Bronchial hyperreactivity to histamine and methacholine in asthmatic children after inhalation of SCH 1000 and chlorpheniramine maleate Roland Woenne, M.D., Meyer Kattan, M.D., CM., Robert P. Orange, M.D.,t and Henry Levison, M.D. Toronto, Onturio, Canada
Nine asthmatic patients with a mean age qf 14 yr received bronchial challenges with histamine and methacholine. The challenges were repeated after inhalation of 80 /~g of SCH 1000 (ipratropium bromide) and 5 mg of chlorpheniramine maleate. The provocation doses which produced a 20% fall in ,forced expiratory volume in I set (FEV,) and the slopes of the dose-response curves were analyzed. SCH 1000 prevented methacholine-induced bronchoconstriction and chlorpheniramine prevented methacholine-induced bronchoconstriction. There was no significant change in the dose-response curve of histamine ajer SCH 1000 or in the dose-response curve of methacholine after chlorpheniramine. The jindings indicate that the mechanisms and receptor sites involved in bronchial provocarion by histamine and methacholine are distinctly different. The histamine response is unlikely to be vagally mediated because histamine-induced bronchoconstriction was not prevented by SCH 1000. Both SCH 1000 and chlorpheniramine caused significant bronchodilatation, suggesting the presence of both histamine-and vugal-dependent bronchomotor tone.
It has been demonstrated in the experimental animal’, * and in human asthmatic patients3that the responseof the airways to inhaled histamine and antigen is dependenton intact vagal pathways. In animals the vagal responseis of reflex characte? in which the afferent pathway originates at the lung irritant receptors.* A similar reflex mechanismhas been suggested in human beings.” It has also been shown that histamine is releasedtogether with other mediatorsfrom sensitizedmast cells if incubatedwith specific antigen in vitroa Several authors235 have attemptedto combine the above data into a unified schema, whereby antigen exposurein the sensitizedasthmaticpatients results in histamine release from mast cells, which then stimulates lung irritant receptors to cause reflex bronchoconstriction. In this hypothesis the vagus plays a major role and a direct action of histamine on bron-
chial smooth muscle is considered to be of minor importance. Histamine and methacholine are both well-established nonallergenic triggers of bronchoconstriction in the asthmatic subject.7, 8 If the effects of these drugs are predominantly vagally mediated, then the blockade of vagal efferent pathways by a drug such as ipratropium bromide (SCH 1000)) an anticholinergic agent, should prevent a responseto both histamine and methacholine. However, recent evidence casts some doubt on the role of the vagus in histamine provocation,g-‘2 raising the possibility that distinctly separate receptor sites and/or pathways exist for methacholine and histamine. To investigate this possibility we attempted to differentially block methacholine and histamine bronchoconstriction by use of specific antagonists, i.e., an anticholinergic agent (SCH 1000)and an Hl antagonist(chlorpheniramine). SUBJECTS
From the Department of Paediatrics and The Research Institute, The Hospital for Sick Children, University of Toronto. Received for publication Jan. I& 1978. Accepted for publication April 18, 1978. Reprint requeststo: H. Levison, M.D., Hospital for Sick Children, 555 University Ave., Toronto, Ontario, Canada MS3 I X8. “TDeceased. 0091-6749/78/0262-0119$00.60/O
@ 1978 The C. V. Mosby
Co.
Nine moderate to mild asthmatic patients between the
agesof 9 to 16 yr (mean, 14 yr) participated in this study. All were perennial asthmatic patients and had positive skin tests to a large variety of allergens. The mild asthmatic patients (3) were on no therapy; the rest of the patients regularly took beta agents. One patient was also on sodium Vol. 62, No. 2, pp. 119-124
120
Woenne
et al.
J. ALLERGY
TABLE
I. Baseline
Patient
50’
,
,
,
,,
1
,
,
IO CUMULATIVE
,
,,
,
,
,
100
,, loo0
pulmonary
function
% predicted FEV,*
P. P. M. G. H. L. M. C. L. c. M. D. L. L. B. M. M. P.
57 2 16* 69k 7 67 t 11 79* 3 92k 3 97+ 5
81? 41* 81-’
CLIN. IMMUNOL. AUGUST 1978
3 4 5
w3w 0.056 -r- 0.033t 0.080 k 0.062
0.106 + 0.020 0.065 0.124 0.129 0.198 0.038 0.093
+ k k + ? +
0.028 0.056 0.037 0.080 0.012 0.012
DOSE OF METHACHOUNE
*Expressed as percent predicted from normal subjects studied in our laboratory. I3 TMean 2 1 SD of pulmonary function for 6 days of the study.
---’ -.-
Ml-
50
t .I
CHLORPH. CONTROL SCHlmD I
Subiect
III
I
1 CUMULATIVE
1111
I
IO
MC
III1
100
DOSE OF HISTAMINE
FIG. 1. The dose-response curves for one subject with histamine and methacholine. Each graph shows the dose-response curve with pretreatment with SCH 1000 and chlorpheniramine and a control.
cromoglycate, 2 were on theophylline, and 3 patients inhaled steroids from a metered dose inhaler. All patients were regularly attending our laboratory for evaluation of asthma and were thus familiar with the tests used in this study. The baseline pulmonary function data are given in Table I. The subjects were selected on their ability to discontinue medication (except for steroids) for at least 12 hr before the study. Informed consent was obtained in all cases.
