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(Chest. 2005;128:1248-1251.)
© 2005 American College of Chest Physicians

Lack of Tachyphylaxis to Methacholine at 24 h*

Donald W. Cockcroft, BSc, MD, FCCP and Beth E. Davis, BSc

Department of Medicine, Division of Respirology, Critical Care, and Sleep Medicine, University of Saskatchewan, Royal University Hospital, Saskatoon, SK, Canada.

Correspondence to: Donald W. Cockcroft, BSc, MD, FCCP, Royal University Hospital, Division of Respirology, Critical Care and Sleep Medicine, 103 Hospital Dr, Ellis Hall, Fifth Floor, Saskatoon, SK, S7N 0W8 Canada; e-mail: cockcroft{at}sask.usask.ca


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Objective: To examine for tachyphylaxis to methacholine at 24 h and to use these data to assess the repeatability of the provocative concentration of a substance causing a 20% fall in FEV1 (PC20) for methacholine and to obtain statistical power calculations.

Design: Retrospective review of four double-blind, placebo-controlled studies with two methacholine PC20 values measured at 24-h intervals.

Setting: Tertiary university hospital bronchoprovocation laboratory.

Patients: Thirty-two subjects with mild-to-moderate well-controlled asthma.

Interventions: The placebo arms of the four studies were examined.

Measurements: Methacholine PC20 (using 2-min tidal breathing method) initial determination and repeat testing at 24 h.

Results: The geometric mean PC20 values were 1.57 mg/mL (95% confidence interval [CI], 1.0 to 2.4 mg/mL) and 1.62 mg/mL (95% CI, 1.0 to 2.6 mg/mL NSD; p = 0.64). The mean absolute difference between the two measurements was < 0.4 doubling concentrations, and 31 of 32 measurements had both values within one doubling concentration. These data provide a statistical power of 92% for 10 subjects to show a one-half concentration {Delta}PC20 and a mean power of 99 ± 1% to show a one-concentration {Delta}PC20.

Conclusions: There is no evidence for methacholine tachyphylaxis at 24 h in subjects with asthma. At 24 h, the average repeatability was well within a one-half concentration change, and individually 31 of 32 measurements (97%) were within one doubling concentration.

Key Words: methacholine • power calculation • repeatability • tachyphylaxis • tolerance


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Subjects with airway responsiveness in the borderline to normal range exhibit tolerance to the bronchoconstrictive effect of methacholine when tests are repeated within a few hours.123 Minor tolerance has been shown in healthy subjects at 24 h.1 In clinical research protocols, we are frequently asked whether we are certain that there is no tachyphylaxis to methacholine-induced bronchoconstriction in subjects with asthma when methacholine challenges are repeated at 24 h. Reviewers of protocols and manuscripts have also requested information, if not data, on the repeatability of the methacholine challenges performed in our laboratory and on the statistical power of our studies, the latter being dependent on repeatability. In order to address these questions, we have done a retrospective analysis on all double-blind, placebo-controlled clinical trials in which methacholine challenges were repeated at 24-h intervals in our laboratory.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Study Design
We identified four clinical trials4567 in which methacholine challenges were performed at 24-h intervals after double-blind administration of placebo. In the studies involving several methacholine challenges performed at 24-h intervals,456 we chose the first two measurements to avoid any possibility of selection bias. The three published studies were evaluated in reverse chronologic order456 followed by the study by Davis and Cockcroft,7 which had not been published at the time of the analysis. Subjects who participated in more than one trial were selected on their first encounter, again to avoid any possibility of selection bias.

Methacholine Challenge
Methacholine challenge was performed using the standardized 2-min tidal breathing method.89 Aerosols were generated with a nebulizer (Twin jet nebulizer; Puritan Bennett Corporation; Carlsbad, CA) that was calibrated to deliver an output of 0.13 mL/min. The nose was clipped, and the aerosols were administered via a loose-fitting facemask and inhaled for 2 min with tidal breathing. Saline solution inhalation was followed by the inhalation of doubling concentrations of methacholine from 0.03 to 32 mg/mL. Full spirograms were performed in triplicate at baseline. Following each inhalation, a single technically acceptable truncated spirogram to obtain FEV1 was obtained at 30 and 90 s. The timing between inhalations was constant at 5 min from the start of one to the start of the next concentration. The percentage decline in FEV1 was calculated from the lowest post-saline solution inhalation value to the lowest post-methacholine inhalation value, and the provocative concentration of a substance causing a 20% fall in FEV1 (PC20) was calculated from an algebraic formula.10 Starting concentrations were determined based on the approximate known airway responsiveness and were identical for the two challenges.

