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Comparative protective effect of the inhaled β2-agonist salbutamol (albuterol) on bronchoconstriction provoked by histamine, methacholine, and adenosine 5′-monophosphate in asthma
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Methacholine
12. Furukawa CT, Shapiro GG, Bierman CW, et al. A doubleblind study comparing the effectivenessof cromolyn sodium and susta:.ned-release theophylline in childhood asthma. Pediatrics. 1984;74:453-9. 13. Cockcroft DW, Killian DN, Mellon JJA, Hargreave FE. Protective effect of drugs on histamine-inducedasthma. Thorax 1917;32:429-31.
sensitivity
14. Cockcroft DW, Murduck BA, Gore PM, et al. Theophylline does not inhibit allergen-induced increase in airway responsiveness to methacholine. J ALLERGYCLIN IMMUNOL1989; 83:913-20.
Comparative protective effect of the inhaled P,-agonist satbutamol (albuterol) on bronchoconstriction provoked by histamine, methacholine, and adenosine 5’-monophosphate in asthma G. D. Phillips, MA, MRCP, J. P. Finnerty, MRCP, and S. T. Holgate, BSc, FRCP Southampton,England We have investigated the ability of salbutamol to protect against bronchoconstriction induced by methacholine, histamine, and adenosine 5’-monophosphate (AMP) in nine subjects with asthma. In a double-blind, placebo-controlled study, salbutamol, 2.5 mg administered by nebulization, increased the geometric mean provocation concentrations of me&choline, histamine, and AMP required to produce a 20% decrease in FEV, from 0.3 to 2.2, 0.4 to 3.8, and 4.0 to 106.7 mglml after placebo and active treatment, respectively (p < 0.01). Thus, this dose of /3,-adrenoceptor agonist displaced the concentration-response curves for me&choline, histamine, and AMP to the right in a parallel fashion by 8.8 (0.6 to 29.3)-, 10.3 (I .4 to 33)-, and 26.6 (1.5 to 76.6)-fold, respectively, the difSerence between the results for AMP and those for histamine and methacholine being statistically sign&ant (p < 0.01). For six of the nine subjects studied, salbutamol displaced the concentration-response curve for AMP to the right by >.50-fold. There was no correlation between bronchodilatation and protection against bronchoconstriction induced by any of the agonists. We conclude that salbutamol protects against bronchoconstriction provoked by methacholine and histamine by functional antagonism, whereas with AMP, an additional activity is demonstrable, possibly involving inhibition of mast cell-mediator release. (J ALLERGYCLIN IMMUNOL1990;85:755-62.)
When subjects with asthma’ and atopic subjects without asthma’ inhale adenosine and its related nu-
Abbreviations used
AMP: Adenosine 5’-monophosphate ANOVA: Analysis of variance From the Departmentof Immunopharmacology,SouthamptonGeneral Hospital, Southampton,England. Supportedby a grant from the Chest, Heart, andStrokeAssociation. Received for publication June 22, 1989. Revised Nov. 6, 1989. Accepted for publication Nov. 9, 1989. Reprint requests: S. T. Holgate, BSc, Dept. of Immunopharmacology, Level D, Center Block, SouthamptonGeneral Hospital, Tremona Road, Southampton,SO9 4XY, England. Correspondenceaddress:G. D. Phillips, MA, Dept. of Immunopharmacology, Level D, Center Block, SouthamptonGeneral Hospital, Tremona Road, Southampton,SO9 4XY, England. l/1/18140
PCzO: Provocative
concentration
required to
produce a 20% fall in FEV, from postsaline baseline
SCG: Sodium cromoglycate BAL: Bronchoalveolar lavage
cleotide, AMP, this causes dose-related bronchoconstriction. Since the effect of these purines on asthmatic airways can be antagonized by inhibitors of mast cellmediator release3 and by selective HI-histamine re755
756 Phillips et al.
J. ALLERGY
TABLE 1. Details of characteristics Subject
1 2 3 4 5 6 7 8 9 Mean SEM
No.
Sex
M M F M F
M F M F 5M,4F
CLIN. IMMUNOL. APRIL 1990
of patients Age W
43 22 24 17 48 45 22 41 32 32.7 3.9
Baseline FEV, 1% predicted)
64 98 86 98 113 65 115 82 106
Atopic
+ + + + + + + +
Treatment
S s s S, B S, Bf S, B S S, B S, Bf
S, Salbutamol;B, beclomethasone dipropionate,50 pg per actuation;EJ beclomethasone dipropionate,250 pg Peractuation.
ceptor antagonists,4.5 both adenosineand AMP have beenproposedto provoke bronchoconstriction by potentiating mediator releasefrom immunologically activated airway mast cells.4*5 In an in vivo model of adenosine-inducedbronchoconstriction in inbred rats, Pauwels et a1.6have demonstratedan approximately twofold increasein the concentration of histamine in BAL fluid during bronchoconstriction provoked by intravenous injection of the adenosine analogue, N-ethylcarboxamide adenosine. Mast cells cultured from the bone marrow of rodents’ or dispersedfrom human lung tissue,8 respond to adenosine and synthetic analoguesby enhancedIgE-stimulated mediator release via a mechanism involving cell surface A2 purinoceptors. Mast cells also possessa distinct population of &adrenoceptorsgthat, when they are activated by agonists suchassalbutamol, up regulateadenylatecyclase activity and are 2 to 30 x 103-foldmore potent than SCG at inhibiting mediator release.loFurther evidence for P,-adrenoceptor agonists suppressing mast cell function in asthmaderives from the observation that salbutamol, inhaled before allergen provocation, inhibits both the airways and circulating mediator responses.” In a previous study, we have reported that inhaled salbutamol was approximately twofold more effective at inhibiting immediate bronchoconstriction provoked by inhaled allergen comparedwith methacholine. I2Although salbutamol may be consideredas a functional antagonistat human airways smoothmuscle, the increased activity expressed by this drug against the airway responseto allergen could be accounted for by an inhibitory action on mast cellmediator release.I2In the presentstudy, we have used this approach to examine the contribution of endogenous mediator releaseto bronchoconstriction that is producedby AMP by comparing the protective effect
of inhaled salbutamol on the airway responsesthat is induced by inhaled methacholine, histamine, and AMP. METHODS Subjects Nine subjects (five male subjects) with a mean (SEM) ageof 32.7 years(3.9 years),participatedin thestudy(Table I). All subjectswere nonsmokerswith asthma,and all except one subject were atopic as defined by positive skin prick tests (>2 mm wheal response) to two or more common aeroallergens (house dust, Dermatophagoides pteronyssinus, D. farinae, mixed grasspollen, mixed tree pollen, cat fur, dog hair, mixed feathers, Aspergillus fumigatus, Candia’a albicans; BencardAllergy Unit, Brentfoid, Middlesex, U.K.). All patients had baseline FEV, >60% of their predicted values or > 1.5 L and had previously been demonstratedto have an increasein FEV, of > 15% after inhaling salbutamol, 200 pg from a metered-doseinhaler, and none was receiving oral corticosteroids, theophylline, or SCG on a regular basis. Bronchodilators were withheld for 8 hours before each visit to the laboratory, although subjectswere allowed to continue inhaled corticosteroids as usual. No patient was studied within 4 weeks of an upper respiratory tract infection or exacerbation of asthma. Subjects gave written, informed consent, and the study was approved by the Southampton University and Hospitals Ethical Committee. Bronchial
provocation
Pulmonary function was measuredbefore and during the provocation as the FEV, with a dry-wedge spirometer (Vitalograph Ltd., Buckingham, U.K.), the first of two consecutive measurementsbeing used for analysis. On each challenge day, methacholine (Sigma Chemical Co., Poole,
Dorset,U.K.), histamine,acidphosphate (BDHChemicals, Poole, Dorset, U.K.), and yeast-derivedAMP (product No. A1752, Sigma Chemical Co., St. Louis, MO.) were made up freshly in 0.9% (wt/vol) sodium chloride to produce a range of increasing doubling concentrationsof 0.03 to 64