METHODS The trial was done single blind at the same hour on 6 days. Each subject completed the study within IO days. Separate days for each challenge were used to avoid drug interference.‘4Pulmonary function studies were obtained with a 9-L Collins water-sealed spirometer and a variablepressure body plethysmograph. A baseline forced expiratory volume in one second (FEVI), thoracic gas volume (TGV), and airways resistance (R,,) were obtained initially and repeated after inhalations of saline, ipratropium bromide (SCH lOOO), and chlorpheniramine maleate, respectively. Specific conductance (SG,,) was determined from R,, and the simultaneously measured TGV. All the measurements were repeated when a 20% or greater drop in FEV, was recorded after the histamine or methacholine
challenge. Methacholine (acetyl-betamethyl choline chloride) and histamine (histamine acid phosphate) challenges were done as recommended by the standardization panel of the Asthma and Allergic Disease Centers, National Institute of Allergy and Infectious Diseases.‘” The concentrations of methacholine used were 0.31, 0.62, 1.25, 2.5, 5.0, 10.0, and 25.0 mg/ml and the concentrations of histamine used were 0.12, 0.25, 0.5, I .O, 2.5, 5.0, and 10.0 mg/ml. Units of inhaled methacholine or histamine were arbitrarily obtained by multiplying inhaled concentrations by the number of breaths taken. Five successive inspiratoty capacity breaths of each concentration were administered. Repeat spirometry was performed 3 min later and immediately the procedure was continued with the next higher concentration until a 20% or greater drop in FEVl was observed with respect to the baseline after saline, SCH 1000, or chlorpheniramine inhalations. From the cumulative amount of methacholine or histamine inhaled (cumulative breaths units) and the accompanying drop in FEVl a dose-response curve for methacholine and histamine was constructed. The provocation dose of methacholine or histamine which induced a 20% drop in FEV, (PDZO-FEV1) was used to compare the bronchial sensitivity to the different challenges. The slope of the dose-response curve was calculated as the rate of fall from a 12% decrease to a 20% decrease in FEV1. A 12% fall was chosen because, from previous experience, this is the point up to which normal children drop.16 The coefficient a from the formula, y = a log10 x + b, was used to reflect this slope. This slope is an index of bronchial reactivity.‘?
EXPERIMENTAL Day 1
PROTOCOL
The subject took 5 successive inspiratoty capacity breaths of saline from a DeVilbiss No. 40 nebulizer (the same nebulizer was used in each subject during the whole study) with an air-flow set at 8 L/min. Three minutes later FEV1, TGV, and R,, were reassessed to detect any response to saline. Subsequently a methacholine challenge was done as described in the section on methods.
VOLUME
62
NUMBER
2
TABLE
Bronchial hyperreactivity
II. Sensitivity
(PD,,-FEV,)
HIS
and reactivity
SCH WOO-HIS
Subject
Phi FEV,
Slope (a)
PDmFEV,
Slope (a)
P. P. M. G. H. L. M. C. L. c. M. D. L. L. B. M. M. P.
24.2 0.26 18.5 13.7 0.26 27.3 3.78 0.26 5.2
21.3 48.4 42.8 58.5 47.7 54.6 51.6 48.5 23.2
21.3 0.32 5.52 11.3 0.24 12.4 4.65 0.13 14.6
50.6 40.0 35.0 36.2 52.0 25.4 39.0 170.6 25.1
Means
10.4
44.1
7.8
52.7
(coefficient
a) with each bronchial
CHL-HIS PDaFEV,
MCH
Slope (al
* * 86.0
20.0 * * * t * *
121
challenge
CHL-MCH
PD*(y FEV,
Slope (a)
PD,,FEV,
Slope (a)
18.15 7.13 1.47 26.5 23.4 23.4 7.3 1.58 9.4
11.4 57.8 113.6 51.3 50.1 7.0 54.7 80.2 33.6
4.65 1.48 1.48 11.8 54.2 5.8 t 2.97 4.65
30.7 126.8 113.6 25.9 167.5 13.6
13.9
50.6
10.9
69.5
61.4 16.8
SCH lOUO-MCH P&y FEV,
Slops (a)
* * * * * 3 * * *
HIS, histamine; CHL, chlorpheniramine; MCH, methacholine. *PD,,,-FEV, not achieved. t Did not complete study. TABLE
Ill. Response
Patient
FEV, base
FEV, SCH
SGaw base
SGw SCH
FEV, base
P. P. M. G. H. L. M. C. L. c. M. C. L. L. B. M. M. P.
2.61 2.21 1.96 3.06 2.82 3.02 2.41 1.33 1.95
2.93 2.9 2.02 3.57 2.91 3.21 2.46 2.0 2.31
0.09 0.043 0.104 0.043 0.119 0.184 0.135 0.033 0.096
0.166 0.128 0.206 0.144 0.18 0.212 0.209 0.083 0.195
1.56 2.31 1.9 3.11 2.75 2.91 1.54 1.99
Mean
to SCH 1000 and chlorpheniramine
2.37ir0.57 2.7kO.54 p < 0.01
0.094-eO.049 0.169&0.044 p < 0.0001
Day 2
FEV, Chlorphenirsmine
1.79 2.57 1.99 3.28 2.91 3.06 2.57 2.08
2.26kO.61 2..53+0.54 p < 0.05
SG.w base
0.049 0.066 0.094 0.045 0.156 0.124 0.035 0.09
Gnw Chlorpheniramine
0.077 0.093 0.109 0.061 0.197 0.17 0.107 0.105
0.082t0.042 0.115~0.046 p < 0.01
The histamine challenge was performed as described in the section on methods.
reported to occur within 30 to 45 min.‘* We therefore obtained a new baseline 30 min after completion of the inhalation and subsequently did a methacholine challenge.
Day 3
Day 6
The subject took 4 puffs of SCH 1000 (80 mcg) from a metered dose inhaler and 30 min later, after a new baseline (FEV,, TGV, R,,) had been obtained, a histamine challenge as on day 2 was done.
