg after GR32191 ; F,,,, = 0.59, P > 0.1) . We conclude that thromboxane is not important in the maintenance of airway hyperresponsiveness in chronic asthma and that thromboxane receptor antagonists are unlikely to provide effective treatment for this group of patients. INTRODUCTION Prostaglandin and leukotriene mediators derived from arachidonic acid are present in the airways of asthmatic subjects. Thromboxane A2 (TxA2) and prostaglandin D2 (PGD2) are released from human lungs during anaphyllactic reactions' while PGD2, leukotriene C4 (LTC 4 ) and TxA 2 levels are all increased in the airways of asthmatic subjects follow- ing allergen challenge . 2 These mediators which have a variety of proinflammatory actions might also be important in the maintenance of airway hyperrespon- siveness and symptoms in chronic asthma . In a dog model, exposure to oxone causes an influx of neutrophils into the airways and leads to the development of airway hyperresponsiveness . 3 This can be prevented by the administration of the cycloox- ygenase inhibitor indomethacin 3 or the TxA2 synthe- tase inhibitor OKY0464 suggesting that it is the release of TxA2 which is directly responsible for the develop- ment of airway hyperresponsiveness in this situation . Airway hyperresponsiveness induced by allergen,' platelet activating factor (PAF), 6 and LTB4' are also inhibited by OKY046. Furthermore, the contractile responses of airway smooth muscle to PGD2 8 and PGF2a9 are inhibited by thromboxane receptor antag- onists . This raises the possibility that TxA 2 release or stimulation of thromboxane receptors might be the final common mechanism through which a number of asthmagenic agents exert their effect . 0952-0600/92/030199+04 $08 .00/0 PULMONARY PHARMACOLOGY 199 An important role for TxA 2 in human asthma has been suggested by studies in which treatment with OKY04610 •" and AA2414 (a TxA 2 receptor antagon- ist) 12 markedly lowered airway responsiveness to ace- tylcholine. However, other TxA2 receptor antagonists (CGS12970 and GR32191) have been ineffective in preventing bronchoconstriction provoked by exercise challenge13 or the inhalation of PAF . 14 A small effect on the immediate response to allergen has been found by some"' but not other workers 16 and there appears to be no effect on the late asthmatic reaction or allergen induced airway hyperresponsiveness . 15 The aim of the present study was therefore to clarify the importance of TxA2 in human asthma by studying the effect of the potent and specific TxA 2 receptor antag- onist GR32191 17 on airway hyperresponsiveness . MATERIALS AND METHODS Twelve asthmatic subjects (22-53 years, nine male) were entered into the study. Seven were atopic as judged by skin prick tests to common aeroallergens . All had a well documented clinical diagnosis of asthma, a prestudy FEV, of at least 60% of the predicted value and a documented 15% variability of ventilatory function . All were using inhaled broncho- dilators as required and regular inhaled corticoster- oids (beclomethasone, less than 1000 .tg daily; mean daily dose 256 pg) but no other medication and they © 1992 Academic Press Limited 200 S. C. Stenton et al w LL 0 N Fig . 1 Individual PD 20FEV, measurements and geometric means (arrows) are shown . The values were: run-in period 32 µg ; pre-treatment 30 µg ; immediately post-treatment 24 µg ; post wash-out period 29 µg . None of these changes in PD ZOFEV, were statistically significant (P>0 .1) . all had measurable airway hyperresponsiveness to methacholine. The study was approved by the local Ethics Committee . A randomized, double-blind, cross-over design was employed. Following a run-in period of 2 weeks, the subjects were randomly assigned to receive either GR32191, 40 mg four times daily, or identical placebo capsules for 3 weeks . After a wash-out period of 2 weeks, and a further run-in period, treatment was repeated with the alternative medication . Airway re- sponsiveness to methacholine was measured on seven occasions; at the start of each of the two run-in periods, at the start of the two treatment periods, at the end of each treatment period, and at the end of the two wash-out periods. The end of the first wash-out period coincided with the beginning