Inhibition by Ro 31-6930 of agonist and allergen induced bronchoconstriction in anaesthetised guinea-pigs and cats

Pulmonary Pharmacology (1991) 4, 225-232 © 1991 Longman Group UK Ltd

PULMONARY PHARMACOLOGY

Inhibition by Ro 31-6930 of Agonist and Allergen Induced Bronchoconstriction in Anaesthetised Guinea-pigs and Cats P . M . Paciorek, D . T. Burden, P . R . Gater, Y . M . Hawthorne, A . M . Spence, J . C . Taylor, J . F. Waterfall Department of Biology, Roche Products Limited, Welwyn Garden City, Hertfordshire, UK SUMMARY. Ro 31-6930, a potent smooth muscle relaxant from the novel class of potassium channel openers, has been compared with BRL 38227, salmeterol and theophylline in a range of models of airway function . Ro 316930 relaxed isolated tracheal muscle from sensitised guinea-pigs which had been contracted by ovalbumin and was equipotent with salmeterol in inhibiting antigen-induced bronchospasm in anaesthetised, sensitised guinea-pigs . In both anaesthetised guinea-pig and cat, Ro 31-6930, BRL 38227 and theophylline were more potent against 5-HT evoked increases in lung resistance than they were on falls in dynamic compliance . Although salmeterol had equivalent activity on both parameters it is unlikely that the small difference seen with the other compounds reflect a preferential effect on large airways . In addition, Ro 31-6930 was an effective bronchodilator when given by inhalation to the anaesthetised guinea-pig. In view of the protective activity of Ro 31-6930 against antigen challenge in the sensitised guinea-pig and its potency in relation to other bronchodilators, it is considered that compounds which relax airway smooth muscle by the opening of plasmalemmal potassium channels may have a role in the treatment of asthma .

pigs have been used as a model of human asthma for many years. 12 One aim of this study, therefore, was to further evaluate the bronchodilator properties of Ro 31-6930 in comparison with BRL 38227, salmeterol and theophylline on smooth muscle contractility in vitro and airway function in vivo using ovalbuminsensitised guinea-pigs . In addition, the actions of these compounds on small and large airways of both guinea-pig and cat have been investigated using measurements of dynamic compliance and lung resistance after 5-HT challenge . The actions of aerosolised Ro 31-6930 have also been compared with those of the other bronchodilators in the anaesthetised guineapig . A preliminary account of some of these findings has been given ."

INTRODUCTION Ro 31-6930, 2-(6-cyano-2,2-dimethyl-2H-l-benzopyran-4-yl)-pyridine 1-oxide, is a potassium channel opener possessing potent smooth muscle relaxant and bronchodilator properties. 1-3 A number of other studies have also shown potassium channel openers to be effective in reducing agonist induced contractions of guinea-pig tracheal preparations both in vivo and in vitro . 4-6 Since these compounds are effective against a wide range of spasmogens on airway smooth muscle it is considered that potassium channel openers may be useful in asthma therapy . Indeed, cromakalim is an effective bronchodilator in man .' Of the current therapy for asthma, inhaled R 2 agonists are the most effective bronchodilators with few associated side effects, although tremor and tachycardia are observed following oral administration .' New longer acting (3 2 -agonists such as salmeterol9 may herald improved bronchodilator therapy . In a previous study, salbutamol was found to be more potent and theophylline less potent than Ro 31-6930 and cromakalim as bronchodilators in both anaesthetised and conscious guinea-pigs following challenge with either 5-HT or histamine .' However, the asthmatic response is a consequence of the action of a number of mediators including products of the lipoxygenase and cyclooxygenase pathways . 1o .11 Anaphylactic responses in the airway of sensitised guinea-

METHODS Sensitisation procedure Female Dunkin Hartley guinea-pigs (350-400 g) were sensitised by simultaneous intraperitoneal and subcutaneous administration of ovalbumin (50 mg in an injection volume of I ml per site) and used for experimentation 14-28 days later ." Measurement of tracheal tone in vitro Strips of trachea from sensitised guinea-pigs containing 2 or 3 cartilage rings were cut along the longitudinal axis directly opposite the smooth muscle layer and suspended for isometric recording at 37°C under