The initial procedure was as on day 5. After chlorpheniramine inhalation however, a histamine challenge was done The order of these days was altered at random. Challenges involving chlorpheniramine were done at the end of the study because of the prolonged effect of this drug.18 The doses of 5 mg of chlorpheniramine and 80 pg of SCH 1000 were chosen as pilot experiments had shown it to be sufficient to prevent bronchial response to histamine and methacholine respectively with the method used.
Day 4 The procedure was the same as on day 3, except that a methacholine challenge was performed instead of histamine.
Day 5 The subjects inhaled 5 mg of chlorpheniramine maleate dissolved in 3.0 ml of saline. This inhalation took 15 min. The maximal bronchodilatation with chlorpheniramine is
RESULTS Fifty-two bronchial challenges were performed in the 9 asthmatic patients. One subject did not complete
122 Woenne et al.
TABLE
IV.
J. ALLERGY
Reproducibility
of the PD,,-FEV,
after
3wk Patient
1 2 3 4 5
Methacholine
23.7 5.49 1.55 41.5 7.3
18.15 7.13 1.47 26.5 20.4
Patient
6 7 8 9 10
Histamine
4.7 14.7 4.38 8.8 0.57
4.9 24.0 5.65 25.8 0.296
the chlorpheniramine part of the study. The results are summarized in Table II. A typical example of the dose-response curves obtained is given in Fig. 1 for one of our subjects. There was no significant shift in the slope or the PDZO-FEVr histamine after SCH 1000 inhalation for the whole group with the use of a paired t test. There was also no significant shift in the slope or the PD2,,-FEVi methacholine after chlorpheniramine inhalation. SCH 1000, 80 pg, blocked the response to methacholine inhalation in all 9 subjects, the maximum dose given being 223.4 cumulative breath units. Chlorpheniramine, 5 mg, blocked the response to 96.85 cumulative breath units of histamine in 7 of 8 subjects. One subject was not completely blocked, but his threshold (PD2,,-FEVJ dose still increased 4.6-fold. There was significant bronchodilatation after SCH 1000 and chlorpheniramine (Table III), although chlorpheniramine proved to be irritating in some patients initially. SCH 1000 produced greater bronchodilatation than chlorpheniraamine. A paired t test taking the mean FEVi and SG,, from the 2 days on which each subject either received SCH 1000 or chlorpheniramine was applied for statistical analysis. There was no significant saline response in our group with respect to FEVr and SG,, (paired t test). None of the subjects showed a change in FEVi larger than 10%. No significant correlation could be detected between baseline FEVi and SG,, and threshold dose necessary to induce a 20% drop in FEVr. Because it was necessary to do the challenges on separate days we looked at the reproducibility of the method in 10 asthmatic subjects. Table IV shows that the PDZOFEVr for both histamine and methacholine is reproducible during a 3-wk period. DISCUSSION The results show that in asthmatic patients bronchial hyperreactivity to methacholine and histamine are mediated by different pathways. SCH 1000 blocks bronchial muscarinic cholinergic receptors and there-
CLIN. IMMUNOL. AUGUST 1978
fore should block the bronchoconstricting effect of vagally induced acetylcholine release. Is One could question whether the dose of SCH 1000 used was sufficient to produce effective blockade. However, the dose decreased bronchomotor tone which has been shown to be partly due to efferent cholinergic influences.*’ Second, the dose was sufficient to block a methacholine challenge. Third, the dose in these children was twice an effective adult dose.*l Finally SCH 1000 is considered to have greater effect than atropine in equivalent doses on bronchial smooth muscle.lg The latter drug by inhalation in small doses has been shown to prevent the bronchoconstrictor response to citric acid9 which is mediated through reflex vagal pathways.” Despite effective vagal blockade, the SCH 1000 did not alter the bronchial response to histamine. Similarly, the bronchial response to methacholine remained unchanged after an amount of antihistamine sufficient to block the bronchial response to histamine had been inhaled. The unaltered sensitivity and reactivity to histamine and methacholine with SCH 1000 and chlorpheniramine, respectively, implies that there is no significant antihistamine effect with the dose of SCH 1000 used and no significant anticholinergic effect with the dose of chlorpheniramine inhaled. The latter is in vivo confirmation of what has been previously found in vitro.** We can only speculate about the precise sites of drug action. Histamine could act on the upper airway via lung irritant receptors23 or directly on the effector organ. 24 We conclude from our data that in human asthmatic patients the important mechanism is direct stimulation of bronchial smooth muscle. Vagal pathways are unlikely to be involved because SCH 1000 had no effect on the dose-response curve of histamine in any of the subjects. If receptors in the upper airways are involved, one would then have to postulate the existence of nonvagally mediated irritant-type receptors for histamine-induced bronchoconstriction because an anticholinergic agent did not alter the response. The method used for inhalation would tend to deposit particles in both the peripheral and the central airways.26 With the higher airway resistance that the subjects had, more of the aerosol would be deposited centrally. It is unlikely that the aerosol was deposited only in the areas in which there is no functional vagal innervation, namely, the terminal bronchioles and alveolar ducts.*’ Therefore the inability to demonstrate a vagal pathway cannot be explained by postulating that vagally mediated upper airway receptors were bypassed.