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mgiml (0.2 to 327 mmol/L), 0.03 to 16 mg/ml (0.1 to 52 mmol/L), and 0.04 to 400 mg/ml (1.12 to 1151.4 mmol/ L) , respectively. The solutions were administeredas aerosols generated from a starting volume of 3 ml in a disposable Inspiron mini-nebulizer (C. R. Bard International, Sunderland, U.K.) driven by compressedair at 8 L/mitt. Under theseconditions, the nebulizer hasan output of 0.48 ml/min and generatesan aerosolwith a massmedian particle diameter of 4.7 p,rn.13Wearing a noseclip, subjects inhaled the aerosolized solutions in five breathsfrom end tidal volume to full inspiratory capacity via a mouthpiece.“. Is Subjectswere trained to take 3 secondsto reach full inspiratory capacity. All the bronchial provocations were carried out at the sametime of day. Study design Subjects attended on six occasions to undertake concentration-responsestudies with each of the three agonists, the paired visits with each agonist being conducted within 1 week of each other. On each occasion, after a 15 minute rest, three baseline measurementsof FEV, were recorded at intervals of 3 minutes, followed by inhalation of neublized !;albutamol,2.5 mg (Allen and Hanbury’s Ltd., Greenford, Middlesex, U.K.) or 0.9% (wt/vol) of sodium chloride plaaebo.Salbutamoland placebowere randomized separatelyfor each of the three agonists and administered double blind. The aerosolizedsolutions were generatedfrom a starting volume of 2.5 ml in an Inspiron mini-nebulizer driven by compressedair at 8 Llmin and inhaled to dryness by deeptidal breathing during a 2- to 3minute time period. The samenebulizer was usedfor all studieson all subjects. Thirty minutes later, a concentration-responsestudy with one of the three agonists was performed. On each occasion, three postdrugmeasurementsof FEV, were recorded at intervals of 3 minutes, followed by inhalation of neublized 0.9% (wt/vol) sodium chloride and repeat measurementsof FEV, at 1 and 3 minutes. If FEV, did not fall by >lO% of the postdrug baseline value, the concentration-responsestudy was undertaken. After inhaling each agonist concentration, FEV, was measured at 1 and 3 minutes, the higher value being recorded.Increasing doubling concentrationsof agonist were then inhaled at 5minute intervals until FEV, had fallen by >20% of the postsaline value or the highest concentration had been administered. Data analysis Figures refer to the mean -+ SEM unless it is otherwise stated,and the p < 0.05 level of significance was accepted. Pre- and postdrugbaselinevaluesof FEV, were compared within each study day with Student’s t test for paired data and between study days with two-factor ANOVA followed by the Newman Keuls’ procedure to determine the source of the variance. For each of the three study days, when active drug was administered, the ability of salbutamol to cause bronchodilatation was determined by expressing the difference betweenthe pre- and postsalbutamolFEV, values asa percentageof the predrugbaselinerecording. The values thus obtained were comparedbetweenstudy days with two-
Salbutamol
and AMP-induced
bronchoconstriction
757
factor ANOVA and the Newman Keuls’ procedure. Since postsaline FEV, values after salbutamol were significantly higher than values after placeboadministration, the agonistconstrictor responsewas expressedas a percentageof the postdrug baseline.16The fall in FEV, after each concentration of agonist was expressedas a percentageof the higher of the two postsaline baseline FEV, recordings, the higher of two measured values being used in each case. The percentagefall in FEV, was plotted against the cumulative concentration of agonist administered on a logarithmic scale, and the PC, was determinedby linear interpolation. The slopes of the concentration-responsecurves were determinedby least-squares,linear-regressionanalysis and comparedbetween agonists, and for the same agonist between postplaceboand postsalbutamolstudy days by use of two-factor ANOVA followed by the Newman Keuls’ procedure and Student’s t test for paired data, respectively. Concentration ratios for the protective effect of salbutamol against bronchoprovocation with each agonist were calculated by dividing the PC,, value obtainedafter administration of active drug by that obtained after administration of placebo for the same agonist. The relative potency of salbutamol in protecting against bronchoconstriction induced by the three agonists was analyzed by comparing the concentration ratios with two-factor ANOVA, followed by the Newman Keuls’ procedure. Any relationship between PC, AMP and PC, methacholine and histamine, betweenthe concentrationratios for AMP and those for methacholine and histamine, between the concentrationratio for AMP and the PC,, methacholine and histamine, and betweenthe concentrationratio for each agonist and the percentageincrease in FEV, produced by salbutamol was investigated with least-squares, linearregression analysis of the data that were logarithmically transformedwhere this was appropriate. RESULTS There were no significant differences in predrug, postplacebo, or postsalbutamol baseline values of FEV, between any of the study days. On the methacholine, histamine, and AMP study days, salbutamol produced 15.9 2 3.8%, 11.8 + 2..5%, and 12.9 + 1.9% increases in FEV, from predrug baseline (p < 0.01 for each) with no significant differences between these values (Table II). No significant differences were found between the slopes of the postplacebo concentration-response curves for the three agonists, and for each agonist the concentration-response curves after placebo and salbutamol administration did not depart significantly from parallel (Table III). Except for one subject with methacholine (No. 4), salbutamol produced a displacement to the right of the concentration-response curve for all three agonists. Thus, the geometric mean (range) concentrations of methacholine, histamine, and AMP required to produce a 20% decrease in FEV, after placebo
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758 Phillips et al.
TABLE II. Baseline FEV, values before and after inhalation Methacholine Subject
No.
1 2 3 4 5 6 7 8 9 Mean SEM
P&rug
2.0* 5.9 3.3 6.5 2.3 2.9 3.5 3.3 3.5 3.7 0.5
of nebulized
salbutamol
Histamine
Postdrug
Predrug
2.9 6.4 3.9 6.8 2.6 3.5 4.0 3.7 3.8 4.2 0.5
2.1 5.6 3.3 6.2 2.6 2.9 3.6 3.4 3.8 3.7 0.4
CLIN. IMMUNOL. APRIL 1990
AMP Postdrug
2.7 6.4 3.9 6.2 2.9 3.2 3.9 3.7 4.0 4.1 0.4
Predrug
2.3 5.7 3.3 5.9 2.9 2.7 3.7 3.3 3.4 3.7 0.4
Postdrug
2.7 6.1 3.8 6.5 3.1 3.2 3.9 3.9 3.9 4.1 0.4
*Values in liters.
administration in the nine subjects studied were 0.3 (0.1 to 1.2), 0.4 (0.1 to 1.2), and 4.0 (2.5 to 7.3) mg/ml, respectively (Fig. 1) (Table IV). Thus, AMP was 7.6 (1.3 to 34)-fold and 9.0 (3.0 to 30.7)-fold less potent than methacholine and histamine, respectively, on a molar basisin reducing FFV, in this group of subjectswith asthma.For methacholine,histamine, and AMP, the mean PC, values after administration of salbutamolwere 2.2 (0.7 to 7.0), 3.8 (1.1 to 12.4), and 106.7 (4.0 to 408.2) mg/ml, respectively, the difference between each of these values and the corresponding postplacebovalue being statistically significant (p < 0.001) (Fig. 1) (Table IV). Expressed as concentration ratios, salbutamol produced a protection of the airways of 8.8 (0.6 to 29.3)-fold, 10.3 (1.4 to 33)-fold, and 26.6 (1.5 to 76.6)-fold against bronchoconstriction provoked by methacholine, histamine, and AMP, respectively, the concentration ratios for AMP being significantly greaterthan the concentration ratios for methacholine(p < 0.01) and histamine (p < 0.01). No significant correlation was found between the concentration ratio for AMP and that for histamine (r = 0.1; p = 0.7), or methacholine (r = 0.4; p = 0.2), or between the concentration ratio for each of the agonists and the predrug baseline FEV,, or the percentagebronchodilatation produced by nebulized salbutamolon the correspondingstudy day. There was a strong relationship, however, between the postplacebolog PC, methacholineandthe concentrationratio for methacholine(r = -0.8;~ = 0.02) andbetween the concentration ratios for histamine and methacholine (r = 0.9;~ = 0.006). DISCUSSION
The presentstudy demonstratesthat salbutamol, 2.5 mg, administeredby nebulization, producesa similar
ninefold or tenfold protection of the airways against bronchoconstriction provoked by methacholine and histamine. However,despiteresulting in a similar 12% to 16% bronchodilatation on all three agonist study days on which active drug was administered, salbutamol produceda significantly greatergeometricmean 27-fold protection for the airways against decreases in FEV, produced by AMP and displaced the nucleotide concentration-responsecurve to the right by >50fold in six of the subjects studied. Since salbutamol is 2 to 30 x lo’-fold more potent than SCG in inhibiting anti-IgE-provoked releaseof histamine from human-dispersed lung mast cells in vitro,” our present data may be interpreted as supporting this &adrenoceptor agonist protecting against the airway effects of methacholine and histamine via a mechanism involving its action as a functional antagonist but having an additional activity against AMP, possibly in preventing degranulation of bronchial mast cells by the nucleotide. We deliberately opted to include patients who were highly reactive to both histamine and methacholineso that the effectsof inhaled salbutamolin displacing the concentration-responsecurves could be described in every case by obtaining true postdrug PC?,,values without having recourse to data manipulations necessary to overcome the problem of censoredvalues. In this study, the greater inhibition afforded by nebulized salbutamol against AMP than against methacholine- and histamine-provoked decreasesin FEV, is unlikely to be causedby differences in functional antagonismon the respectivestudy days, since the degreeof bronchodilatation producedwas not significantly different. One possibility is that, in being limited to smooth muscle, the action of AMP in the airways is more completely reversed by salbutamol than is that of provocants like histamine, which exact their effects, in addition, on neural reflexes and
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Salbutamol
TABLE III. Gradients of the concentration-response and placebo
NO.