Correspondence to : Dr. P. M . Paciorek, Department of Biology, Roche Products Limited, P .O. Box 8, Welwyn Garden City, Hertfordshire AL7 3AY, UK . 225

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initial tension of 1 .5 g in Krebs solution of the following composition (mM) : NaCl 118, NaHCO 3 25, D-glucose 10 .5, KCl 4 .7, MgSO 4 0.49, KH2PO 4 1 .2, CaC1 2 2 .5, gassed with 95%O 2/5%CO 2 . Tissues were equilibrated for 90 min during which indomethacin (1 µM) was added to eliminate spontaneous tone . Contraction of trachealis muscle from sensitised guinea-pigs was evoked by the addition of a concentration of ovalbumin which represented approximately an EC 75 (10 ng ml - ') . Once the contracture had stabilised, relaxant drugs were administered in cumulative concentration increments with each addition being made when the response to the previous concentration had reached a plateau . Responses were expressed as a percentage of that caused by aminophylline (1 mM) added at the end of the experiment . In a separate study, cumulative concentration-effect curves to ovalbumin were constructed in tissues which had been incubated for 20 min with drug or vehicle . Responses were calculated as a percentage of the maximum contraction attainable with ovalbumin (10 .tg ml - ') .

Measurement of pulmonary inflation pressure Female Dunkin Hartley guinea-pigs (450-600 g) were anaesthetised with urethane (1 .75 g kg - ' i.p.) followed by atropine (1 .0 mg kg - ' i.p .) . The trachea was cannulated and animals were respired with room air (10 ml kg - ' body weight : 55 inflations min"') . Pulmonary inflation pressure (PIP) was measured using a Statham P231D pressure transducer attached to a side arm of the afferent limb of the respiratory circuit. The left jugular vein was cannulated for the injection of drugs and the right carotid artery for the measurement of mean arterial pressure (MAP) via a Statham P231 D pressure transducer . Deep body temperature was maintained at 37°C using a thermostatically controlled heating blanket . Bronchoconstriction was evoked by either x ED 65 doses of 5-HT (2-20 .tg kg - ' i .v .) or by the administration of increasing doses of 5-HT (1-300 µg kg - ' i .v .) . Test agents were administered cumulatively i .v . or by inhalation from a DeVilbiss ultrasonic nebulizer attached to the afferent limb of the respiratory circuit, 10 min before each agonist challenge . The median particle size generated by the nebulizer under these conditions was approximately 5 µm as determined by a Malvern 2600 particle sizer . 15 In a separate series of experiments, anaesthetised, ovalbumin-sensitised guinea-pigs were bilaterally vagotomised and prepared for measurement of pulmonary inflation pressure as described above . Test agents or vehicle were administered i .v . as a single bolus dose 10 min prior to antigen challenge . Animals were challenged with an aerosol of ovalbumin

(10 mg ml -' : 5 sec) . PIP and MAP were monitored for a 20 min period following antigen exposure . Measurement of resistance and compliance Guinea-pigs

Female Dunkin Hartley guinea-pigs (450-600 g) were anaesthetised with urethane (1 .75 g kg - ' i .p .) followed by atropine (1 .0 mg kg - ' i.p .) . The trachea was cannulated and animals were respired with room air (10 ml kg - ' body weight: 55 inflations min - ') . Tracheal airflow was measured using a heated Gould model 000 pneumotachograph attached to a Validyne model 45-14 differential pressure transducer . Transpulmonary pressure was measured using a Validyne model 45-24 differential pressure transducer, one side of which was attached to an 18 gauge needle inserted into the tracheal cannula with the other side attached to an identical needle inserted through the chest at the fifth or sixth intercostal space and into the intrapleural space . Airflow and transpulmonary pressure signals were fed into a Buxco model 6 Pulmonary Analyser which integrated the flow signal to give tidal volume . Total lung resistance (R L) and dynamic lung compliance (Cdy„) were then calculated at isovolumetric points as per the method of Amdur and Mead ." Permanent recordings of airflow, transpulmonary pressure, tidal volume, R L and C dy„ were made on a Brush model 480 polygraph . A submaximal dose of 5-HT (2-20 µg kg - ' i .v .) which increased RL by approximately 250% and decreased C dy„ by approximately 70% was determined and repeated at 10 min intervals until constant responses were obtained . Test agents were then administered cumulatively i .v . 10 min prior to each 5-HT challenge . In order to prevent atelectasis, the lungs were re-inflated after each bronchoconstrictor response by clamping the outflow tubing to the respiratory pump for three tidal volumes . Cats