Statistical Analysis
Statistical analysis was done using a statistical software package (STATISTIX for Windows; Analytical Software; Tallahassee, FL). Since methacholine PC20 values are log normally distributed,11 the PC20 values were log-transformed prior to analysis. We compared the first (baseline) PC20 value with the 24-h PC20 value using a paired t test. Using standard statistical methods, we looked at repeatability by determining the coefficient of variability.12 We also looked at the practical issue of the average and maximal absolute difference in duplicate determinations. Finally, we used standard statistical methods to apply these data to power calculations.13


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We identified 32 subjects who had duplicate methacholine challenge results at 24-h intervals. There were 13 men, 19 women, with a mean (± SEM) age of 26.9 ± 1.5 years (age range, 17 to 54 years), and a mean height of 170 ± 2 cm (range, 138 to 185 cm). The mean baseline FEV1 was 3.47 ± 0.15 L (range, 2.17 to 5.85 L) or 92.6 ± 2.1% predicted (range, 68 to 122% predicted), and the baseline geometric mean pretrial PC20 was 1.1 mg/mL (95% confidence interval [CI], 0.72 to 1.7 mg/mL; range, 0.07 to 7.5 mg/mL).

The geometric mean PC20 was 1.57 mg/mL (95% CI, 1.0 to 2.4 mg/mL) at baseline and 1.62 mg/mL (95% CI, 1.0 to 2.6 mg/mL) at 24 h (p = 0.64). A regression plot of the baseline vs the 24-h methacholine PC20 is shown in Figure 1 .



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  		Figure 1. Baseline methacholine PC20 (horizontal axis) vs 24-h methacholine PC20 both in milligrams per milliliter log scale. Dotted line = 95% CI of the sample; solid lines = predicted 95% CI of the population (plus or minus one doubling concentration approximately).

 
The coefficient of variability was 31%. The mean absolute difference in log PC20 was 0.139, equating to a 0.38 doubling concentration. An inspection of the individual data (Fig 1) showed that both determinations within one doubling concentration in 31 of the 32 subjects (97%).

The SD of the difference between the two determinations of log PC20 was 0.169. From this value, we calculated that a study size of 10 subjects has a 92% power to detect a one-half concentration change in PC20 (ie, {Delta} log PC20, 0.18) and a 99% (+) power to detect a one doubling dose change in methacholine PC20 (ie, {Delta} log PC20, 0.30).


   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
These data provide objective documentation that there is no tolerance to methacholine at 24 h across a wide range of airway responsiveness values between borderline responsiveness (ie, PC20, 4 to 8 mg/mL) and marked responsiveness (ie, PC20, < 0.25 mg/mL). The data also show that the test has a reasonable repeatability with a mean difference between two determinations of less than one doubling concentration and a difference in individual values of less than one doubling concentration in 97% of the subjects. A sample size of 10 subjects has a 92% power to detect a one-half doubling concentration change in PC20 and a 99% power to detect a full doubling concentration change in PC20.

There are some previous data to suggest tolerance to methacholine at 24 h. Beckett et al1 performed five methacholine challenges in 24 h in subjects without asthma. Challenges were performed at 0, 4, 8, 12, and 24 h, and a significant reduction in the response to methacholine was seen at all four time periods when compared to time 0.1 They also demonstrated a statistically significant increase, but likely not a clinically significant increase, in the methacholine-induced rise in specific airway resistance when methacholine challenges were repeated at 24-h intervals, again in nonasthmatic subjects. Stevens et al,2 using the same methacholine method described in this article, were able to demonstrate tolerance to the effect of methacholine at 3 and 6 h only in subjects whose methacholine PC20 was > 16 mg/mL but not in subjects (similar to those reported here) whose methacholine PC20 was < 8 mg/mL. In contrast to methacholine (and acetylcholine), short-term tolerance to histamine-induced bronchoconstriction is relatively easy to demonstrate in subjects with mild asthma and mild airway hyperresponsiveness.14 This does not occur in subjects with asthma and more significant airway hyperresponsiveness.15

The data presented here refer only to PC20 challenge tests that were repeated at 24 h. There is a possibility that challenges repeated at shorter intervals might show tachyphylaxis. This study did not address methacholine PC20 challenge tests that were repeated for several days at 24-h intervals. However, three of the four studies from which these data were extracted involved daily challenges for 4 days45 or 5 days.6 A review of these publications did not suggest any trend for reduced responsiveness.

These data do not preclude the possibility that other aspects of the methacholine dose-response curve (eg, the slope and plateau) might have changed. However, the PC20 (or provocative dose of a substance causing a 20% fall in FEV1) is the most commonly used end point both for research and for clinical studies.

These data have practical value. They will provide useful published data that validate the common practices in research laboratories. For example, we have long stated that a one-half concentration change in the mean PC20 was the minimum clinically significant difference within a group of subjects and that a one-concentration change in PC20 was the minimum clinically significant difference within an individual. These arbitrary cutpoints are given reasonable support by the data presented here.