1 2 3 4 5 6 7 8 9 Mean (SEW
Salbutamol
Placebo
-29.2 -35.5 - 22.6 -45.8 -48.4 -33.1 -59.7 -28.0 - 19.9 -35.8
-24.4 -43.0 -27.5 -38.0 -66.5 -27.4 -29.3 - 25.4 - 24.2 - 34.0
-18 -30.1 -66.1 -40.6 -33.1 -40.2 -26.0 - 30.7 -20.8 - 34.0
(4.4)
(4.6)
(4.8)
TABLE IV. PC, values for methacholine, and salbutamol
No.
1 2 3 4 5 6 7 8 9 Geometric mean (ran&
Placebo
0.2 0.9 0.1 1.2 0.1 0.1 0.6 0.2 0.8 0.3 (0.1-1.2)
- 22.4 -40 -37.3 -52.1 -40.4 -44.6 - 24.0 - 26.6 - 22.9 - 34.5 (3.6)
Placebo
Salbutamol
- 26.7 -31.5 - 32.4 - 24.9 -45.6 -24.4 -43.2 -33.7 - 18.4 -31.2 (3.0)
- 34.3 - 36.4 -35.3 - 29.5 -39.6 - 32.9 - 26.3 -33.9 -24.8 -32.6
(1.6)
histamine, and AMP after inhalation of nebulized placebo Histamine
Methacholine Subject
759
AMP
Histamine
Salbutamol
Placebo
bronchoconstriction
curves after inhalation of nebulized salbutamol
Methacholine Subject
and AMP-induced
Salbutamol
2.4 7.0 1.8 0.7 0.7 0.9 6.9 3.8 3.7 2.2 (0.7-7.0)
Placebo
0.2 1.1 0.1 0.8 0.2 0.2 1.2 0.2 1.2 0.4 (0.1-1.2)
postcapillary venular permeability. However, this latter explanation is unlikely, since the effects of adenosine on ahway smooth muscle in vitro are weak and inconsistent.” Although: we have been unable to demonstrate a significant protective effect of the inhaled muscarinic antagonist, ipratropium bromide, on adenosineinduced bronchoconstriction, Okayama et al. I8 were able to demonstrate some reversal of adenosineinduced bronchoconstriction by inhaled atropine. In addition, in vitroI and in vivozo studies in the rabbit lend somesupportto a possible neurologic mechanism for adenosine-inducedbronchoconstriction. However, since histamine also stimualtes neural reflexes in the airways,2’ it is unlikely that an action of salbutamol in protecting against such reflexes accounts for its greater prorective effect against AMP. Although the mode of action of AMP in provoking bronchocon-
AMP
Salbutamol
1.6 12.4 3.3 1.1 2.8 2.4 9.9 3.6 8.8 3.8 (1.1-12.4)
Placebo
Salbutamol
2.5 5.2 3.2 2.7 5.9 3.6 7.3 3.9 4.1 4.0 (2.5-7.3)
191.6 284.5 199.2 4.0 34.1 52.0 408.2 204.3 279.2 106.7 (4.0-408.2)
striction in subjectswith asthmaremains to be determined, one suggestion involves the potentiation of preformed mediator release, principally histamine, from activated airway mast cells.4,* Thus, adenosine and related analoguesactive at the A, purinoceptor potentiate the immunologically stimulated release of the preformed mediators, histamine and phexosaminidase,from rodent’ and human’ mast cells in vitro. In vivo, selective H,-histamine-receptor antagonists produce >80% protection of the airways against bronchoconstriction provoked by AMP,4. 5 whereas the mast cell membrane-stabilizing drugs, SCG and nedocromil sodium, displacethe AMP doseresponsecurve to the right by tenfold to twentyfold,’ although this latter effect may be due to a combination of inhibition of mast cell mediator-releaseand C fiber afferent reflexes.** In addition, after intravenous injection of the adenosine analogue, N-
760
Phillips
et al.
J. ALLERGY
Methachdine
Histamine s
r
P
CLIN. IMMUNOL. APRIL 1990
AMP 5
P
s
1
0 FIG. 1. Effect of inhaled nebulized salbutamol, 2.5 mg (S) and placebo (P) on bronchoconstriction provoked by methacholine, histamine, and AMP in nine subjects with asthma. The horizontal bars denote geometric mean values.
ethylcarboxamide adenosine, Pauwels et a1.6 have demonstratedan approximately twofold increase in BAL fluid histamineconcentrationin the rabbit. Thus, one possibility is that the additional inhibitory effect of salbutamol againstAMP-induced bronchoconstriction involves the prevention of adenosine-inducedenhancementof preformed mediator release from activated airway mast cells. Although the present data may be interpreted as supporting such a mechanism, this evidence is only indirect and capable of alternative interpretations, as already discussed. Moreover, the similarity in the slopesof the postsalbutamol concentration-responsecurves for all three agonists may be interpreted as suggesting that the B,-agonist protects against them all by a similar mechanism. Recently, however, we have been able to demonstrate an approximately twofold increase in the concentration of histamine in peripheral blood after bronchoprovocation with AMP in 10 atopic subjectswithout asthmawith no similar elevations being observed after inhalation of concentrationsof methacholine sufficient to provoke equivalent decreasesin pulmonary function.23
Evidence in supportof an additional mastcell effect of salbtuamol includes the observation that, whereas oral and inhaled salbutamol produced a similar threefold to fourfold protection for the airways against metbacholine-induced bronchoconstriction, a small dose(10 pg) of inhaled salbutamolwastwice aspotent in inhibiting decreasesin pulmonary function provoked by allergen than was an oral dose of 8 mg,‘* suggesting an additional local effect at the level of epithelial mastcells. Similarly, Andersonet a1.24have demonstratedthat, whereas salbutamol administered via both tbe oral and inhaled routes produced bronchodilatation, only the inhaled drug protected against exercise-induced bronchoconstriction, another stimulus purported to involve mast cell-mediator release.“, 26 Although B,-adrenoceptor agonists, such as salbutamol, are consideredto exert their clinical effects by relaxing bronchial smooth muscle, there is evidence to suggestthat they also possess“antiallergic” activity. Thus, catecholaminesinhibit the release of histamine from guinea pig-sensitized lung*’ and of histamine and slow-reacting substanceof anaphylaxis
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from human sensitized lung fragments.**Salbutamol inhibits Igt-dependent histamine release from mast cells recovered from subjects with asthmaby BAL29 and is up to 30 x 103-foldmore potent than SCG in inhibiting anti-IgE-provoked releaseof histamine and prostaglandin D, from human-dispersed lung mast cells.” In the nose, the P,-adrenoceptoragonist, fenoterol, protects against the increasein nasal resistance provoked by challenge with specific allergen,3o whereas in the lower airways, terbutaline3’ and salbutamol” inhibit exercise- and allergen-provoked bronchoconstriction, respectively, as well as the associated increase in the concentrations of mast cellderived mediators in the peripheral circulation. Although an additional effect of salbutamol in inhibiting mast cell-mediator release might explain its more potent protective action against AMP, an alternative possibility is P,-receptor down regulation. All the participating subjects took inhaled P,-adrenergic agonists on a regular basis and may therefore have experienced such an effect,32which, if inflammatory cells are affectedto a greaterextent than smoothmuscle, might accountfor the magnitudeof the salbutamol effect against AMP. In the present study, no correlation was found between the extent of bronchodilatation produced by salbutamol and the ability of this drug to protect against bronchoconstriction by any of the three agonists administered, suggesting that, even for methacholine and histamine, thesetwo drug effects are mediated by separatemechanisms.Similar findings have previously been reported for methacholine and histamine independently, but not for the two agonists in the samesubjects.Thus, Salomeet a1.33 have reported a dissociation betweenthe ability of inhaled fenoterol to bronchodilate and to protect against the bronchoconstrictive action of histamine. In this study, bronchodilatatbon, measured as the increase in FEV,, lasted longer than protection against histamine. This finding has been confirmed in a separatestudy by demonstratingthat bronchodilatation produced by the inhaled P,-agonists, salbutamol and metaproterenol, lastedup to 4 hours, whereasattenuationof histamineinduced bronchoconstriction reachedmaximum at 30 minutes and thereafter declined, although the drugs were still highly active at 1 hour, covering the time course use:din the present study.34Further evidence for a dissociation between bronchodilatation and protection againstbronchoconstriction derives from studies with drugs that cause bronchodilatation through different mechanisms. Thus, Chung and Snashal13s demonstrated that inhaled salbutamol produced a greaterincreasein the baselinevalue of specificairway conductancethan atropine but afforded lessprotection
Salbutamol
and AMP-induced
bronchoconstriction
761
against methacholine challenge. In a further study in which similar degrees of bronchodilatation were achieved by inhalation of nebulized salbutamol and ipratropium bromide, only the P,-agonist significantly displaced the histamine dose-responsecurve to the right.36 Conversely, calcium antagonists have been found to produce a small but significant protection against histamine challenge without causing any changes in resting pulmonary function,37 and both salbutamoP* and calcium antagonists3’can protect against exercise-inducedbronchoconstriction without bronchodilatation. In the present study there was an inverse relationship betweenbaselineresponsivenessto methacholine and the extent of protection afforded by salbutamol against this agonist, although no such relationship could be demonstratedfor histamine or AMP. Our observations differ from observations reported by Bandouvakiset a1.39who observeda strongcorrelation between the protective effect of the P,-adrenergicagonist, fenoterol, on the responseto histamine and the level of responsivenessto this amine at baseline but found no similar relationship for methacholine. We conclude that inhaled salbutamol protects against methacholine- and histamine-induced bronchoconstriction by a mechanism predominantly involving functional antagonismand which is independent of the degree of bronchodilatation produced. However, an enhancedeffect of this drug is evident against AMP. One possible explanation for this finding involves inhibition of adenosine-inducedenhancement of preformed mediator release from activated airway mastcells. We further suggestthat the efficacy of P,-adrenoceptor agonists in asthma may not be solely restricted to their bronchodilator activity but may also involve direct antibronchoconstrictor activity, as well as inhibition of inflammatory mediator release. REFERENCES 1. Cushley MJ, Tattersfield AE, Holgate ST. inhaled adenosine and guanosine on airway resistance in normal and asthmatic subjects. Br J Clin Pharmacol 1983;15:161-5. 2. Chan W, Cushley MJ, Holgate ST. The effect of inhaled adenosine 5’-monophosphate on airway calibre in normal and asthmatic subjects. Clin Sci 1986;70:65-6P. 3. Phillips GD, Richards R, Scott VL, Holgate ST. Sodium cromoglycate and nedocromil sodium inhibit bronchoconstriction provoked by adenosine 5’-monophosphate in atopic and nonatopic asthma [Abstract]. Am Rev Respir Dis 1988;137 (suppl):29. 4. Rafferty P, Bedsley CRW, Holgate ST. The contribution of histamine to immediate bronchoconstriction provoked by inhaled allergen and adenosine 5’-monophosphate in atopic asthma. Am Rev Respir Dis 1987;136:369-73. 5. Phillips GD, Rafferty P, Beasley CRW, Holgate ST. Effect of