Mongrel cats of either sex (2 .4-4 .3 kg) were anaesthetised with a-chloralose (80 mg kg - ' i .p .) and pentobarbitone (6 mg kg - ' i .p .) . MAP was recorded from the left carotid artery using a Statham P231 D pressure transducer and drugs were administered via a jugular vein . The trachea was cannulated and animals were respired with room air (13 ml kg - ' body weight : 25 inflations min - ') . Animals were bilaterally vagotomised and deep body temperature was maintained at 37°C using a thermostatically controlled heating blanket . Tracheal airflow (Gould model 00 pneumotachograph) and transpulmonary pressure were recorded as described above. Tidal volume, RL, and Cdy „ were then derived from these signals using a Buxco model 6 Pulmonary Analyser . Submaximal bronchoconstriction was produced by the infusion of 5-HT (2-20 µg kg - ' min - ') via a

Bronchodilator properties of Ro 31-6930

cannulated femoral vein . The dose of 5-HT was adjusted to produce an increase in R L of approximately 500% and a decrease in C d3,f of approximately 35% ." , " When a constant level of bronchoconstriction was produced test agents were administered cumulatively i .v .

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Drugs Aminophylline, atropine sulphate, indomethacin, ovalbumin (Grade V) and theophylline were obtained from Sigma. Ro 31-6930, BRL 38227 and salmeterol were synthesised at Roche Products . Ro 31-6930, indomethacin, BRL 38227 and salmeterol were dissolved in absolute ethanol and diluted to the required concentrations in 0 .9% w/v NaCl for in vivo studies or distilled water for tissue bath experiments . Atropine, ovalbumin and theophylline were dissolved in 0 .9% w/v NaCl . Aminophylline was dissolved in distilled water. For administration by the inhaled route, test agents were dissolved in dimethylsulphoxide (DMSO) and diluted to the required concentration in distilled water (<_ 10% DMSO) .

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Statistical analysis Results are expressed as mean+S .E.M derived from 4-6 experiments, unless otherwise stated . Statistical evaluation was performed by an unpaired t-test and significance accepted where P < 0 .05 .

RESULTS

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Fig . 1-(A) The effect of Ro 31-6930 ( •), BRL 38227 ( •), salmeterol (A) and theophylline (V) on ovalbumin-contracted trachea from sensitised guinea-pigs. (B) Control responses ( •), and the effect of Ro 31-6930, 30 µM (V), BRL 38227, 30 µM ( •) and salmeterol, 1 µM (A) on the concentration-effect curve

for ovalbumin on trachea from sensitised guinea-pigs . Each data point represents the mean value derived from 4 experiments, vertical bars show S .E.M.

Effects on tracheal tone in vitro Ro 31-6930 (0 .01-1 µM), BRL 38227 (0 .1-10 µM), salmeterol (0.001-0 .3 µM) and theophylline (11000 µM) caused concentration-dependent relaxation of trachea from sensitised guinea-pigs which had been -1 ) contracted by ovalbumin (10 ng ml with IC 50 values of 0 .065±0 .016, 0 .7+-0 .34, 0 .029+0.006 and 56 .4 ± 22 .0 gM respectively (Fig. IA) . Preliminary experiments indicated that high concentrations of Ro 31-6930 (3 .tM) and BRL 38227 (30 µM) did not cause a greater degree of relaxation than the highest concentrations quoted above for these agents (data not shown) . Whereas the maximum responses which could be achieved with Ro 31-6930 (1 .tM) and BRL 38227 (10 µM) were significantly less (P < 0 .05) than that following aminophylline (1 mM), both salmeterol and theophylline completely relaxed the trachealis muscle . In a separate series of experiments, the concentration-effect curve for ovalbumin (1-10 000 ng ml -1 ) was shifted approximately 10-fold to the right following pretreatment with Ro 31-6930 (30 µM) and BRL 38227 (30 tM) . In contrast, pretreatment with salmet-

erol (1 1tM), completely abolished ovalbumin-induced contractions (Fig. 1 B) .