of the second run-in period . Airway responsiveness to methacholine was measured using a previously evaluated dosimeter technique." Inhaled bronchodilators were avoided for 6 h before each test and all the tests for each subject were performed at the same time of day . Following baseline measurements of FEV,, methacholine was inhaled in doubling doses from 3 to 6400 µg at 5-min intervals. FEV, was recorded after each dose and further doses were given until there was a greater than 20% fall. The dose causing exactly a 20% fall in FEV, was determined by linear interpolation from the dose- response curve and expressed as PD 20FEV, . Changes in FEV, and PD20FEV, were tested for statistical significance using a two-way analysis of variance . RESULTS Three subjects failed to complete the study, two because of intercurrent respiratory infections and one because of the possibility of pregnancy . Their results are not included in the analysis . The mean prestudy FEV, was 2 .841 (77% of predicted). There were no statistically significant changes in the baseline FEV, measurements at any of the subsequent visits (2 .82 and 2.86 pre- and post-GR32191, and 2 .95 and 2 .94 pre- and post-placebo ; F,,, = 1 .02, P > 0.1) . Individual PD20FEV, measurements before and after placebo and GR32191 are shown in Figure 1 together with the geometric means. The repeatability of the measure- ments of PD20FEV, in these subjects was determined from the coefficient of repeatability based on the measurements at the first two visits . Its numerical value was 2.54 suggesting that in 95% of cases, the second of a pair of replicate measurements of PD20FEV, was within the range x 2 .54 to x 1/2 .54 of the first measurement . In comparison with this, the magnitudes of the changes in mean PD 20FEV, during the treatment periods were all small (29 and 38 gg pre- and post-GR32191 and 30 and 24 pre- and post- placebo) . None were statistically significant (F,,,==0.59, P>0.1) . DISCUSSION Airway responsiveness is frequently used as a measure of asthma activity both in clinical practice and in research studies . It correlates with asthmatic symp- toms and the use of medication 19 and it is increased by exposure to asthmagenic agents such as allergen or occupational agents in sensitized subjects . 20' 21 Oral and inhaled corticosteroids which are effective pro- phylactic treatments in asthma cause a reduction in airway responsiveness ." All these features suggest that it provides a useful measurement of the underly- ing pathophysiological process in asthma which is presumed to be related to inflammation in the air- ways . However, the mechanisms which are important in maintaining airway responsiveness in asthmatic sub- jects are incompletely understood . Geometric factors secondary to airway inflammation and mucosal oedema may have a role but chemical mediators acting directly to increase the responsiveness of the neuromuscular apparatus in the airways are also likely to be important . Previous animal and human studies have suggested that TxA2 might be particu- larly important in this regard but this view is not supported by the results of this study which demon- strated that the specific TxA 2 receptor antagonist GR32191 had no significant effect on airway respons- iveness in a group of moderately severe asthmatic subjects . Studies using similar numbers of subjects have demonstrated significant reductions in airway re- sponsiveness when inhaled corticosteroids are used 23,24 and it is unlikely that a clinically significant effect of the drug would have been missed by a study of this size. However, these results do not exclude the possi- bility that a small effect might have been demon- strated had larger numbers been studied . It is unlikely that our negative results were due to an inadequate dose of GR32191 or a failure of the drug to reach the appropriate intrapulmonary receptors . The doses employed were more than those required to inhibit platelet aggregation in vitro 25 and it has previously been shown that a single 80 mg dose of GR32191 can effectively antagonize airway TxA 2 receptors .' Being a receptor antagonist, GR32191 has no effect on circu- lating TxA2 levels and these were not measured . Other studies examining