762 Phillips et al.
oral terfenadineon the bronchoconstrictorresponseto inhaled histamine and adenosine 5’-monophosphate in non-atopic asthma. Thorax 1987;42:939-45. 6. Pauwels R, Joos Cl, Kips J, Van der StraetenM. Adenosine and neurokinin A causehistamine release in the airways [abstract]. Am Rev Respir Dis 1988;137(suppl):212. 7. Marquardt DL, Walker LL, WassermanSI. Adenosine receptors on mouse bone marrow-derived mast cells. Functional significance and regulation by aminophylline. J Immunol 1984;133:932-7. 8. Hughes PJ, Holgate ST, Church MK. Adenosine inhibits and potentiatesIgE-dependenthistamine releasefrom human lung mast cells by an A,-purinoceptor-mediatedmechanism. Biothem Pharmacol 1984;33:3847-52. 9. Skidmore IF. B-agonistsas mast cell stabilizers. In: Kay AB, ed, Asthma: clinical, pharmacological, and therapeutic progress. Oxford: Blackwell Scientific, 1986:171-9. 10. Church MK, Hiroi J. Inhibition of IgE-dependenthistamine release from human dispersedlung mast cells by anti-allergic drugs and salbutamol. Br J Pharmacol 1987;90:421-9. 11. Howarth PH, Durham SR, Lee TH, Kay AB, Church MK, Holgate ST. Influence of albuterol, cromolyn sodium, and ipratropium bromide on the airway and circulating mediator responsesto antigen bronchial provocation in asthma.Am Rev Respir Dis 1985;132:986-92. 12. Holgate ST, Benyon RC, Howarth PH, et al. Relationship between mediator release from human lung mast cell in vitro and in vivo. Int Arch Allergy Appl Immunol 1985;77:47-56. 13. Lewis RA. Therapeuticaerosols.In: Cumming Cl, Bonsignore C, eds. Drugs and the lung. London: Plenum Publishing, 1984:63-86. 14. Chai H, Farr RS, Froehlich LA, et al. Standardizationof bronchial challenge procedures.J ALLERGYCLIN IMMUNOL1975; 56~323-7. 15. Ryan Cl, Dolovich MR, Roberts RS, et al. Standardizationof inhalation provocation tests: two techniques of aerosol generation and inhalation compared. Am Rev Respir Dis 1981; 123:195-9. 16. ChungKF, Morgan B, Keyes SJ, SnashallPD. Histaminedoseresponserelationships in normal and asthmatic subjects: the importance of starting airway calibre. Am Rev Respir Dis 1982;126:849-54. 17. Holgate ST, Cushley MJ, Mann JS, Hughes PJ, Church MK. The actions of purine on human airways. Arch Int Pharmacodyn Ther 1986;28O(suppl):240-52. 18. Okayama M, Ma J-Y, Mataoka I, et al. Role of vagal nerve activity on adenosine-inducedbronchoconstrictionin asthma [Abstract]. Am Rev Respir Dis 1986;133(suppl):A93. 19. GustafssonLE, Wiklund NP, CederqvistB. Apparent enhancement of cholinergic transmissionin rabbit bronchi via adenosine A, receptors. Eur J Pharmacol 1986;120:179-85. 20. Pauwels RA, Van der Straeten ME. An animal model for adenosine-inducedbronchoconstriction. Am Rev Respir Dis 1987;136:374-8. 21. Cockcroft DW, Killian DN, Mellon JJ, Hargreave FE. Protective effect of drugs on histamine-inducedasthma. Thorax 1977~321429-37. 22. Dixon M, JacksonDM, Richards IM. The action of sodium cromoglycateon “C” fibre endings in the dog lung. Br J Pharmacol 1980;70:11-13.
J. ALLERGY
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23. Phillips GD, Ng WH, Church MK, Holcate ST. The response of plasmahistamineto bronchoprovocationwith methacholine, adenosine5’-monophosphate(AMP) and allergen in atopic, nonasthmaticsubjects [in press]. Am Rev Respir Dis. 24. Anderson SD, Seale JP, Rozea P, Bandler L, Theobald G, Lindsay DA. Inhaled and oral salbutamol in exercise-induced asthma. Am Rev Respir Dis 1976;114:493-500. 25. Lee TH, Brown MJ, Nagy L, Causon R, Walport MJ, Kay AB. Exercise-inducedreleaseof histamineand neutrophil chemotactic factor in atopic asthmatics.J ALLERGYCLINIMMUNOL 1982;70:73-81. 26. Anderson SD, Bye PTP, SchoefferRE, Seale JP, Taylor KM, Ferris L. Arterial plasma histamine levels at rest and during and after exercise in patients with asthma:effectsof terbutaline aerosol. Thorax 1981;36:259-67. 27. Schild HO. Histamine release and anaphylactic shock in isolated lungs of guinea pigs. Q J Exp Physiol 1936;26:165. 28. ButchersPR, Fullerton JR, Skidmore IF, ThompsonLE, Vardey CJ, Wheeldon A. A comparisonof the anti-anaphylactic activities of salbutamol and disodium cromoglycatein the rat, the rat mast cell, and in human lung tissue. Br J Pharmacol 1979;67:23-32. 29. Flint KC. Bronchoalveolarmast cells and asthma.Berlin, Heidelberg: Springer-Verlag, 1987:39-41. 30. SchumacherMJ. Effect of a B-adrenergicagonist, fenoterol, on nasal sensitivity to allergen. J ALLERGYCLIN IMMUNOL 1980;66:33-6. 31. Ferris L, Anderson SD, Temple DM. Histamine release in exercise-inducedasthma. Br Med J 1978;1:1697. 32. VathenenAS, Knox AJ, Higgins BG, Britton JR, Tattersfield AE. Reboundincreasein bronchial responsivenessafter treatment with inhaled terbutaline. Lancet 1988;1:554-8. 33. Salome CM, Schoeffel RE, Yan K, Woolcock AJ. Effect of aerosol fenoterol on the severity of bronchial hyperreactivity in patients with asthma. Thorax 1983;38:854-8. 34. Ahrens RC, Harris JB, Milavetz G, Annis L, Ries R. Use of bronchial provocation with histamine to compare the pharmacodynamicsof inhaled albuterol and metaproterenolin patients with asthma. J ALLERGYCLIN IMMUNOL1987;79:87682. 35. Chung KF, Snashall PD. Methacholine dose-responsecurves in normal and asthmatic man: effect of starting conductance and pharmacologicalantagonism.Clin Sci 1984;66:665-73. 36. Britton I, Hanley SP, Garrett HV, Hadfield JW, Tattersfield AE. Dose related effects of salbutamol and ipratropium bromide on airway calibre and reactivity in subjectswith asthma. Thorax 1988;43:300-5. 37. Barnes PJ, Wilson WM, Brown MJ. A calcium antagonist, nifedipine, modifies exercise-induced asthma. Thorax 1981; 36:726-30. 38. Hetzel MR, Baten JC, Clark TJH. Do sympathomimetic amines prevent exercise-inducedasthmaby bronchodilatation alone? Br J Dis Chest 1977;71:109-14. 39. BandouvakisJ, Carder A, RobertsR, Ryan G, HargreaveFE. The effect of ipratropium and fenoterol on methacholine-and histamine-inducedbronchoconstriction.Br J Dis Chest 1981; 75:295-305.