Effects on pulmonary inflation pressure Antigen bronchoconstriction

In ovalbumin-sensitised guinea-pigs, PIP before administration of vehicle was 6 .5±0 .5 mmHg (n=10). Antigen challenge evoked a sharp increase in PIP (maximal change of 13 .0 ± 2 .7 mmHg ; n = 10) which reached a peak at 2 min and then declined to approximately 50% of its initial value by 10 min with little or no further decline over a subsequent 10 min period . Intravenous administration of Ro 31-6930 (100µg kg -1 ), salmeterol (100µg kg -1 ) and theophylline (100 000 tg kg -1 ) caused inhibition of the change in PIP following antigen challenge at each of the measured time points, whereas BRL 38227 (300 and 1000 gg kg -1 ) had no significant effect (Figs 2 & 3) .

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Pulmonary Pharmacology A

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Fig. 2-(A) The effect of vehicle, 1 ml kg - ' i .v . ( •) , Ro 316930, 30 µg kg - ' i .v . (U) and 100 gg kg - ' i .v. (A) and (B) the effect of vehicle, 1 ml kg -' i.v. (0), salmeterol, 30 gg kg - ' i .v . (U) and 100 µg kg"' i .v, (A) on the bronchoconstriction evoked by ovalbumin in anaesthetised, sensitised guinea-pigs . C= Predose, OA =ovalbumin challenge at time 0 . Each data point represents the mean value derived from 6 experiments, vertical bars show S.E .M .

Time (min)

Fig . 3-(A) The effect of vehicle, 1 ml kg - ' i .v . ( •) , BRL 38227, 300 µg kg -' i .v. (U) and 1000 µg kg - ' i .v . (A) and (B) the effect of vehicle, I ml kg - ' i .v . ( •) , theophylline, 30 000 µg kg - ' i .v . (∎) and 100 000 µg kg - ' i .v . (A) on the bronchoconstriction evoked by ovalbumin in anaesthetised, sensitised guinea-pigs . C = Pre-dose, OA =ovalbumin challenge at time 0 . Each data point represents the mean value derived from 6 experiments, vertical bars show S .E .M .

5-HT bronchoconstriction

Following i.v . administration Ro 31-6930 (3100 µg kg -1 ), BRL 38227 (10-1000 tg kg -1 ), salmet-1 ) erol (0 .3-30 gg kg and theophylline (100030 000 µg kg -1 ) evoked dose related reductions in the submaximal responses to 5-HT with ID 50 values of 36 .8±11 .1, 169±44 .0, 7.6±2 .9 and 11000 ± 2700 µg kg -1 respectively (Fig . 4) . At the largest tested doses, Ro 31-6930 and BRL 38227 reduced MAP from 41 .5+10 .7 to 17 .5+2 .3 and 27 .9 ± 2 .4 to 19 .0 ± 1 .5 mmHg respectively . Inhaled Ro 31-6930 (0 .1, 0.3 and 1 mg ml -1 ; 1; 2 min), BRL 38227 (0 .3 and 1 mg ml_ 2 min) and salmeterol (0 .03 and 0 .1 mg ml -1 ; 2 min) evoked dose related depressions of the dose response curves to 5-HT (Fig . 5) which were most marked following the lowest doses of 5-HT (43 .2-100% inhibition) and least at the highest doses (15 .9-75.3% inhibition) . Mean pre-inhalation MAP was 30 .6 ± 1 .2 mmHg (n = 28) . Ro 31-6930 (0 .1, 0.3 and 1 mg ml -1 ) reduced MAP by 5 .4 ± 1 .6, 7.6 ± 1 .7 and 7 .9 ± 1 .2 mmHg and

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Fig. 4-The effect of Ro 31-6930 ( •) , BRL 38227 (U), salmeterol (A) and theophylline ( •) on 5-HT induced bronchoconstriction in anaesthetised guinea-pigs . Each data point represents the mean value derived from 4 experiments, vertical bars show S .E.M .