We have done our best to avoid any selection bias, as outlined in the "Materials and Methods" section. Nevertheless, these subjects have participated in one and sometimes more clinical trials (ie, several of the subjects have been well-trained in the methods). The laboratory also has vast experience with using this method. It is therefore possible that the repeatability of the test might not be quite so good in clinical laboratories routinely testing subjects who have less experience with bronchoprovocation. It is also likely that the repeatability of the test over longer time periods (ie, weeks) might not be as good.1617 Therefore, in clinical practice, we would recommend that the minimum clinically relevant change in methacholine PC20 within an individual should probably be arbitrarily established at two or more doubling concentrations.

In summary, we have presented data to support the lack of tachyphylaxis to methacholine administered at 24-h intervals in subjects with asthma, and have used these results to provide repeatability data and power calculations for the measurement of methacholine PC20.


   Acknowledgements
 
The authors thank Jacquie Bramley for assisting in the preparation of this manuscript.


   Footnotes
 
Abbreviations: CI = confidence interval; NSD = no significant difference; PC20 = provocative concentration of a substance causing a 20% fall in FEV1

Received for publication January 17, 2005. Accepted for publication February 7, 2005.


   References
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Beckett, WS, McDonnell, WF, III, Wong, ND (1988) Tolerance to methacholine inhalation challenge in nonasthmatic subjects. Am Rev Respir Dis 137,1499-1501[ISI][Medline]
  2. Stevens, WH, Manning, PJ, Watson, RM, et al Tachyphylaxis to inhaled methacholine in normal but not asthmatic subjects. J Appl Physiol 1990;69,875-879[Abstract/Free Full Text]
  3. Haber, AD, Beckett, WS Indomethacin and methacholine tolerance in normal subjects: role of prostaglandin synthesis in the attenuation of methacholine’s effect during repeated inhalation challenges. Chest 1992;102,856-863[Abstract/Free Full Text]
  4. Bhagat, R, Kalra, S, Swystun, VA, et al Rapid onset of tolerance to the bronchoprotective effect of salmeterol. Chest 1995;108,1235-1239[Abstract/Free Full Text]
  5. Kalra, S, Swystun, VA, Bhagat, R, et al Inhaled corticosteroids do not prevent the development of tolerance to the bronchoprotective effect of salmeterol. Chest 1996;109,953-956[Abstract/Free Full Text]
  6. Davis, BE, Reid, JK, Cockcroft, DW Formoterol thrice weekly does not result in the development of tolerance to bronchoprotection. Can Respir J 2003;10,23-26[Medline]
  7. Davis, BE, Cockcroft, DW Effect of single dose of montelukast sodium on methacholine chloride PC20. Can Respir J 2005;12,26-28[Medline]
  8. Cockcroft, DW, Killian, DN, Mellon, JJ, et al Bronchial reactivity to inhaled histamine: a method and clinical survey. Clin Allergy 1977;7,235-243[CrossRef][ISI][Medline]
  9. Crapo, RO, Casaburi, R, Coates, AL, et al Guidelines for methacholine and exercise challenge testing-1999: this official statement of the American Thoracic Society was adopted by the ATS Board of Directors, July 1999. Am J Respir Crit Care Med 2000;161,309-329[Free Full Text]
  10. Cockcroft, DW, Murdock, KY, Mink, JT Determination of histamine PC20: comparison of linear and logarithmic interpolation. Chest 1983;84,505-506[Free Full Text]
  11. Cockcroft, DW, Berscheid, BA, Murdock, KY Unimodal distribution of bronchial responsiveness to inhaled histamine in a random human population. Chest 1983;83,751-754[Abstract/Free Full Text]
  12. Bland, M An introduction to medical statistics. 1995,267-268 Oxford University Press. Oxford, UK:
  13. Bland, M An introduction to medical statistics. 1995,143-144 Oxford University Press. Oxford, UK:
  14. Manning, PJ, Jones, GL, O’Byrne, PM Tachyphylaxis to inhaled histamine in asthmatic subjects. J Appl Physiol 1987;63,1572-1577[Abstract/Free Full Text]
  15. Connolly, MJ, Stenton, SC, Avery, AJ, et al Refractory period following bronchoconstriction provoked by histamine in asthmatic subjects. Thorax 1989;44,146-150[Abstract]
  16. Dehaut, P, Rachiele, A, Martin, RR, et al Histamine dose-response curves in asthma: reproducibility and sensitivity of different indices to assess response. Thorax 1983;38,516-522[Abstract]
  17. Trigg, CJ, Jhalli, N, Herdman, MJ, et al The daily variability of bronchial responsiveness to methacholine. Eur Respir J 1990;3,867-871[Abstract]




This Article
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