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Methacholine
12. Furukawa CT, Shapiro GG, Bierman CW, et al. A doubleblind study comparing the effectivenessof cromolyn sodium and susta:.ned-release theophylline in childhood asthma. Pediatrics. 1984;74:453-9. 13. Cockcroft DW, Killian DN, Mellon JJA, Hargreave FE. Protective effect of drugs on histamine-inducedasthma. Thorax 1917;32:429-31.
sensitivity
14. Cockcroft DW, Murduck BA, Gore PM, et al. Theophylline does not inhibit allergen-induced increase in airway responsiveness to methacholine. J ALLERGYCLIN IMMUNOL1989; 83:913-20.
Comparative protective effect of the inhaled P,-agonist satbutamol (albuterol) on bronchoconstriction provoked by histamine, methacholine, and adenosine 5’-monophosphate in asthma G. D. Phillips, MA, MRCP, J. P. Finnerty, MRCP, and S. T. Holgate, BSc, FRCP Southampton,England We have investigated the ability of salbutamol to protect against bronchoconstriction induced by methacholine, histamine, and adenosine 5’-monophosphate (AMP) in nine subjects with asthma. In a double-blind, placebo-controlled study, salbutamol, 2.5 mg administered by nebulization, increased the geometric mean provocation concentrations of me&choline, histamine, and AMP required to produce a 20% decrease in FEV, from 0.3 to 2.2, 0.4 to 3.8, and 4.0 to 106.7 mglml after placebo and active treatment, respectively (p < 0.01). Thus, this dose of /3,-adrenoceptor agonist displaced the concentration-response curves for me&choline, histamine, and AMP to the right in a parallel fashion by 8.8 (0.6 to 29.3)-, 10.3 (I .4 to 33)-, and 26.6 (1.5 to 76.6)-fold, respectively, the difSerence between the results for AMP and those for histamine and methacholine being statistically sign&ant (p < 0.01). For six of the nine subjects studied, salbutamol displaced the concentration-response curve for AMP to the right by >.50-fold. There was no correlation between bronchodilatation and protection against bronchoconstriction induced by any of the agonists. We conclude that salbutamol protects against bronchoconstriction provoked by methacholine and histamine by functional antagonism, whereas with AMP, an additional activity is demonstrable, possibly involving inhibition of mast cell-mediator release. (J ALLERGYCLIN IMMUNOL1990;85:755-62.)
When subjects with asthma’ and atopic subjects without asthma’ inhale adenosine and its related nu-
Abbreviations used
AMP: Adenosine 5’-monophosphate ANOVA: Analysis of variance From the Departmentof Immunopharmacology,SouthamptonGeneral Hospital, Southampton,England. Supportedby a grant from the Chest, Heart, andStrokeAssociation. Received for publication June 22, 1989. Revised Nov. 6, 1989. Accepted for publication Nov. 9, 1989. Reprint requests: S. T. Holgate, BSc, Dept. of Immunopharmacology, Level D, Center Block, SouthamptonGeneral Hospital, Tremona Road, Southampton,SO9 4XY, England. Correspondenceaddress:G. D. Phillips, MA, Dept. of Immunopharmacology, Level D, Center Block, SouthamptonGeneral Hospital, Tremona Road, Southampton,SO9 4XY, England. l/1/18140
PCzO: Provocative
concentration
required to
produce a 20% fall in FEV, from postsaline baseline
SCG: Sodium cromoglycate BAL: Bronchoalveolar lavage
cleotide, AMP, this causes dose-related bronchoconstriction. Since the effect of these purines on asthmatic airways can be antagonized by inhibitors of mast cellmediator release3 and by selective HI-histamine re755
756 Phillips et al.
J. ALLERGY
TABLE 1. Details of characteristics Subject
1 2 3 4 5 6 7 8 9 Mean SEM
No.
Sex
M M F M F
M F M F 5M,4F
CLIN. IMMUNOL. APRIL 1990
of patients Age W
43 22 24 17 48 45 22 41 32 32.7 3.9
Baseline FEV, 1% predicted)
64 98 86 98 113 65 115 82 106
Atopic
+ + + + + + + +
Treatment
S s s S, B S, Bf S, B S S, B S, Bf
S, Salbutamol;B, beclomethasone dipropionate,50 pg per actuation;EJ beclomethasone dipropionate,250 pg Peractuation.
ceptor antagonists,4.5 both adenosineand AMP have beenproposedto provoke bronchoconstriction by potentiating mediator releasefrom immunologically activated airway mast cells.4*5 In an in vivo model of adenosine-inducedbronchoconstriction in inbred rats, Pauwels et a1.6have demonstratedan approximately twofold increasein the concentration of histamine in BAL fluid during bronchoconstriction provoked by intravenous injection of the adenosine analogue, N-ethylcarboxamide adenosine. Mast cells cultured from the bone marrow of rodents’ or dispersedfrom human lung tissue,8 respond to adenosine and synthetic analoguesby enhancedIgE-stimulated mediator release via a mechanism involving cell surface A2 purinoceptors. Mast cells also possessa distinct population of &adrenoceptorsgthat, when they are activated by agonists suchassalbutamol, up regulateadenylatecyclase activity and are 2 to 30 x 103-foldmore potent than SCG at inhibiting mediator release.loFurther evidence for P,-adrenoceptor agonists suppressing mast cell function in asthmaderives from the observation that salbutamol, inhaled before allergen provocation, inhibits both the airways and circulating mediator responses.” In a previous study, we have reported that inhaled salbutamol was approximately twofold more effective at inhibiting immediate bronchoconstriction provoked by inhaled allergen comparedwith methacholine. I2Although salbutamol may be consideredas a functional antagonistat human airways smoothmuscle, the increased activity expressed by this drug against the airway responseto allergen could be accounted for by an inhibitory action on mast cellmediator release.I2In the presentstudy, we have used this approach to examine the contribution of endogenous mediator releaseto bronchoconstriction that is producedby AMP by comparing the protective effect
of inhaled salbutamol on the airway responsesthat is induced by inhaled methacholine, histamine, and AMP. METHODS Subjects Nine subjects (five male subjects) with a mean (SEM) ageof 32.7 years(3.9 years),participatedin thestudy(Table I). All subjectswere nonsmokerswith asthma,and all except one subject were atopic as defined by positive skin prick tests (>2 mm wheal response) to two or more common aeroallergens (house dust, Dermatophagoides pteronyssinus, D. farinae, mixed grasspollen, mixed tree pollen, cat fur, dog hair, mixed feathers, Aspergillus fumigatus, Candia’a albicans; BencardAllergy Unit, Brentfoid, Middlesex, U.K.). All patients had baseline FEV, >60% of their predicted values or > 1.5 L and had previously been demonstratedto have an increasein FEV, of > 15% after inhaling salbutamol, 200 pg from a metered-doseinhaler, and none was receiving oral corticosteroids, theophylline, or SCG on a regular basis. Bronchodilators were withheld for 8 hours before each visit to the laboratory, although subjectswere allowed to continue inhaled corticosteroids as usual. No patient was studied within 4 weeks of an upper respiratory tract infection or exacerbation of asthma. Subjects gave written, informed consent, and the study was approved by the Southampton University and Hospitals Ethical Committee. Bronchial
provocation
Pulmonary function was measuredbefore and during the provocation as the FEV, with a dry-wedge spirometer (Vitalograph Ltd., Buckingham, U.K.), the first of two consecutive measurementsbeing used for analysis. On each challenge day, methacholine (Sigma Chemical Co., Poole,
Dorset,U.K.), histamine,acidphosphate (BDHChemicals, Poole, Dorset, U.K.), and yeast-derivedAMP (product No. A1752, Sigma Chemical Co., St. Louis, MO.) were made up freshly in 0.9% (wt/vol) sodium chloride to produce a range of increasing doubling concentrationsof 0.03 to 64
VOLUME NUMBER
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mgiml (0.2 to 327 mmol/L), 0.03 to 16 mg/ml (0.1 to 52 mmol/L), and 0.04 to 400 mg/ml (1.12 to 1151.4 mmol/ L) , respectively. The solutions were administeredas aerosols generated from a starting volume of 3 ml in a disposable Inspiron mini-nebulizer (C. R. Bard International, Sunderland, U.K.) driven by compressedair at 8 L/mitt. Under theseconditions, the nebulizer hasan output of 0.48 ml/min and generatesan aerosolwith a massmedian particle diameter of 4.7 p,rn.13Wearing a noseclip, subjects inhaled the aerosolized solutions in five breathsfrom end tidal volume to full inspiratory capacity via a mouthpiece.