Bronchodilator properties of Ro 31-6930

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(10-1000 gg kg -1 i .v .), salmeterol (0 .1-10 pg kg - t i.v.) and theophylline (300-30 000 gg kg -1 i .v .) evoked dose related inhibition (Fig . 6) of the effects of 5-HT on R L and C d,,,, (IDso values : 7 .4+1 .5 and 14 .2±3 .7; 103 .4±25 .6 and 157 .8±33 .6 ; 4.2± 1 .9 and 4.9 ± 2 .0; 7600 ± 1800 and 12 900 ± 2500 µg kg - t respectively) . Vehicle administration had no significant effect on the 5-HT induced changes in R L and Cd,,H. With the exception of salmeterol the other bronchodilators were 1 .5- to 2-fold more effective in reversing 5-HT induced changes in RL than CdO . Ro 31-6930 (1-100µg kg -1 ) and BRL 38227 (10-1000 gg kg -1 ) evoked dose related reductions in MAP with decreases from 32 .9 _ +3 .2 to 23 .6±2 .8 and 39 .4_+4.6 to 26.6 ± 2 .2 mmHg respectively at the largest tested _ 1) doses . Theophylline (30 000 µg kg evoked a reduction in MAP from 43 .5 ± 4 .7 to 31 .9 ± 2 .1 mmHg but lower doses were without effect . Salmeterol (0 .1-10 µg kg - t) had no effect on MAP . Cats

Mean resting RL and C dr „ were 5 .87±0 .53 cmH2O 1-1 s-1 (n=23) and 14 .3±0 .89 ml cmH 20 -1 (n= 17) respectively . Ro 31-6930 (3-100 gg kg -1 i .v .), BRL 38227 (10-300 pg kg -1 i .v .), salmeterol

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Fig . 5-Control responses (∎), the effects of vehicle 10% DMSO/H 20 ( •) and (A) Ro 31-6930, 0 .1 mg ml -1 (A), 0 .3 mg ml -1 (V) and 1 .0 mg ml -1 ( •) inhaled for 2 min, (B) BRL 38227, 0.3 mg ml -1 (A) and 1 .0mgml -1 (V) inhaled for -1 2 min and (C) salmeterol, 0 .03 mg ml (A) and 0.1 mg ml -1 (7) inhaled for 2 min on 5-HT induced bronchoconstriction in anaesthetised guinea-pigs . Each data point represents the mean value derived from 4 experiments, vertical bars show S .E .M .

BRL 38227 (0 .3 and 1 mg ml -1 ) by 7 .2±2 .4 and 7.1 ± 1 .2 mmHg respectively. Effects on resistance and compliance

Guinea-pigs

Mean resting R L and Cd,. o were 0 .16±0 .01 cmH2O m1 -1 s -1 and 0 .28 ± 0 .01 ml cmH2O - 1 respectively (n=25) . Ro 31-6930 (1-100 pg kg -1 i .v.), BRL 38227

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Fig . 6-The effect of Ro 31-6930 (0), BRL 38227 (∎), salmeterol (A) and theophylline (7) on 5-HT induced changes in (A) lung resistance (R L ) and (B) dynamic lung compliance (Cd0n) in anaesthetised guinea-pigs . Each data point represents the mean value derived from 5 experiments, vertical bars show S .E .M .

2 30

Pulmonary Pharmacology

(0 .3-30 µg kg -1 i.v .) and theophylline (1000100 000 µg kg -1 i.v .) evoked dose related inhibition (Fig . 7) of the effects of 5-HT on R, and C d,,,, (ID50 values : 16 .7 ± 1 .5 and 60 .1 ± 22 .9 ; 61 .1 ± 16 .9 and 147 .7 ± 56 .4 ; 2 .6 ± 0 .8 and 3 .4 ± 2 .0 ; 12 400 ± 3800 and 37 300±20 100 gg kg -1 respectively) . As before, with the exception of salmeterol, the other bronchodilators were more effective in reversing the effects of 5-HT on RL than Cd,,f . The simultaneous assessment of the cardiovascular actions of the bronchodilators could not be carried out in this study as the continuous 5-HT infusion employed to produce a sustained bronchoconstriction evoked a tachycardia and hypotension.