VOLUME
62
NUMBER
2
The action of methacholine is predominantly muscarinic at postganglionic receptors which are very close to smooth muscle. Thus, in the lung an action at bronchial end organ cholinergic receptors is likely. Because the bronchodilating effect of chlorpheniramine cannot be explained by an anticholinergic action, the increased bronchial tone seen in the asthmatic patients must be, in part, related to increased amounts of histamine present at the Hl receptor sites or to increased sensitivity of these receptors. Bronchodilatation was greater with SCH 1000 than with chlorpheniramine, suggesting that vagal mechanisms are of greater importance for resting bronchomotor tone in asthmatic subjects. Theoretically, the use of a percent decrease in FEV, could account for some error in the results when the baseline is already abnormal. However, Rubinfeld and Painz8 were unable to show a relationship between airway caliber and bronchial reactivity. Studies in our laboratory have also failed to show any correlation between the FEVl and SG,, and the threshold dose for provocation. I62 2g We confirmed this poor correlation in the present study. Despite a bronchodilator effect of SCH 1000 no change in the reactivity or sensitivity to histamine was observed. Similarly there was no alteration in the methacholine dose-response curve when chlorpheniramine was used. These findings make it unlikely that the change in baseline pulmonary function after SCH 1000 and chlorpheniramine altered the sensitivity to the bronchial challenges. The possibility exists that the interpretation of the results might be affected by the fact that the study was done on 6 separate days. However, daily variation could not account for the differences because we have demonstrated that the provocation is highly reproducible even over 3 wk. Although different doses of histamine and methacholine were used they were considered to have a biologically equivalent effect on the airway. In 46 normal subjects studied in our laboratory the percent change in FEVl was the same with the maximum amount of either histamine or methacholine.2g In addition the percentage of asthmatic patients who respond to the two agents is similar.‘6, 2g There have been partial attempts with similar methods to answer the questions we raised with respect to the effects of SCH 1000 and atropineg-I2 on the PD,,-FEV, histamine. However, there has been no attempt to evaluate the possible mechanisms of bronchial hyperreactivity to histamine and methacholine in the same group of asthmatic subjects. Casterline and Evansso used diphenhydramine to dem-
Bronchial
hyperreactivity
123
onstrate that it specifically blocked histamine. Diphenhydramine, however, has strong anticholinergic effect?” making it difficult to draw reliable conclusions on its receptor-blocking capacities. The inability to demonstrate a vagally mediated pathway in this chemically induced bronchoconstriction supports the data of Fish and co-workers’0 with respect to antigen-induced bronchoconstriction. Much of the data that assign a central role to the vagus in the asthmatic response have been obtained in animals. Vagal function in animals is in several respects very different from that in man (e.g., the very strong Hering Breuer reflex in dogs and cats compared with man”‘). We conclude from these data that the major action of histamine and methacholine is directly on bronchial smooth muscle. In view of the differential blockade we observed in human asthmatic subjects, the mechanisms and receptor sites involved in bronchial provocation by histamine and methacholine must be distinctly different. The specific antagonists, chlorpheniramine and SCH 1000, both result in bronchodilatation by acting at these separate sites. Beta agonists on the other hand act at the effector organ that is common to both these pathways and this may explain the observation that these agents are more potent in relieving bronchial obstruction. REFERENCES I
2
3.
4.
5.
6.
7. 8.
9.
De Kock, M. A., Nadel, J. A., Zwi, S., Colebatch, H. J., and Olsen, C. P.: New method for perfusing bronchial arteries: Histamine bronchoconstriction and apnea, J. Appl. Physiol. 21:185, 1966. Gold, W. M., Kessler, G. F., and Yu, D. Y. C.: Role of the vagus nerves in experimental asthma in dogs. J. Appt. Physiol. 33719, 1972. Yu, D. T. C., Galant, S. P., and Gold, W. M.: Inhibition of antigen-induced bronchoconstriction by atropine in asthmatic patients, J. Appl. Physiol. 32:823, 1972. Mills. J. F., Seltick, H., and Widdicombe, J. G.: Activity of lung irritant receptors in pulmonary microembolism, anaphylaxis and drug-induced bronchoconstrictions. J. Physiol. 203:337, 1969. Simonsson, B. G., Jacobs, F. M.. and Nadel, J. A.: Role of autonomicnervoussystem and the cough reflex in the increased responsiveness of airways in patients with obstructive airway disease, J. Clin. Invest. 46: 1812, 1967. Orr, T. S. C., Pollard, M. C., Gwilliam. J., and Cox, J. S. G.: Mode of action of disodium cromoglycate; studies on immediate type hypersensitivity reactions using “double sensitization” with two antigenically distinct reagins, Clin. Exp. Immunol: 7:745, 1970. Itkin, U. H.: Bronchial hypersensitivity to mecholyl and histamine in asthma subjects, J. ALLERGY 40~245, 1967. Parker, C. D., Bilbo, R. E., and Reed, C. E.: Methacholine aerosol as a test for bronchial asthma, Arch. Intern. Med. 1194.52, 1965. Rosenthal. R. R.. Norman. P. S., Summer. W. E., and Per-
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mutt, S.: Role of the parasympathetic system in antigeninduced bronchospasm, J. Appl. Physiol. 42:600, 1977. Fish, J. E., Rosenthal, R. R., Summer, W. R., Menkes, H., Norman, P. S., and Permutt, S.: The effect of atropine on acute antigen-mediated airway constriction in subjects with allergic asthma, Am. Rev. Respir. Dis. 1152371, 1977. Casterline, C. L., Evans, R., III, and Ward, Cl. W.: Theeffect of atropine and albuterol aerosols on the human bronchial response to histamine, J. ALLERGY CLIN. IMMUNOL. 58:607, 1976. Nair, N. S., Bewtra, A. K., Watt, G. D., Burke, K. M., and Townley, R. Cl.: Protection by SCH 1000 and metaproterenol and against bronchoconstriction, Am. Rev. Respir. Dis. 11568, 1977. Weng, T. R., and Levison, H.: Standards of pulmonary function in children, Am. Rev. Respir. Dis. 99:879, 1969. Mitchell, C., Bouhuys, A.: Interaction between bronchoconstrictor stimuli on human airway smooth muscle, Yale J. Biol. Med. 49:317, 1976. Chai, H., et al.: Standardization of bronchial inhalation procedures, J. ALLERGY CLIN. IMMUNOL. 56~323, 197.5. Mitchell, I., Corey, M., Woenne, R., Krastins, I. R. B., and Levison, H.: Bronchial hyperreactivity in cystic fibrosis and asthma, J. Pediatr. (Accepted for publication.) Grohek, J., Gayrard, P., Smith, A. P., Grimaud, C., and Charpin, J.: Airway response to carbachol in normal and asthmatic subjects, Am. Rev. Respir. Dis. 115937, 1977. Popa, V. T.: Bronchodilating activity of an HI blocker, chlorpheniramine, J. ALLERGY CLIN. IMMUNOL. 56~323, 1975. Engelhardt, A., and Klupp, H.: The pharmacology of a new tropane alkaloid derivative, Postgrad. Med. J. Sl(Supp1. 7):82, 1975. Macklem, P. T., Woolcock, A. J., Hogg, J. C., Nadel, J. A., and Wilson, N. J. Partitioning of pulmonary resistance in the dog, J. Appl. Physiol. 26~798, 1969.