“. Is Subjectswere trained to take 3 secondsto reach full inspiratory capacity. All the bronchial provocations were carried out at the sametime of day. Study design Subjects attended on six occasions to undertake concentration-responsestudies with each of the three agonists, the paired visits with each agonist being conducted within 1 week of each other. On each occasion, after a 15 minute rest, three baseline measurementsof FEV, were recorded at intervals of 3 minutes, followed by inhalation of neublized !;albutamol,2.5 mg (Allen and Hanbury’s Ltd., Greenford, Middlesex, U.K.) or 0.9% (wt/vol) of sodium chloride plaaebo.Salbutamoland placebowere randomized separatelyfor each of the three agonists and administered double blind. The aerosolizedsolutions were generatedfrom a starting volume of 2.5 ml in an Inspiron mini-nebulizer driven by compressedair at 8 Llmin and inhaled to dryness by deeptidal breathing during a 2- to 3minute time period. The samenebulizer was usedfor all studieson all subjects. Thirty minutes later, a concentration-responsestudy with one of the three agonists was performed. On each occasion, three postdrugmeasurementsof FEV, were recorded at intervals of 3 minutes, followed by inhalation of neublized 0.9% (wt/vol) sodium chloride and repeat measurementsof FEV, at 1 and 3 minutes. If FEV, did not fall by >lO% of the postdrug baseline value, the concentration-responsestudy was undertaken. After inhaling each agonist concentration, FEV, was measured at 1 and 3 minutes, the higher value being recorded.Increasing doubling concentrationsof agonist were then inhaled at 5minute intervals until FEV, had fallen by >20% of the postsaline value or the highest concentration had been administered. Data analysis Figures refer to the mean -+ SEM unless it is otherwise stated,and the p < 0.05 level of significance was accepted. Pre- and postdrugbaselinevaluesof FEV, were compared within each study day with Student’s t test for paired data and between study days with two-factor ANOVA followed by the Newman Keuls’ procedure to determine the source of the variance. For each of the three study days, when active drug was administered, the ability of salbutamol to cause bronchodilatation was determined by expressing the difference betweenthe pre- and postsalbutamolFEV, values asa percentageof the predrugbaselinerecording. The values thus obtained were comparedbetweenstudy days with two-
Salbutamol
and AMP-induced
bronchoconstriction
757
factor ANOVA and the Newman Keuls’ procedure. Since postsaline FEV, values after salbutamol were significantly higher than values after placeboadministration, the agonistconstrictor responsewas expressedas a percentageof the postdrug baseline.16The fall in FEV, after each concentration of agonist was expressedas a percentageof the higher of the two postsaline baseline FEV, recordings, the higher of two measured values being used in each case. The percentagefall in FEV, was plotted against the cumulative concentration of agonist administered on a logarithmic scale, and the PC, was determinedby linear interpolation. The slopes of the concentration-responsecurves were determinedby least-squares,linear-regressionanalysis and comparedbetween agonists, and for the same agonist between postplaceboand postsalbutamolstudy days by use of two-factor ANOVA followed by the Newman Keuls’ procedure and Student’s t test for paired data, respectively. Concentration ratios for the protective effect of salbutamol against bronchoprovocation with each agonist were calculated by dividing the PC,, value obtainedafter administration of active drug by that obtained after administration of placebo for the same agonist. The relative potency of salbutamol in protecting against bronchoconstriction induced by the three agonists was analyzed by comparing the concentration ratios with two-factor ANOVA, followed by the Newman Keuls’ procedure. Any relationship between PC, AMP and PC, methacholine and histamine, betweenthe concentrationratios for AMP and those for methacholine and histamine, between the concentrationratio for AMP and the PC,, methacholine and histamine, and betweenthe concentrationratio for each agonist and the percentageincrease in FEV, produced by salbutamol was investigated with least-squares, linearregression analysis of the data that were logarithmically transformedwhere this was appropriate. RESULTS There were no significant differences in predrug, postplacebo, or postsalbutamol baseline values of FEV, between any of the study days. On the methacholine, histamine, and AMP study days, salbutamol produced 15.9 2 3.8%, 11.8 + 2..5%, and 12.9 + 1.9% increases in FEV, from predrug baseline (p < 0.01 for each) with no significant differences between these values (Table II). No significant differences were found between the slopes of the postplacebo concentration-response curves for the three agonists, and for each agonist the concentration-response curves after placebo and salbutamol administration did not depart significantly from parallel (Table III). Except for one subject with methacholine (No. 4), salbutamol produced a displacement to the right of the concentration-response curve for all three agonists. Thus, the geometric mean (range) concentrations of methacholine, histamine, and AMP required to produce a 20% decrease in FEV, after placebo
J. ALLERGY
758 Phillips et al.
TABLE II. Baseline FEV, values before and after inhalation Methacholine Subject
No.
1 2 3 4 5 6 7 8 9 Mean SEM
P&rug
2.0* 5.9 3.3 6.5 2.3 2.9 3.5 3.3 3.5 3.7 0.5
of nebulized
salbutamol
Histamine
Postdrug
Predrug
2.9 6.4 3.9 6.8 2.6 3.5 4.0 3.7 3.8 4.2 0.5
2.1 5.6 3.3 6.2 2.6 2.9 3.6 3.4 3.8 3.7 0.4
CLIN. IMMUNOL. APRIL 1990
AMP Postdrug
2.7 6.4 3.9 6.2 2.9 3.2 3.9 3.7 4.0 4.1 0.4
Predrug
2.3 5.7 3.3 5.9 2.9 2.7 3.7 3.3 3.4 3.7 0.4
Postdrug
2.7 6.1 3.8 6.5 3.1 3.2 3.9 3.9 3.9 4.1 0.4
*Values in liters.
administration in the nine subjects studied were 0.3 (0.1 to 1.2), 0.4 (0.1 to 1.2), and 4.0 (2.5 to 7.3) mg/ml, respectively (Fig. 1) (Table IV). Thus, AMP was 7.6 (1.3 to 34)-fold and 9.0 (3.0 to 30.7)-fold less potent than methacholine and histamine, respectively, on a molar basisin reducing FFV, in this group of subjectswith asthma.For methacholine,histamine, and AMP, the mean PC, values after administration of salbutamolwere 2.2 (0.7 to 7.0), 3.8 (1.1 to 12.4), and 106.7 (4.0 to 408.2) mg/ml, respectively, the difference between each of these values and the corresponding postplacebovalue being statistically significant (p < 0.001) (Fig. 1) (Table IV). Expressed as concentration ratios, salbutamol produced a protection of the airways of 8.8 (0.6 to 29.3)-fold, 10.3 (1.4 to 33)-fold, and 26.6 (1.5 to 76.6)-fold against bronchoconstriction provoked by methacholine, histamine, and AMP, respectively, the concentration ratios for AMP being significantly greaterthan the concentration ratios for methacholine(p < 0.01) and histamine (p < 0.01). No significant correlation was found between the concentration ratio for AMP and that for histamine (r = 0.1; p = 0.7), or methacholine (r = 0.4; p = 0.2), or between the concentration ratio for each of the agonists and the predrug baseline FEV,, or the percentagebronchodilatation produced by nebulized salbutamolon the correspondingstudy day. There was a strong relationship, however, between the postplacebolog PC, methacholineandthe concentrationratio for methacholine(r = -0.8;~ = 0.02) andbetween the concentration ratios for histamine and methacholine (r = 0.9;~ = 0.006). DISCUSSION
The presentstudy demonstratesthat salbutamol, 2.5 mg, administeredby nebulization, producesa similar
ninefold or tenfold protection of the airways against bronchoconstriction provoked by methacholine and histamine. However,despiteresulting in a similar 12% to 16% bronchodilatation on all three agonist study days on which active drug was administered, salbutamol produceda significantly greatergeometricmean 27-fold protection for the airways against decreases in FEV, produced by AMP and displaced the nucleotide concentration-responsecurve to the right by >50fold in six of the subjects studied. Since salbutamol is 2 to 30 x lo’-fold more potent than SCG in inhibiting anti-IgE-provoked releaseof histamine from human-dispersed lung mast cells in vitro,” our present data may be interpreted as supporting this &adrenoceptor agonist protecting against the airway effects of methacholine and histamine via a mechanism involving its action as a functional antagonist but having an additional activity against AMP, possibly in preventing degranulation of bronchial mast cells by the nucleotide. We deliberately opted to include patients who were highly reactive to both histamine and methacholineso that the effectsof inhaled salbutamolin displacing the concentration-responsecurves could be described in every case by obtaining true postdrug PC?,,values without having recourse to data manipulations necessary to overcome the problem of censoredvalues. In this study, the greater inhibition afforded by nebulized salbutamol against AMP than against methacholine- and histamine-provoked decreasesin FEV, is unlikely to be causedby differences in functional antagonismon the respectivestudy days, since the degreeof bronchodilatation producedwas not significantly different. One possibility is that, in being limited to smooth muscle, the action of AMP in the airways is more completely reversed by salbutamol than is that of provocants like histamine, which exact their effects, in addition, on neural reflexes and
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Salbutamol
TABLE III. Gradients of the concentration-response and placebo
NO.