DISCUSSION Asthma is characterised by a reversible bronchoconstriction and airway hyperreactivity to a variety of stimuli . In addition, inflammation of the lungs is an important feature of chronic asthma, contributing to the hyperreactivity, oedema, mucus plugging, cellular infiltration and damage frequently observed with this disease ."

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Fig. 7-The effect of Ro 31-6930 ( •), BRL 38227 (0), salmeterol (A) and theophylline (Y) on 5-HT induced changes in (A) lung resistance (RL ) and (B) dynamic lung compliance (C d,C) in anaesthetised cats . Each data point represents the mean value derived from 3-5 experiments, vertical bars show S .E .M .

The bronchoconstriction associated with asthma is currently treated with three classes of bronchodilators, the Q2 -adrenoceptor agonists, methylxanthines and anticholinergics . More recently attention has focused on a novel class of relaxants of bronchial smooth muscle, the potassium channel openers, 20 which are currently being evaluated as bronchodilators for the treatment of asthma . In this study we have compared Ro 31-6930 with the novel long acting R 2 -agonist salmeterol, 9 BRL 38227, 5 a potassium channel opener under clinical evaluation, and theophylline, in a variety of allergen and agonist induced models of bronchoconstriction . Ro 31-6930 has been shown to be a potent relaxant of guinea-pig isolated trachea following contraction induced by a number of individual spasmogens although, in common with other potassium channel openers, potency is reduced when tone is raised by a muscarinic agonist . 3 Since it is clear that the asthmatic response is dependent on the action of more than one mediator, the effects of Ro 31-6930 have now been studied in tracheae from sensitised guinea-pigs which were challenged by exposure to antigen . In this tissue, contraction occurs as the result of release of anaphylactic mediators including histamine and leukotrienes.21 ° 22 All the agents tested were potent relaxants of sustained tracheal tone when administered during the plateau phase of an antigen contraction, although the potassium channel openers achieved only 60-80% of maximal relaxation . In contrast, when tissues were pretreated with the test agents, at concentrations which were equivalent multiples of IC 50 on maintained contraction, only salmeterol totally inhibited the antigen induced increase in tracheal tone, whereas the potassium channel openers were markedly less effective . These findings may indicate that potassium channel openers are relatively more effective on the tonic phase of antigen-induced contraction of trachealis from sensitised guinea-pigs than against the initial phase of the response . It has previously been suggested" , " that potassium channel openers may exert some of their pharmacological actions by indirectly inhibiting calcium entry via dihydropyridinesensitive calcium channels or by the inhibition of refilling of intracellular calcium stores, actions which may be required to maintain smooth muscle contraction . However, a component of the initial rise in tracheal tone following antigen challenge probably involves the formation of 1P3 and subsequent intracellular calcium release, this certainly being the case for any leukotrienes released following antigen challenge . 25 Although it may be expected that the actions of IP 3 would be attenuated under conditions in which Ca" stores are depleted following inhibition of the refilling of those stores by potassium channel openers, it could equally be argued that the refilling process is only fully activated once Cat+ is mobilised through