J. ALLERGY
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21. Storms, W. W., DePeco, G. A., and Reed, C. E.: Aerosol SCH 1000, Am. Rev. Respir. Dis. 111:419, 1975. 22. Tozzi, S., Roth, F. E., and Tabachnik, I. I. A.: The pharmacology of azatadine, a potential anti-allergic drug, Agents Actions 4:264, 1974. 23. Vidruck, E., Hahn, H. L., Nadel, J. A., and Sampson, S. R.: Location and stimulation of rapidly adapting airway receptors in dogs with a fiberoptic bronchoscope, Physiologist 18~432, 1975. 24. Douglas, W. W.: Histamine and antihistamines, in Goodman, L. S., and Gilman, A., editors: The pharmacological basis of therapeutics, ed. 5, New York, 1975, MacMillan Publishing Company, p. 594 25. Douglas, W. W.: Histamine and antihistamines, in Goodman, L. S., and Gilman, A., editors: The Pharmacologic Basis of Therapeutics, ed. 5, New York, 1975, MacMillan Publishing Company, p. 608. 26. Dolovich, M. B., Sanchis, J., Rossman, C., and Newhouse, M. T.: Aerosol penetrance: A sensitive index of peripheral airway obstruction, J. Appl. Physiol. 40~468, 1976. 27. Widdicombe, J. G.: Regulation of tracheobronchial smoothmuscle, Physiol. Rev. 43: 1, 1963. 28. Rubinfeld, A. R., and Pain, M. C. F. Relationship between bronchial reactivity, airway caliber and severity of asthma, Am. Rev. Respir. Dis. 115:381, 1977. 29. Mellis, C. M., Kattan, M., Keens, T., and Levison, H.: Comparative study of histamine and exercise in asthmatic children, Am. Rev. Respir. Dis. 117~91 I, 1978. 30. Casterline, C. L., and Evans, R.: Further studies on the mechanism of human histamine-induced asthma. The effect of aerosolized HI-receptor antagonist (diphenhydramine), J. ALLERGY CLIN. IMMUNOL. 59~420, 1977. 31. Widdicombe, J. G.: Respiratory reflexes in man and other mammalian species, Clin. Sci. 21: 163, 1961.
Nine asthmatic patients with a mean age qf 14 yr received bronchial challenges with histamine and methacholine. The challenges were repeated after inhalation of 80 /~g of SCH 1000 (ipratropium bromide) and 5 mg of chlorpheniramine maleate. The provocation doses which produced a 20% fall in ,forced expiratory volume in I set (FEV,) and the slopes of the dose-response curves were analyzed. SCH 1000 prevented methacholine-induced bronchoconstriction and chlorpheniramine prevented methacholine-induced bronchoconstriction. There was no significant change in the dose-response curve of histamine ajer SCH 1000 or in the dose-response curve of methacholine after chlorpheniramine. The jindings indicate that the mechanisms and receptor sites involved in bronchial provocarion by histamine and methacholine are distinctly different. The histamine response is unlikely to be vagally mediated because histamine-induced bronchoconstriction was not prevented by SCH 1000. Both SCH 1000 and chlorpheniramine caused significant bronchodilatation, suggesting the presence of both histamine-and vugal-dependent bronchomotor tone.
It has been demonstrated in the experimental animal’, * and in human asthmatic patients3that the responseof the airways to inhaled histamine and antigen is dependenton intact vagal pathways. In animals the vagal responseis of reflex characte? in which the afferent pathway originates at the lung irritant receptors.* A similar reflex mechanismhas been suggested in human beings.” It has also been shown that histamine is releasedtogether with other mediatorsfrom sensitizedmast cells if incubatedwith specific antigen in vitroa Several authors235 have attemptedto combine the above data into a unified schema, whereby antigen exposurein the sensitizedasthmaticpatients results in histamine release from mast cells, which then stimulates lung irritant receptors to cause reflex bronchoconstriction. In this hypothesis the vagus plays a major role and a direct action of histamine on bron-
chial smooth muscle is considered to be of minor importance. Histamine and methacholine are both well-established nonallergenic triggers of bronchoconstriction in the asthmatic subject.7, 8 If the effects of these drugs are predominantly vagally mediated, then the blockade of vagal efferent pathways by a drug such as ipratropium bromide (SCH 1000)) an anticholinergic agent, should prevent a responseto both histamine and methacholine. However, recent evidence casts some doubt on the role of the vagus in histamine provocation,g-‘2 raising the possibility that distinctly separate receptor sites and/or pathways exist for methacholine and histamine. To investigate this possibility we attempted to differentially block methacholine and histamine bronchoconstriction by use of specific antagonists, i.e., an anticholinergic agent (SCH 1000)and an Hl antagonist(chlorpheniramine). SUBJECTS
From the Department of Paediatrics and The Research Institute, The Hospital for Sick Children, University of Toronto. Received for publication Jan. I& 1978. Accepted for publication April 18, 1978. Reprint requeststo: H. Levison, M.D., Hospital for Sick Children, 555 University Ave., Toronto, Ontario, Canada MS3 I X8. “TDeceased. 0091-6749/78/0262-0119$00.60/O
@ 1978 The C. V. Mosby
Co.