1 2 3 4 5 6 7 8 9 Mean (SEW
Salbutamol
Placebo
-29.2 -35.5 - 22.6 -45.8 -48.4 -33.1 -59.7 -28.0 - 19.9 -35.8
-24.4 -43.0 -27.5 -38.0 -66.5 -27.4 -29.3 - 25.4 - 24.2 - 34.0
-18 -30.1 -66.1 -40.6 -33.1 -40.2 -26.0 - 30.7 -20.8 - 34.0
(4.4)
(4.6)
(4.8)
TABLE IV. PC, values for methacholine, and salbutamol
No.
1 2 3 4 5 6 7 8 9 Geometric mean (ran&
Placebo
0.2 0.9 0.1 1.2 0.1 0.1 0.6 0.2 0.8 0.3 (0.1-1.2)
- 22.4 -40 -37.3 -52.1 -40.4 -44.6 - 24.0 - 26.6 - 22.9 - 34.5 (3.6)
Placebo
Salbutamol
- 26.7 -31.5 - 32.4 - 24.9 -45.6 -24.4 -43.2 -33.7 - 18.4 -31.2 (3.0)
- 34.3 - 36.4 -35.3 - 29.5 -39.6 - 32.9 - 26.3 -33.9 -24.8 -32.6
(1.6)
histamine, and AMP after inhalation of nebulized placebo Histamine
Methacholine Subject
759
AMP
Histamine
Salbutamol
Placebo
bronchoconstriction
curves after inhalation of nebulized salbutamol
Methacholine Subject
and AMP-induced
Salbutamol
2.4 7.0 1.8 0.7 0.7 0.9 6.9 3.8 3.7 2.2 (0.7-7.0)
Placebo
0.2 1.1 0.1 0.8 0.2 0.2 1.2 0.2 1.2 0.4 (0.1-1.2)
postcapillary venular permeability. However, this latter explanation is unlikely, since the effects of adenosine on ahway smooth muscle in vitro are weak and inconsistent.” Although: we have been unable to demonstrate a significant protective effect of the inhaled muscarinic antagonist, ipratropium bromide, on adenosineinduced bronchoconstriction, Okayama et al. I8 were able to demonstrate some reversal of adenosineinduced bronchoconstriction by inhaled atropine. In addition, in vitroI and in vivozo studies in the rabbit lend somesupportto a possible neurologic mechanism for adenosine-inducedbronchoconstriction. However, since histamine also stimualtes neural reflexes in the airways,2’ it is unlikely that an action of salbutamol in protecting against such reflexes accounts for its greater prorective effect against AMP. Although the mode of action of AMP in provoking bronchocon-
AMP
Salbutamol
1.6 12.4 3.3 1.1 2.8 2.4 9.9 3.6 8.8 3.8 (1.1-12.4)
Placebo
Salbutamol
2.5 5.2 3.2 2.7 5.9 3.6 7.3 3.9 4.1 4.0 (2.5-7.3)
191.6 284.5 199.2 4.0 34.1 52.0 408.2 204.3 279.2 106.7 (4.0-408.2)
striction in subjectswith asthmaremains to be determined, one suggestion involves the potentiation of preformed mediator release, principally histamine, from activated airway mast cells.4,* Thus, adenosine and related analoguesactive at the A, purinoceptor potentiate the immunologically stimulated release of the preformed mediators, histamine and phexosaminidase,from rodent’ and human’ mast cells in vitro. In vivo, selective H,-histamine-receptor antagonists produce >80% protection of the airways against bronchoconstriction provoked by AMP,4. 5 whereas the mast cell membrane-stabilizing drugs, SCG and nedocromil sodium, displacethe AMP doseresponsecurve to the right by tenfold to twentyfold,’ although this latter effect may be due to a combination of inhibition of mast cell mediator-releaseand C fiber afferent reflexes.** In addition, after intravenous injection of the adenosine analogue, N-
760
Phillips
et al.
J. ALLERGY
Methachdine
Histamine s
r
P
CLIN. IMMUNOL. APRIL 1990
AMP 5
P
s
1
0 FIG. 1. Effect of inhaled nebulized salbutamol, 2.5 mg (S) and placebo (P) on bronchoconstriction provoked by methacholine, histamine, and AMP in nine subjects with asthma. The horizontal bars denote geometric mean values.
ethylcarboxamide adenosine, Pauwels et a1.6 have demonstratedan approximately twofold increase in BAL fluid histamineconcentrationin the rabbit. Thus, one possibility is that the additional inhibitory effect of salbutamol againstAMP-induced bronchoconstriction involves the prevention of adenosine-inducedenhancementof preformed mediator release from activated airway mast cells. Although the present data may be interpreted as supporting such a mechanism, this evidence is only indirect and capable of alternative interpretations, as already discussed. Moreover, the similarity in the slopesof the postsalbutamol concentration-responsecurves for all three agonists may be interpreted as suggesting that the B,-agonist protects against them all by a similar mechanism. Recently, however, we have been able to demonstrate an approximately twofold increase in the concentration of histamine in peripheral blood after bronchoprovocation with AMP in 10 atopic subjectswithout asthmawith no similar elevations being observed after inhalation of concentrationsof methacholine sufficient to provoke equivalent decreasesin pulmonary function.23
Evidence in supportof an additional mastcell effect of salbtuamol includes the observation that, whereas oral and inhaled salbutamol produced a similar threefold to fourfold protection for the airways against metbacholine-induced bronchoconstriction, a small dose(10 pg) of inhaled salbutamolwastwice aspotent in inhibiting decreasesin pulmonary function provoked by allergen than was an oral dose of 8 mg,‘* suggesting an additional local effect at the level of epithelial mastcells. Similarly, Andersonet a1.24have demonstratedthat, whereas salbutamol administered via both tbe oral and inhaled routes produced bronchodilatation, only the inhaled drug protected against exercise-induced bronchoconstriction, another stimulus purported to involve mast cell-mediator release.“, 26 Although B,-adrenoceptor agonists, such as salbutamol, are consideredto exert their clinical effects by relaxing bronchial smooth muscle, there is evidence to suggestthat they also possess“antiallergic” activity. Thus, catecholaminesinhibit the release of histamine from guinea pig-sensitized lung*’ and of histamine and slow-reacting substanceof anaphylaxis
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from human sensitized lung fragments.**Salbutamol inhibits Igt-dependent histamine release from mast cells recovered from subjects with asthmaby BAL29 and is up to 30 x 103-foldmore potent than SCG in inhibiting anti-IgE-provoked releaseof histamine and prostaglandin D, from human-dispersed lung mast cells.” In the nose, the P,-adrenoceptoragonist, fenoterol, protects against the increasein nasal resistance provoked by challenge with specific allergen,3o whereas in the lower airways, terbutaline3’ and salbutamol” inhibit exercise- and allergen-provoked bronchoconstriction, respectively, as well as the associated increase in the concentrations of mast cellderived mediators in the peripheral circulation. Although an additional effect of salbutamol in inhibiting mast cell-mediator release might explain its more potent protective action against AMP, an alternative possibility is P,-receptor down regulation. All the participating subjects took inhaled P,-adrenergic agonists on a regular basis and may therefore have experienced such an effect,32which, if inflammatory cells are affectedto a greaterextent than smoothmuscle, might accountfor the magnitudeof the salbutamol effect against AMP. In the present study, no correlation was found between the extent of bronchodilatation produced by salbutamol and the ability of this drug to protect against bronchoconstriction by any of the three agonists administered, suggesting that, even for methacholine and histamine, thesetwo drug effects are mediated by separatemechanisms.Similar findings have previously been reported for methacholine and histamine independently, but not for the two agonists in the samesubjects.Thus, Salomeet a1.33 have reported a dissociation betweenthe ability of inhaled fenoterol to bronchodilate and to protect against the bronchoconstrictive action of histamine. In this study, bronchodilatatbon, measured as the increase in FEV,, lasted longer than protection against histamine. This finding has been confirmed in a separatestudy by demonstratingthat bronchodilatation produced by the inhaled P,-agonists, salbutamol and metaproterenol, lastedup to 4 hours, whereasattenuationof histamineinduced bronchoconstriction reachedmaximum at 30 minutes and thereafter declined, although the drugs were still highly active at 1 hour, covering the time course use:din the present study.34Further evidence for a dissociation between bronchodilatation and protection againstbronchoconstriction derives from studies with drugs that cause bronchodilatation through different mechanisms. Thus, Chung and Snashal13s demonstrated that inhaled salbutamol produced a greaterincreasein the baselinevalue of specificairway conductancethan atropine but afforded lessprotection