Bronchodilator properties of Ro 31-6930

excitation/contraction coupling . Therefore, during the tonic phase of the response 1P3 may be acting on a relatively full intracellular Ca t + store. Thus, in the isolated trachea, potassium channel openers may be exerting a relatively minor effect on intracellular calcium release or its resequestration and thereby showing low efficacy when used as a pretreatment to antigen challenge . In the anaesthetised guinea-pig Ro 31-6930, salmeterol and theophylline were capable of inhibiting the anaphylactic bronchoconstriction when administered prior to antigen challenge although the doses required were higher than those which inhibited 5-HT bronchoconstriction . In this model Ro 31-6930 was as effective as salmeterol as a bronchodilator, whereas BRL 38227 showed no significant efficacy at the doses tested . This was an unexpected result in view of the relative potencies of Ro 31-6930 and BRL 38227 in other in vivo and in vitro models of asthma . Interestingly, none of the agents were capable of reversing the antigen-induced bronchoconstriction once it had been established (data not shown) . In a similar study, Daffonchio et a1 26 showed that infusion of isoprenaline partially reversed an established bronchospasm but that subsequent administration of a high, intravenous bolus dose had no further effect . It is envisaged that, in addition to the spasmogenic action on airway smooth muscle evoked by the release of anaphylactic mediators, oedema formation and mucus secretion are contributing to the bronchoconstriction . Thus, under these circumstances relaxation of airway smooth muscle alone is not sufficient to reduce PIP, and the reduced effect of the bronchodilators may reflect their inability to modify oedema formation or mucus secretion . p2-agonist bronchodilators are frequently employed for the acute relief of bronchoconstriction and for the prevention of exercise or cold air induced bronchospasm . The preferred route of administration, at least in the UK, 27 is by inhalation, providing a rapid onset of action with reduced dosage, hence fewer side effects. When administered by inhalation to anaesthetised guinea-pigs Ro 31-6930 inhibited 5-HT induced bronchoconstriction . BRL 38227 was also effective by the inhaled route but was less potent than Ro 31-6930 . Salmeterol was the most effective bronchodilator by this route of administration . Both Ro 31-6930 and BRL 38227 reduced MAP following inhalation although the falls were smaller than those observed following i .v . administration . We have previously demonstrated that Ro 31-6930 and cromakalim prevent histamine and 5-HT induced bronchoconstriction in pithed, vagotomised guineapigs with potencies which were consistent with those reported in the present study .3 Since the hypotensive actions of the potassium channel openers were prevented in the pithed, vagotomised animals, it is unlikely that their bronchodilator effects result from

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catecholamine release due to adrenergic reflexes stimulated by falls in blood pressure . Although cardiovascular effects are observed in these experimental models, there is evidence in man that at bronchodilator doses potassium channel openers cause little or no changes in heart rate or blood pressure . 28,29 An evaluation of PIP in experimental animals provides a simple assessment of resistance to air flow which is generated predominantly in the large airways . However, asthma is a disease characterised not only by bronchoconstriction but also mucus secretion, oedema and cellular infiltration, actions which would affect not only the large airways but also the terminal bronchioles, alveoli and lung parenchyma . We have therefore evaluated Ro 31-6930 in a model of 5-HT induced changes in lung resistance (RL) and dynamic compliance (Cd,,,) to determine whether potassium channel openers also have an effect on lung elasticity and small airway calibre . The effects of bronchodilators on 5-HT induced changes in R L and Cd,,,, were similar in both anaesthetised guinea-pig and cat . Salmeterol was up to 10fold more potent than Ro 31-6930 which was, in turn, more potent than BRL 38227 . Theophylline was markedly less potent than any other bronchodilator tested in these systems . In both species, Ro 316930, BRL 38227 and theophylline displayed between 2- and 4-fold greater potency on increases in R L than on falls in Cd,, f following 5-HT . In contrast, salmeterol was equipotent on both parameters in the guineapig and the cat . Since the doses of 5-HT which were employed in these experiments produced a submaximal increase in RL but a near-maximal decrease in C dyn, such relatively small differences in potency do not imply that any of these compounds has a preferential effect on upper or lower airways . In support of our findings in the anaesthetised cat, Drewett and Rodger1s showed cromakalim to be 2-fold more effective in reversing 5-HT induced changes in RL than Cd,.f . In the anaesthetised guinea-pig, other workers have reported that cromakalim is equieffective on 5-HT induced submaximal changes in both RL and Cd,,f . 30 However, similar data has been used to conclude that inhaled BRL 38227 has a relatively marked effect on smaller airways in comparison to salbutamol following histamine challenge . 31 Details of the magnitude of the rise in RL and fall in C dyn are not recorded for the latter study and it is clear that there are differences in detailed methodology . Further studies of the actions of potassium channel openers and other bronchodilators on upper and lower airway function are required . In summary, Ro 31-6930 is more potent as a bronchodilator than other compounds from the novel class of potassium channel openers . Ro 31-6930 is effective following inhalation and intravenous administration against allergen and agonist induced bronch-

232 Pulmonary Pharmacology

oconstriction through action at both large and small airways . Crucially, Ro 31-6930 has equivalent potency to salmeterol in an animal model of allergic bronchospasm. These data further suggest that potassium channel openers may be useful in the treatment of asthma .

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