Nine moderate to mild asthmatic patients between the
agesof 9 to 16 yr (mean, 14 yr) participated in this study. All were perennial asthmatic patients and had positive skin tests to a large variety of allergens. The mild asthmatic patients (3) were on no therapy; the rest of the patients regularly took beta agents. One patient was also on sodium Vol. 62, No. 2, pp. 119-124
120
Woenne
et al.
J. ALLERGY
TABLE
I. Baseline
Patient
50’
,
,
,
,,
1
,
,
IO CUMULATIVE
,
,,
,
,
,
100
,, loo0
pulmonary
function
% predicted FEV,*
P. P. M. G. H. L. M. C. L. c. M. D. L. L. B. M. M. P.
57 2 16* 69k 7 67 t 11 79* 3 92k 3 97+ 5
81? 41* 81-’
CLIN. IMMUNOL. AUGUST 1978
3 4 5
w3w 0.056 -r- 0.033t 0.080 k 0.062
0.106 + 0.020 0.065 0.124 0.129 0.198 0.038 0.093
+ k k + ? +
0.028 0.056 0.037 0.080 0.012 0.012
DOSE OF METHACHOUNE
*Expressed as percent predicted from normal subjects studied in our laboratory. I3 TMean 2 1 SD of pulmonary function for 6 days of the study.
---’ -.-
Ml-
50
t .I
CHLORPH. CONTROL SCHlmD I
Subiect
III
I
1 CUMULATIVE
1111
I
IO
MC
III1
100
DOSE OF HISTAMINE
FIG. 1. The dose-response curves for one subject with histamine and methacholine. Each graph shows the dose-response curve with pretreatment with SCH 1000 and chlorpheniramine and a control.
cromoglycate, 2 were on theophylline, and 3 patients inhaled steroids from a metered dose inhaler. All patients were regularly attending our laboratory for evaluation of asthma and were thus familiar with the tests used in this study. The baseline pulmonary function data are given in Table I. The subjects were selected on their ability to discontinue medication (except for steroids) for at least 12 hr before the study. Informed consent was obtained in all cases.
METHODS The trial was done single blind at the same hour on 6 days. Each subject completed the study within IO days. Separate days for each challenge were used to avoid drug interference.‘4Pulmonary function studies were obtained with a 9-L Collins water-sealed spirometer and a variablepressure body plethysmograph. A baseline forced expiratory volume in one second (FEVI), thoracic gas volume (TGV), and airways resistance (R,,) were obtained initially and repeated after inhalations of saline, ipratropium bromide (SCH lOOO), and chlorpheniramine maleate, respectively. Specific conductance (SG,,) was determined from R,, and the simultaneously measured TGV. All the measurements were repeated when a 20% or greater drop in FEV, was recorded after the histamine or methacholine
challenge. Methacholine (acetyl-betamethyl choline chloride) and histamine (histamine acid phosphate) challenges were done as recommended by the standardization panel of the Asthma and Allergic Disease Centers, National Institute of Allergy and Infectious Diseases.‘” The concentrations of methacholine used were 0.31, 0.62, 1.25, 2.5, 5.0, 10.0, and 25.0 mg/ml and the concentrations of histamine used were 0.12, 0.25, 0.5, I .O, 2.5, 5.0, and 10.0 mg/ml. Units of inhaled methacholine or histamine were arbitrarily obtained by multiplying inhaled concentrations by the number of breaths taken. Five successive inspiratoty capacity breaths of each concentration were administered. Repeat spirometry was performed 3 min later and immediately the procedure was continued with the next higher concentration until a 20% or greater drop in FEVl was observed with respect to the baseline after saline, SCH 1000, or chlorpheniramine inhalations. From the cumulative amount of methacholine or histamine inhaled (cumulative breaths units) and the accompanying drop in FEVl a dose-response curve for methacholine and histamine was constructed. The provocation dose of methacholine or histamine which induced a 20% drop in FEV, (PDZO-FEV1) was used to compare the bronchial sensitivity to the different challenges. The slope of the dose-response curve was calculated as the rate of fall from a 12% decrease to a 20% decrease in FEV1. A 12% fall was chosen because, from previous experience, this is the point up to which normal children drop.16 The coefficient a from the formula, y = a log10 x + b, was used to reflect this slope. This slope is an index of bronchial reactivity.‘?
EXPERIMENTAL Day 1
PROTOCOL
The subject took 5 successive inspiratoty capacity breaths of saline from a DeVilbiss No. 40 nebulizer (the same nebulizer was used in each subject during the whole study) with an air-flow set at 8 L/min. Three minutes later FEV1, TGV, and R,, were reassessed to detect any response to saline. Subsequently a methacholine challenge was done as described in the section on methods.