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against methacholine challenge. In a further study in which similar degrees of bronchodilatation were achieved by inhalation of nebulized salbutamol and ipratropium bromide, only the P,-agonist significantly displaced the histamine dose-responsecurve to the right.36 Conversely, calcium antagonists have been found to produce a small but significant protection against histamine challenge without causing any changes in resting pulmonary function,37 and both salbutamoP* and calcium antagonists3’can protect against exercise-inducedbronchoconstriction without bronchodilatation. In the present study there was an inverse relationship betweenbaselineresponsivenessto methacholine and the extent of protection afforded by salbutamol against this agonist, although no such relationship could be demonstratedfor histamine or AMP. Our observations differ from observations reported by Bandouvakiset a1.39who observeda strongcorrelation between the protective effect of the P,-adrenergicagonist, fenoterol, on the responseto histamine and the level of responsivenessto this amine at baseline but found no similar relationship for methacholine. We conclude that inhaled salbutamol protects against methacholine- and histamine-induced bronchoconstriction by a mechanism predominantly involving functional antagonismand which is independent of the degree of bronchodilatation produced. However, an enhancedeffect of this drug is evident against AMP. One possible explanation for this finding involves inhibition of adenosine-inducedenhancement of preformed mediator release from activated airway mastcells. We further suggestthat the efficacy of P,-adrenoceptor agonists in asthma may not be solely restricted to their bronchodilator activity but may also involve direct antibronchoconstrictor activity, as well as inhibition of inflammatory mediator release. REFERENCES 1. Cushley MJ, Tattersfield AE, Holgate ST. inhaled adenosine and guanosine on airway resistance in normal and asthmatic subjects. Br J Clin Pharmacol 1983;15:161-5. 2. Chan W, Cushley MJ, Holgate ST. The effect of inhaled adenosine 5’-monophosphate on airway calibre in normal and asthmatic subjects. Clin Sci 1986;70:65-6P. 3. Phillips GD, Richards R, Scott VL, Holgate ST. Sodium cromoglycate and nedocromil sodium inhibit bronchoconstriction provoked by adenosine 5’-monophosphate in atopic and nonatopic asthma [Abstract]. Am Rev Respir Dis 1988;137 (suppl):29. 4. Rafferty P, Bedsley CRW, Holgate ST. The contribution of histamine to immediate bronchoconstriction provoked by inhaled allergen and adenosine 5’-monophosphate in atopic asthma. Am Rev Respir Dis 1987;136:369-73. 5. Phillips GD, Rafferty P, Beasley CRW, Holgate ST. Effect of
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oral terfenadineon the bronchoconstrictorresponseto inhaled histamine and adenosine 5’-monophosphate in non-atopic asthma. Thorax 1987;42:939-45. 6. Pauwels R, Joos Cl, Kips J, Van der StraetenM. Adenosine and neurokinin A causehistamine release in the airways [abstract]. Am Rev Respir Dis 1988;137(suppl):212. 7. Marquardt DL, Walker LL, WassermanSI. Adenosine receptors on mouse bone marrow-derived mast cells. Functional significance and regulation by aminophylline. J Immunol 1984;133:932-7. 8. Hughes PJ, Holgate ST, Church MK. Adenosine inhibits and potentiatesIgE-dependenthistamine releasefrom human lung mast cells by an A,-purinoceptor-mediatedmechanism. Biothem Pharmacol 1984;33:3847-52. 9. Skidmore IF. B-agonistsas mast cell stabilizers. In: Kay AB, ed, Asthma: clinical, pharmacological, and therapeutic progress. Oxford: Blackwell Scientific, 1986:171-9. 10. Church MK, Hiroi J. Inhibition of IgE-dependenthistamine release from human dispersedlung mast cells by anti-allergic drugs and salbutamol. Br J Pharmacol 1987;90:421-9. 11. Howarth PH, Durham SR, Lee TH, Kay AB, Church MK, Holgate ST. Influence of albuterol, cromolyn sodium, and ipratropium bromide on the airway and circulating mediator responsesto antigen bronchial provocation in asthma.Am Rev Respir Dis 1985;132:986-92. 12. Holgate ST, Benyon RC, Howarth PH, et al. Relationship between mediator release from human lung mast cell in vitro and in vivo. Int Arch Allergy Appl Immunol 1985;77:47-56. 13. Lewis RA. Therapeuticaerosols.In: Cumming Cl, Bonsignore C, eds. Drugs and the lung. London: Plenum Publishing, 1984:63-86. 14. Chai H, Farr RS, Froehlich LA, et al. Standardizationof bronchial challenge procedures.J ALLERGYCLIN IMMUNOL1975; 56~323-7. 15. Ryan Cl, Dolovich MR, Roberts RS, et al. Standardizationof inhalation provocation tests: two techniques of aerosol generation and inhalation compared. Am Rev Respir Dis 1981; 123:195-9. 16. ChungKF, Morgan B, Keyes SJ, SnashallPD. Histaminedoseresponserelationships in normal and asthmatic subjects: the importance of starting airway calibre. Am Rev Respir Dis 1982;126:849-54. 17. Holgate ST, Cushley MJ, Mann JS, Hughes PJ, Church MK. The actions of purine on human airways. Arch Int Pharmacodyn Ther 1986;28O(suppl):240-52. 18. Okayama M, Ma J-Y, Mataoka I, et al. Role of vagal nerve activity on adenosine-inducedbronchoconstrictionin asthma [Abstract]. Am Rev Respir Dis 1986;133(suppl):A93. 19. GustafssonLE, Wiklund NP, CederqvistB. Apparent enhancement of cholinergic transmissionin rabbit bronchi via adenosine A, receptors. Eur J Pharmacol 1986;120:179-85. 20. Pauwels RA, Van der Straeten ME. An animal model for adenosine-inducedbronchoconstriction. Am Rev Respir Dis 1987;136:374-8. 21. Cockcroft DW, Killian DN, Mellon JJ, Hargreave FE. Protective effect of drugs on histamine-inducedasthma. Thorax 1977~321429-37. 22. Dixon M, JacksonDM, Richards IM. The action of sodium cromoglycateon “C” fibre endings in the dog lung. Br J Pharmacol 1980;70:11-13.
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23. Phillips GD, Ng WH, Church MK, Holcate ST. The response of plasmahistamineto bronchoprovocationwith methacholine, adenosine5’-monophosphate(AMP) and allergen in atopic, nonasthmaticsubjects [in press]. Am Rev Respir Dis. 24. Anderson SD, Seale JP, Rozea P, Bandler L, Theobald G, Lindsay DA. Inhaled and oral salbutamol in exercise-induced asthma. Am Rev Respir Dis 1976;114:493-500. 25. Lee TH, Brown MJ, Nagy L, Causon R, Walport MJ, Kay AB. Exercise-inducedreleaseof histamineand neutrophil chemotactic factor in atopic asthmatics.J ALLERGYCLINIMMUNOL 1982;70:73-81. 26. Anderson SD, Bye PTP, SchoefferRE, Seale JP, Taylor KM, Ferris L. Arterial plasma histamine levels at rest and during and after exercise in patients with asthma:effectsof terbutaline aerosol. Thorax 1981;36:259-67. 27. Schild HO. Histamine release and anaphylactic shock in isolated lungs of guinea pigs. Q J Exp Physiol 1936;26:165. 28. ButchersPR, Fullerton JR, Skidmore IF, ThompsonLE, Vardey CJ, Wheeldon A. A comparisonof the anti-anaphylactic activities of salbutamol and disodium cromoglycatein the rat, the rat mast cell, and in human lung tissue. Br J Pharmacol 1979;67:23-32. 29. Flint KC. Bronchoalveolarmast cells and asthma.Berlin, Heidelberg: Springer-Verlag, 1987:39-41. 30. SchumacherMJ. Effect of a B-adrenergicagonist, fenoterol, on nasal sensitivity to allergen. J ALLERGYCLIN IMMUNOL 1980;66:33-6. 31. Ferris L, Anderson SD, Temple DM. Histamine release in exercise-inducedasthma. Br Med J 1978;1:1697. 32. VathenenAS, Knox AJ, Higgins BG, Britton JR, Tattersfield AE. Reboundincreasein bronchial responsivenessafter treatment with inhaled terbutaline. Lancet 1988;1:554-8. 33. Salome CM, Schoeffel RE, Yan K, Woolcock AJ. Effect of aerosol fenoterol on the severity of bronchial hyperreactivity in patients with asthma. Thorax 1983;38:854-8. 34. Ahrens RC, Harris JB, Milavetz G, Annis L, Ries R. Use of bronchial provocation with histamine to compare the pharmacodynamicsof inhaled albuterol and metaproterenolin patients with asthma. J ALLERGYCLIN IMMUNOL1987;79:87682. 35. Chung KF, Snashall PD. Methacholine dose-responsecurves in normal and asthmatic man: effect of starting conductance and pharmacologicalantagonism.Clin Sci 1984;66:665-73. 36. Britton I, Hanley SP, Garrett HV, Hadfield JW, Tattersfield AE. Dose related effects of salbutamol and ipratropium bromide on airway calibre and reactivity in subjectswith asthma. Thorax 1988;43:300-5. 37. Barnes PJ, Wilson WM, Brown MJ. A calcium antagonist, nifedipine, modifies exercise-induced asthma. Thorax 1981; 36:726-30. 38. Hetzel MR, Baten JC, Clark TJH. Do sympathomimetic amines prevent exercise-inducedasthmaby bronchodilatation alone? Br J Dis Chest 1977;71:109-14. 39. BandouvakisJ, Carder A, RobertsR, Ryan G, HargreaveFE. The effect of ipratropium and fenoterol on methacholine-and histamine-inducedbronchoconstriction.Br J Dis Chest 1981; 75:295-305.