the role of thromboxane in airway hyperresponsiveness have produced conflict- ing results. The thromboxane synthetase inhibitor OKY046 given for 4 days either orally 10 or by inhala- tion26 caused small but significant reductions in air- way responsiveness though there was no effect when a single dose was given by inhalation." A specific TxA2 receptor antagonist AA2414 caused a significant reduction in airway responsiveness when given at a dose of 40 mg/day but not when 20 mg/day was given." However, these studies employed open de- signs with no attempt at blinding or placebo control . When the thromboxane synthetase inhibitor CGS13080 was given orally for 2 days as part of a placebo-controlled trial it did not have any effect on airway responsiveness ." The studies with OKY046 and AA2414 used older subjects (mean ages 57 and 51 years) who were not taking inhaled corticosteroids . Little is known of the effect of these agents on intrapulmonary TxA2 levels but as the subjects in our study were all using inhaled corticosteroids, it is possible that the resulting inhibi- tion of phospholipase A2 had already reduced the Effect of GR32191 on Airway Responsiveness in Asthma 201 TxA2 levels in the airways and masked any potential further benefit of specific TxA 2 receptor antagonism . However, all our subjects had active asthma and marked airway hyperresponsiveness despite being on inhaled corticosteroids, suggesting that either the TxA2 was not being effectively antagonized or factors other than TxA2 were important in maintaining their asthmatic activity . In either case, the results argue against anything other than a minor role for TxA 2 or any other mediator acting through thromboxane receptors in human asthma. This makes it unlikely that TxA2 receptor antagonists will find a useful clinical role in the management of chronic symptoma- tic asthma. At the very least our data imply that a thromboxane antagonist will have no effect greater than a low dose of inhaled corticosteroid and no additive effect . It has been known for some time that cyclo-oxyge- nase inhibitors do not provide effective treatment for asthma though this may be because their effect in reducing TxA 2 and prostaglandin levels is balanced by an increase in leukotrienes which can increase airway responsiveness, or by a decrease of potentially bron- chodilator prostaglandins PGE2 or PGI2 (prostacyc- lin). However, recent results of a double-blind, pla- cebo-controlled trial of a specific TxA 2 synthetase inhibitor (Dazmagrel) also failed to demonstrate any increase in airway responsiveness . 27 This reinforces our argument that TxA2 is not a centrally important mediator in the pathogenesis of airway hyperresponsi- veness in asthma . References 1 . Schulman E S, Newball H H, Demers L M, Fitzpatrick F A, Adkinson N F . Anaphylactic release of thromboxane A2, prostaglandin D2, and prostacycline from human lung parenchyma. Am Rev Respir Dis 1981 ; 124: 402-406 . 2 . Wenzel S E, Westcott J Y, Larsen G L . Bronchoalveolar lavage fluid mediator levels 5 minutes after allergen challenge in atopic subjects with asthma: relationship to the development of late asthmatic responses . J Allergy Clin Immunol 1991 ; 87: 540-548 . 3 . O'Byrne P M, Walters E H, Aizawa H, Fabbri L M, Holtzamn M J, Nadel J A. Indomethacin inhibits the airway hyperresponsiveness but not the neutrophil influx induced by ozone in dogs . Am Rev Respir Dis 1984; 130 : 220-224 . 4 . Aizawa H, Chung K F, Liekauf G D, et al . Significance of thromboxane generation in ozone-induced airway responsiveness in dogs . J Appl Physiol 1985 ; 59: 1941-1946 . 5 . Chung K F, Aizawa H, Becker A B, Frick 0, Gold W M, Nadel J A . Inhibition of antigen-induced airway hyperresponsiveness by a thromboxane synthetase inhibitor (OKY-046) in allergic dogs . Am Rev Respir Dis 1986; 134 : 258-261 . 6. Chung K F, Aizawa H, Leikauf G D, Ueki I F, Evans T W, Nadel J A . Airway hyperresponsiveness induced by platelet-activating factor : role of thromboxane generation. J Pharmacol Exp Ther 1986 ; 236: 580-584 . 7 . O'Byrne P M, Leikauf G D, Aizawa H, et al . Leukotriene B4 induced airway hyperresponsiveness in dogs. J Appl Physiol 1985 ; 59: 1941-1946 . 