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TABLE
Bronchial hyperreactivity
II. Sensitivity
(PD,,-FEV,)
HIS
and reactivity
SCH WOO-HIS
Subject
Phi FEV,
Slope (a)
PDmFEV,
Slope (a)
P. P. M. G. H. L. M. C. L. c. M. D. L. L. B. M. M. P.
24.2 0.26 18.5 13.7 0.26 27.3 3.78 0.26 5.2
21.3 48.4 42.8 58.5 47.7 54.6 51.6 48.5 23.2
21.3 0.32 5.52 11.3 0.24 12.4 4.65 0.13 14.6
50.6 40.0 35.0 36.2 52.0 25.4 39.0 170.6 25.1
Means
10.4
44.1
7.8
52.7
(coefficient
a) with each bronchial
CHL-HIS PDaFEV,
MCH
Slope (al
* * 86.0
20.0 * * * t * *
121
challenge
CHL-MCH
PD*(y FEV,
Slope (a)
PD,,FEV,
Slope (a)
18.15 7.13 1.47 26.5 23.4 23.4 7.3 1.58 9.4
11.4 57.8 113.6 51.3 50.1 7.0 54.7 80.2 33.6
4.65 1.48 1.48 11.8 54.2 5.8 t 2.97 4.65
30.7 126.8 113.6 25.9 167.5 13.6
13.9
50.6
10.9
69.5
61.4 16.8
SCH lOUO-MCH P&y FEV,
Slops (a)
* * * * * 3 * * *
HIS, histamine; CHL, chlorpheniramine; MCH, methacholine. *PD,,,-FEV, not achieved. t Did not complete study. TABLE
Ill. Response
Patient
FEV, base
FEV, SCH
SGaw base
SGw SCH
FEV, base
P. P. M. G. H. L. M. C. L. c. M. C. L. L. B. M. M. P.
2.61 2.21 1.96 3.06 2.82 3.02 2.41 1.33 1.95
2.93 2.9 2.02 3.57 2.91 3.21 2.46 2.0 2.31
0.09 0.043 0.104 0.043 0.119 0.184 0.135 0.033 0.096
0.166 0.128 0.206 0.144 0.18 0.212 0.209 0.083 0.195
1.56 2.31 1.9 3.11 2.75 2.91 1.54 1.99
Mean
to SCH 1000 and chlorpheniramine
2.37ir0.57 2.7kO.54 p < 0.01
0.094-eO.049 0.169&0.044 p < 0.0001
Day 2
FEV, Chlorphenirsmine
1.79 2.57 1.99 3.28 2.91 3.06 2.57 2.08
2.26kO.61 2..53+0.54 p < 0.05
SG.w base
0.049 0.066 0.094 0.045 0.156 0.124 0.035 0.09
Gnw Chlorpheniramine
0.077 0.093 0.109 0.061 0.197 0.17 0.107 0.105
0.082t0.042 0.115~0.046 p < 0.01
The histamine challenge was performed as described in the section on methods.
reported to occur within 30 to 45 min.‘* We therefore obtained a new baseline 30 min after completion of the inhalation and subsequently did a methacholine challenge.
Day 3
Day 6
The subject took 4 puffs of SCH 1000 (80 mcg) from a metered dose inhaler and 30 min later, after a new baseline (FEV,, TGV, R,,) had been obtained, a histamine challenge as on day 2 was done.
The initial procedure was as on day 5. After chlorpheniramine inhalation however, a histamine challenge was done The order of these days was altered at random. Challenges involving chlorpheniramine were done at the end of the study because of the prolonged effect of this drug.18 The doses of 5 mg of chlorpheniramine and 80 pg of SCH 1000 were chosen as pilot experiments had shown it to be sufficient to prevent bronchial response to histamine and methacholine respectively with the method used.
Day 4 The procedure was the same as on day 3, except that a methacholine challenge was performed instead of histamine.
Day 5 The subjects inhaled 5 mg of chlorpheniramine maleate dissolved in 3.0 ml of saline. This inhalation took 15 min. The maximal bronchodilatation with chlorpheniramine is
RESULTS Fifty-two bronchial challenges were performed in the 9 asthmatic patients. One subject did not complete
122 Woenne et al.
TABLE
IV.
J. ALLERGY
Reproducibility
of the PD,,-FEV,
after
3wk Patient
1 2 3 4 5
Methacholine
23.7 5.49 1.55 41.5 7.3
18.15 7.13 1.47 26.5 20.4
Patient
6 7 8 9 10
Histamine
4.7 14.7 4.38 8.8 0.57
4.9 24.0 5.65 25.8 0.296
the chlorpheniramine part of the study. The results are summarized in Table II. A typical example of the dose-response curves obtained is given in Fig. 1 for one of our subjects. There was no significant shift in the slope or the PDZO-FEVr histamine after SCH 1000 inhalation for the whole group with the use of a paired t test. There was also no significant shift in the slope or the PD2,,-FEVi methacholine after chlorpheniramine inhalation. SCH 1000, 80 pg, blocked the response to methacholine inhalation in all 9 subjects, the maximum dose given being 223.4 cumulative breath units. Chlorpheniramine, 5 mg, blocked the response to 96.85 cumulative breath units of histamine in 7 of 8 subjects. One subject was not completely blocked, but his threshold (PD2,,-FEVJ dose still increased 4.6-fold. There was significant bronchodilatation after SCH 1000 and chlorpheniramine (Table III), although chlorpheniramine proved to be irritating in some patients initially. SCH 1000 produced greater bronchodilatation than chlorpheniraamine. A paired t test taking the mean FEVi and SG,, from the 2 days on which each subject either received SCH 1000 or chlorpheniramine was applied for statistical analysis. There was no significant saline response in our group with respect to FEVr and SG,, (paired t test). None of the subjects showed a change in FEVi larger than 10%. No significant correlation could be detected between baseline FEVi and SG,, and threshold dose necessary to induce a 20% drop in FEVr. Because it was necessary to do the challenges on separate days we looked at the reproducibility of the method in 10 asthmatic subjects. Table IV shows that the PDZOFEVr for both histamine and methacholine is reproducible during a 3-wk period. DISCUSSION The results show that in asthmatic patients bronchial hyperreactivity to methacholine and histamine are mediated by different pathways. SCH 1000 blocks bronchial muscarinic cholinergic receptors and there-
CLIN. IMMUNOL. AUGUST 1978
fore should block the bronchoconstricting effect of vagally induced acetylcholine release. Is One could question whether the dose of SCH 1000 used was sufficient to produce effective blockade. However, the dose decreased bronchomotor tone which has been shown to be partly due to efferent cholinergic influences.*’ Second, the dose was sufficient to block a methacholine challenge. Third, the dose in these children was twice an effective adult dose.*l Finally SCH 1000 is considered to have greater effect than atropine in equivalent doses on bronchial smooth muscle.lg The latter drug by inhalation in small doses has been shown to prevent the bronchoconstrictor response to citric acid9 which is mediated through reflex vagal pathways.” Despite effective vagal blockade, the SCH 1000 did not alter the bronchial response to histamine. Similarly, the bronchial response to methacholine remained unchanged after an amount of antihistamine sufficient to block the bronchial response to histamine had been inhaled. The unaltered sensitivity and reactivity to histamine and methacholine with SCH 1000 and chlorpheniramine, respectively, implies that there is no significant antihistamine effect with the dose of SCH 1000 used and no significant anticholinergic effect with the dose of chlorpheniramine inhaled. The latter is in vivo confirmation of what has been previously found in vitro.** We can only speculate about the precise sites of drug action. Histamine could act on the upper airway via lung irritant receptors23 or directly on the effector organ. 24 We conclude from our data that in human asthmatic patients the important mechanism is direct stimulation of bronchial smooth muscle. Vagal pathways are unlikely to be involved because SCH 1000 had no effect on the dose-response curve of histamine in any of the subjects. If receptors in the upper airways are involved, one would then have to postulate the existence of nonvagally mediated irritant-type receptors for histamine-induced bronchoconstriction because an anticholinergic agent did not alter the response. The method used for inhalation would tend to deposit particles in both the peripheral and the central airways.26 With the higher airway resistance that the subjects had, more of the aerosol would be deposited centrally. It is unlikely that the aerosol was deposited only in the areas in which there is no functional vagal innervation, namely, the terminal bronchioles and alveolar ducts.*’ Therefore the inability to demonstrate a vagal pathway cannot be explained by postulating that vagally mediated upper airway receptors were bypassed.
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The action of methacholine is predominantly muscarinic at postganglionic receptors which are very close to smooth muscle. Thus, in the lung an action at bronchial end organ cholinergic receptors is likely. Because the bronchodilating effect of chlorpheniramine cannot be explained by an anticholinergic action, the increased bronchial tone seen in the asthmatic patients must be, in part, related to increased amounts of histamine present at the Hl receptor sites or to increased sensitivity of these receptors. Bronchodilatation was greater with SCH 1000 than with chlorpheniramine, suggesting that vagal mechanisms are of greater importance for resting bronchomotor tone in asthmatic subjects. Theoretically, the use of a percent decrease in FEV, could account for some error in the results when the baseline is already abnormal. However, Rubinfeld and Painz8 were unable to show a relationship between airway caliber and bronchial reactivity. Studies in our laboratory have also failed to show any correlation between the FEVl and SG,, and the threshold dose for provocation. I62 2g We confirmed this poor correlation in the present study. Despite a bronchodilator effect of SCH 1000 no change in the reactivity or sensitivity to histamine was observed. Similarly there was no alteration in the methacholine dose-response curve when chlorpheniramine was used. These findings make it unlikely that the change in baseline pulmonary function after SCH 1000 and chlorpheniramine altered the sensitivity to the bronchial challenges. The possibility exists that the interpretation of the results might be affected by the fact that the study was done on 6 separate days. However, daily variation could not account for the differences because we have demonstrated that the provocation is highly reproducible even over 3 wk. Although different doses of histamine and methacholine were used they were considered to have a biologically equivalent effect on the airway. In 46 normal subjects studied in our laboratory the percent change in FEVl was the same with the maximum amount of either histamine or methacholine.2g In addition the percentage of asthmatic patients who respond to the two agents is similar.‘6, 2g There have been partial attempts with similar methods to answer the questions we raised with respect to the effects of SCH 1000 and atropineg-I2 on the PD,,-FEV, histamine. However, there has been no attempt to evaluate the possible mechanisms of bronchial hyperreactivity to histamine and methacholine in the same group of asthmatic subjects. Casterline and Evansso used diphenhydramine to dem-
Bronchial
hyperreactivity
123
onstrate that it specifically blocked histamine. Diphenhydramine, however, has strong anticholinergic effect?” making it difficult to draw reliable conclusions on its receptor-blocking capacities. The inability to demonstrate a vagally mediated pathway in this chemically induced bronchoconstriction supports the data of Fish and co-workers’0 with respect to antigen-induced bronchoconstriction. Much of the data that assign a central role to the vagus in the asthmatic response have been obtained in animals. Vagal function in animals is in several respects very different from that in man (e.g., the very strong Hering Breuer reflex in dogs and cats compared with man”‘). We conclude from these data that the major action of histamine and methacholine is directly on bronchial smooth muscle. In view of the differential blockade we observed in human asthmatic subjects, the mechanisms and receptor sites involved in bronchial provocation by histamine and methacholine must be distinctly different. The specific antagonists, chlorpheniramine and SCH 1000, both result in bronchodilatation by acting at these separate sites. Beta agonists on the other hand act at the effector organ that is common to both these pathways and this may explain the observation that these agents are more potent in relieving bronchial obstruction. REFERENCES I
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