8 . Beasley R C W, Featherstone R L, Church M K, et al. The effect of a thromboxane receptor antagonist GR32191B on 2 0 2 S. C . Stenton et al PgD2 and allergen induced bronchoconstriction . J Appl Physiol 1989 ; 66 : 1685-1693 . 9 . Jones R L, Peesapati V, Wilson N H . Antagonism of the thromboxane-sensitive contractile systems of the rabbit aorta, dog saphenous vein and guinea-pig trachea . Br J Pharmacol 1982; 76 : 423-428 . 10 . Fujimura M, Sasaki F, Nakatsumi Y, et al . Effects of a thromboxane synthetase inhibitor (OKY-046) and a lipoxygenase inhibitor (AA-861) on bronchial responsiveness to acetylcholine in asthmatic subjects . Thorax 1986 ; 41 : 955-959. 11 . Fujimura M, Sakamoto S, Matsuda T . Attenuating effect of a thromboxane synthetase inhibitor (OKY-046) on bronchial responsiveness to methacholine is specific to bronchial asthma . Chest 1990 ; 98 : 656-660. 12. Fujimura M, Sakamoto S, Saito M, Mikaye Y, Matsuda T . Effect of a thromboxane A2 receptor antagonist (AA-2414) on bronchial hyperresponsiveness to methacholine in subjects with asthma. J Allergy Clin Immunol 1991 ; 87: 23-27 . 13 . Finnerty J P, Twentyman 0 P, Harris A, Palmer J B D, Holgate ST . Effect of GR32191, a potent thromboxane receptor antagonist, on exercise induced bronchoconstriction in asthma. Thorax 1991 ; 46: 190-192 . 14 . Stenton S C, Ward C, Duddridge M, et al . The actions of GR32191B, a thromboxane receptor antagonist, on the effects of inhaled PAF on human airways . Clin Exp Allergy 1990; 20: 311-317 . 15 . Manning P J, Stevens W H, Cockroft D W, O'Byrne P M . The role of thromboxane in allergen-induced asthmatic responses . Eur Respir J 1991 ; 4 : 667-672 . 16. Black P N, Salmon B T, Ewan P, Fuller R W . The effect of CGS 12970, a thromboxane synthetase inhibitor, on the response to inhaled allergen . Thorax 1989; 44 : 316-317P . 17. Lumley P, White B P, Humphrey P PA . GR32191, a highly potent and specific thromboxane A Z receptor blocking drug on platelets and vascular and airways smooth muscle in vitro . Br J Pharmacol 1989 ; 97 : 783-794 . 18 . Connolly M J, Avery A J, Walters E H, Hendrick D J . The relationship between bronchial responsiveness to methacholine and bronchial responsiveness to histamine in asthmatic subjects . Pulmon Pharmacol 1988; 1 : 53-58 . 19 . Cockroft D W, Killian D N, Mellon J J A, Hargreave F E . Bronchial reactivity to histamine : a method and clinical survey . Clin Allergy 1977 ; 7 : 235-243 . 20 . Cockcroft D W, Ruffin R E, Dolovich J, Hargreave F E . Allergen induced increase in non-allergic bronchial reactivity . Clin Allergy 1977 ; 7 : 503-513 . 21, Cartier A, Pineau L, Malo J-L. Monitoring of maximum expiratory peak flow rates and histamine inhalation tests in the investigations of occupational asthma. Clin Allergy 1984 ; 14 : 193-196. 22 . Ryan G, Latimer K M, Juniper E F, Roberts R S, Hargreave F E . Effect of beclomethasone dipropionate on bronchial responsiveness to histamine in controlled non-steroid-dependent asthma . J Allergy Clin Immunol 1985 ; 75 : 25-30. 23 . Ryan G, Latimer K M, Juniper E F, Roberts R S, Hargreave F E. Effect of beclomethasone diproprionate on bronchial responsiveness to histamine in controlled nonsteroid-dependent asthma . J Allergy Clin Immunol 1985 ; 75: 25-30 . 24. Bel E H, Timmers M C, Hermans J, Dijkman J H, Sterk P J . The long-term effects of nedocromil sodium and beclomethasone diproprionate on bronchial responsiveness to methacholine in nonatopic asthmatic subjects . Am Rev Respir Dis 1990 ; 141 : 21-28 . 25. Thomas M, Lumley P . Preliminary assessment of a novel thromboxane A2 receptor-blocking drug, GR32191, in healthy subjects. Circulation 1990 ; 81 : 153-158 . 26 . Fujimura M, Nishioka S, Kumabashiri I, Matsuda T, Mifune J . Effects of aerosol administration of a thromboxane synthetase inhibitor (OKY-046) on bronchial responsiveness to acetylcholine in asthmatic subjects . Chest 1990; 98 : 276-279 . 27 . Gardiner P V, Young C L, Duddridge M, Holmes K, Hendrick D J, Walters E H . Effect of a thromboxane synthetase inhibitor (Dazmagrel) on bronchial reactivity to methacholine in asthmatic subjects . Am Rev Respir Dis 1991 ; 4 : A644 . Date received : 11 September 1991 Date revised : 29 October 1991 Date accepted: 6 January 1992 page 1 page 2 page 3 page 4